Treatments for multiple sclerosis

Abstract

The present invention relates to RG6035, a bispecific antibody against human CD20 and human transferrin receptor, for use in the treatment of multiple sclerosis.

Description

[0001] TREATMENTS FOR MULTIPLE SCLEROSIS

[0002] TECHNICAL FIELD

[0003] The present invention relates to RG6035, a bispecific antibody against human CD20 and human transferrin receptor, for use in the treatment of multiple sclerosis.

[0004] BACKGROUND OF THE INVENTION

[0005] Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) that affects approximately 2.2 million people worldwide. It is the most common inflammatory neurological disease in young adults (GBD 2016 Multiple Sclerosis Collaborators, 2019).

[0006] MS is clinically subcategorized into different clinical phenotypes: relapsing remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS) (Lublin et al. 2014). These three phenotypes are further subdivided into active and non active forms based on the presence or absence of disease activity, defined by the presence of clinical relapses and / or so-called active lesions on a magnetic resonance imaging (MRI) scan compared with those from a previous scan. Relapsing MS (RMS) forms encompass RRMS and active SPMS, and progressive MS (PMS) forms constitute non-active SPMS and PPMS (Lublin et al., 2014). However, all evidence available to date suggest that RRMS, PPMS, and SPMS belong to the same disease spectrum (Lassman, 2019).

[0007] Historically, it was believed that CNS tissue damage in MS was mediated by infiltrating proinflammatory CD4+ T-cells (Furuzawa-Carballeda et al., 2007; Sospedra and Martin, 2005). However, it is now known that B-cells are also key contributors to MS immunopathology, as demonstrated by the observed clinical efficacy of CD20-targeting, peripherally B-cell-depleting therapies in relapsing-remitting MS (Chisari et al., 2021 ; Daneman and Prat, 2015; Gasperi et al., 2016; Freskgard and Urich, 2017; Gelfand et al., 2017; Johnsen et al., 2019; Greenfield and Hauser, 2018; Klein et al., 2013; Jakimovski et al., 2017; Al-Sawaf et al., 2017; Montalban et al., 2017; Herter et al., 2013; Sabatino et al., 2019). Recent publications suggest that CNS-compartmentalized B-cells are particularly detrimental in progressive MS (PMS) (Howell et al., 2011). The presence of B-cell-rich lymphoid-like structures in PMS is associated with an unfavourable disease course, including cortical demyelination, brain atrophy, microglia activation, as well as synaptic and neuronal loss (Howell et al., 2011 ; Reali et al., 2020).

[0008] Most of the more recently approved high efficacy therapies for MS target B-cells. The importance of this mode of action has been reinforced with the successful development of B-cell-depleting monoclonal antibodies (mAbs) that target the CD20 surface antigen, such as ocrelizumab and ofatumumab, both of which are examples of Type I anti-CD20-mAbs (Sabatino et al. 2019). Type I anti-CD20-mAbs indirectly eliminate B-cells in the periphery by engaging a range of effector mechanisms, such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), for example, which may not efficiently work in the CNS due to the lack of complement factors and fewer effector cells (Komori et al.2016). By eliminating peripheral B-cells, however, they efficiently prevent acute attacks (relapses) in MS via the indirect killing of B-cells that wash out of the brain, but this has only a limited effect on disability progression. Therefore, disease progression still represents a high unmet medical need.

[0009] Unlike Type I anti-CD20-mAbs, Type II anti-CD20-mAbs, such as obinutuzumab, predominantly target B-cells through direct cellular cytotoxicity mechanisms, inducing Fc-effector independent, non-apoptotic, direct B cell death (Klein et al. 2013). Type II anti-CD20-mAbs are thought to have better killing potential of B-cells in the CNS due to relying on mechanisms that are more suited to the unique immune environment of the CNS.

[0010] Another key hurdle in the development of novel mAb therapies remains their delivery to the brain, and specifically the compartmentalized B-cells, through the blood brain barrier (BBB). Due to the large size of mAbs, only a small percentage (approximately 0.03% to 0.2%) of peripherally administered antibodies, such as ocrelizumab, reaches the brain (Poduslo et al. 1994; Yu and Watts 2013).

[0011] The Applicant has developed a molecular Brainshuttle™ that effectively engages transferrin receptor 1 (TfR1) on the BBB endothelium to mediate transcytosis to enhance transport of therapeutic antibodies across the BBB, and thus achieve higher exposure and potency within CNS niches (Niewoehner et al.

[0012] 2014).

[0013] As described herein, the Applicant has invented RG6035: a fusion between obinutuzumab and the Brainshuttle™ module, wherein the Fc-region of obinutuzumab was made Fc-region-effector-function-silent by introducing the P329G / L234A / L235A mutation (PGLALA mutation). This improves the safety whilst retaining B-cell depleting properties (Herter et al., 2018). Importantly, obinutuzumab containing the PGLALA mutation is surprisingly still able to deplete B-cells in whole blood and to mediate anti-tumour efficacy in xenograft models in contrast to Fc-region-effector-function-silenced version of other anti-CD20 antibodies that lost B-cell depletion activity in absence of Fc-region-effector-functions. Thus, RG6035 has an advantageous safety profile compared with an Fc-region-effector-function-competent construct, while maintaining the ability to cross the BBB and to deplete B-cells.

[0014] As described herein, the clinical study BP42230 (NCT05704361) is the first study where RG6035 is administered to humans. A non-randomized, open-label, adaptive design was chosen to assess the safety, tolerability, and immunogenicity, as well as characterization of the pharmacokinetics (PK) and pharmacodynamics (PD) following single ascending doses of RG6035 administered intravenously (IV) to patients with MS. Subsequent non-randomized, open-label, adaptive design studies assessing the safety, tolerability, and immunogenicity, as well as characterization of the pharmacokinetics (PK) and pharmacodynamics (PD) were conducted, investigating single ascending doses (SAD) and multiple ascending doses (MAD) of RG6035 administered subcutaneously (SC) to patients with MS.

[0015] Whilst new treatments of MS show promise, there remains a need in the art for optimisation of the treatments, for example the determination of a dose or dosing regimen that optimises treatment outcomes for patients (including effective depletion of B cells whilst minimising adverse events), whilst simultaneouslyaddressing issues such as patient compliance and convenience. Furthermore, prior to the disclosure of the present application, the acute effectiveness of RG6035 and its specific effectiveness at achieving certain clinical outcomes remained to be determined.

[0016] SUMMARY OF THE INVENTION

[0017] The present invention is based, at least in part, on clinical data that has found that RG6035 for use in the treatment of multiple sclerosis results in the surprisingly rapid, direct depletion of B-cells in the central nervous system (CNS), for example as determined by measuring B-cell depletion in the cerebral spinal fluid (CSF). Based on the clinical data, the present invention provides for a treatment regime that is well tolerated and is surprisingly effective at triggering B-cell depletion in the central nervous system (CNS). RG6035 does not cause any profound or sustained B-cell depletion in the blood.

[0018] Accordingly, in one aspect of the present invention there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg, 2000 mg, or 4000 mg.

[0019] The present invention also provides a method of treating multiple sclerosis in a subject in need thereof, wherein the treatment comprises administering RG6035 at a dose of 700 mg, 2000 mg, or 4000 mg to a subject in need thereof.

[0020] In some embodiments of the present invention the 700 mg, 2000 mg, or 4000 mg dose of RG6035 is administered intravenously.

[0021] In some embodiments, the 700 mg, 2000 mg, or 4000 mg dose of RG6035 is effective to deplete B-cells in the CNS of the subject. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 50%, for example when the (first) dose of RG6035 is 700 mg. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 80%, for example when the (first) dose of RG6035 is 2000 mg. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 80%, for example when the (first) dose of RG6035 is 4000 mg. In some embodiments, the depletion of B-cells in the CNS is determined by comparing the number or concentration of B-cells in the CSF prior to the administration of the dose of RG6035, and the number or concentration of B-cells in the CSF after the (first) administration of RG6035. Hence, references to the depletion of B-cells in the CNS herein may be expressed as or measured by the depletion of B-cells in the CSF.

[0022] Accordingly, in some embodiments the 700 mg, 2000 mg, or 4000 mg dose of RG6035 is effective to deplete B-cells in the CSF of the subject. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 50%, for example when the (first) dose of RG6035 is 700 mg. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 80%, for example when the (first) dose of RG6035 is 2000 mg. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 80%, for example when the(first) dose of RG6035 is 4000 mg. In some embodiments, the depletion of B-cells in the CSF is determined by comparing the number or concentration of B-cells in the CSF prior to the administration of the dose of RG6035, and the number or concentration of B-cells in the CSF after the (first) administration of RG6035.

[0023] In some embodiments, the depletion of B-cells in the CNS or in the CSF is achieved within 14 days following administration of the dose of RG6035. In some embodiments, the CSF to serum ratio of RG6035 following the administration of the dose of RG6035 is 0.5% or greater. In some embodiments, the CSF to serum ratio of RG6035 following the administration of the dose of RG6035 is 1% or greater. In some embodiments, the B-cells are CD19 positive B-cells. In some embodiments, the B-cells are CD20 positive B-cells. In some embodiments, the B-cells are CD19 positive and CD20 positive B-cells. In some embodiments, CD19 positive B cells may be used as a proxy for total B-cells.

[0024] In some embodiments, the treatment comprises administering the RG6035 at a first dose on day 1 . The first dose of the RG6035 may be an induction dose. Induction doses may also be referred to as a priming dose.

[0025] In some embodiments, the treatment comprises administering the RG6035 at a first dose, which is an induction dose, followed by one or more subsequent maintenance doses of RG6035. Generally, the amount of RG6035 administered for each maintenance dose is the same. Preferably, the induction dose is administered intravenously, and the one or more subsequent maintenance doses are administered subcutaneously. In some embodiments, the 700 mg, 2000 mg, or 4000 mg dose is the induction dose, and the treatment further comprises administering one or more subsequent maintenance doses of RG6035. In some embodiments, the one or more subsequent maintenance doses of RG6035 are each 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg. Preferably, the induction dose of RG6035 is 2000 mg. Preferably, the one or more subsequent maintenance doses of RG6035 are each 300 mg. Preferably, the one or more subsequent maintenance doses of RG6035 are each 100 mg.

[0026] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 100 mg.

[0027] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 150 mg.

[0028] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 300 mg.

[0029] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 600 mg.

[0030] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 700 mg.In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 100 mg.

[0031] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 150 mg.

[0032] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 300 mg.

[0033] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 600 mg.

[0034] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 700 mg.

[0035] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 100 mg.

[0036] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 150 mg.

[0037] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 300 mg.

[0038] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 600 mg.

[0039] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 700 mg.

[0040] In some embodiments, the one or more subsequent maintenance doses of RG6035 are Q1W. In some embodiments, the one or more subsequent maintenance doses of RG6035 are Q2W. In some embodiments, the one or more subsequent maintenance doses of RG6035 are Q3W. In some embodiments, the one or more subsequent maintenance doses of RG6035 are Q4W. In preferred embodiments, the one or more subsequent maintenance doses of RG6035 may be Q2W.

[0041] In some embodiments, the maintenance doses are administered on day 8 and every 7 days thereafter. In some embodiments, the induction dose is administered on day 1 , and the maintenance doses are administered on day 15 and every 14 days thereafter. In some embodiments, the induction dose is administered on day 1 , and the maintenance doses are administered on day 22 and every 21 days thereafter. In some embodiments, the induction dose is administered on day 1 , and the maintenance doses are administered on day 29 and every 28 days thereafter.In some embodiments the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0042] In some embodiments the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0043] In some embodiments the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every week starting on day 8 and every 7 days thereafter. In a particularly preferred embodiment, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0044] In some embodiments, RG6035 comprises a first polypeptide with the amino acid sequence of SEQ ID NO: 01, a second polypeptide with the amino acid sequence of SEQ ID NO: 02, a third polypeptide with the amino acid sequence of SEQ ID NO: 03 and a fourth polypeptide with the amino acid sequence of SEQ ID NO: 05.

[0045] In some embodiments, the multiple sclerosis is relapsing multiple sclerosis or progressive multiple sclerosis. In some embodiments, the multiple sclerosis is relapsing multiple sclerosis. In some embodiments, the multiple sclerosis progressive multiple sclerosis. In some embodiments, the progressive multiple sclerosis is primary progressive MS (PPMS), or secondary progressive MS (SPMS)In some embodiments, no anti-drug antibodies to RG6035 are detected in the CSF following administration of the dose of RG6035 for at least about 50 days following the administration of the dose of RG6035. In some embodiments, the RG6035 does not induce the production of anti-drug antibodies in the subject for at least about 50 days following the administration of the dose of RG6035. In some embodiments, no antidrug antibodies to RG6035 are detected in the CSF following administration of the dose of RG6035 for at least about 25 days following the administration of the dose of RG6035. In some embodiments, the RG6035 does not induce the production of anti-drug antibodies in the subject for at least about 25 days following the administration of the dose of RG6035.

[0046] In some embodiments, RG6035 is for administration in combination with ocrelizumab. In some embodiments, RG6035 is for subsequent, sequential or separate administration with ocrelizumab. In some embodiments, RG6035 is for subsequent, sequential or separate administration with ocrelizumab, wherein the ocrelizumab is administered before the first dose of RG6035. In some embodiments, ocrelizumab is administered before the first dose of RG6035.

[0047] In embodiments in which ocrelizumab is administered before the first dose of RG6035, no further doses of ocrelizumab are administered after the first dose of RG6035.

[0048] In some embodiments, the depleted B-cells are CNS-compartmentalized and / or meningeal B-cells. In some embodiments, RG6035 induces Fc-effector-independent B-cell death upon binding CD20 on B-cells.

[0049] The present invention also provides RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg, 150 mg, 300 mg, 600 mg or 700 mg.

[0050] The present invention also provides a method of treating multiple sclerosis in a subject in need thereof, wherein the treatment comprises administering RG6035 at a dose of 100 mg, 150 mg, 300 mg, 600 mg or 700 mg to a subject in need thereof.

[0051] The present invention also provides compositions comprising RG6035. The compositions may be used in the dosing regimens or methods of treatment of the invention. For example, the present invention provides a composition comprising RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg, 2000 mg, or 4000 mg. The present invention also provides a method of treating multiple sclerosis in a subject in need thereof, wherein the treatment comprises administering a composition comprising to a subject in need thereof, wherein the treatment comprises administering RG6035 at a dose of 700 mg, 2000 mg, or 4000 mg. The present invention provides a composition comprising RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg. The present invention also provides a method of treating multiple sclerosis in a subject in need thereof, wherein the treatment comprises administering a composition comprising to a subject in need thereof, wherein the treatment comprises administering RG6035 at a dose of 100 mg, 150 mg, 300 mg, 600 mg or 700 mg.The compositions provided and used in the invention may be pharmaceutical compositions, which may further comprise one or more pharmaceutically acceptable excipients.

[0052] The present invention provides compositions, in particular pharmaceutical compositions, that are suitable for use in the medical uses and methods of treatment disclosed herein.

[0053] Medical uses and methods of the present disclosure are performed on a subject indeed thereof, i.e. treatments are administered to a subject in need thereof.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 - (A) Fluorescence-activated cell sorting (FACS) analysis of Z-138 cell binding with Brainshuttle™-CD20 and control mAbs. A fluorescein isothiocyanate-labelled F(ab)’2 goat anti-human Fcy-specific antibody was used for detection, and the median fluorescence intensity (MFI) was measured. (B) Direct cell killing induced by binding of Brainshuttle™ CD20 and control mAbs to Z-138 cells measured by annV assay and PI uptake. Results expressed as uptake of IgG normalised to total cell protein (C) hTfR1-mediated intracellular uptake of Brainshuttle™-CD20 and control mAbs into MDCKII-hTfR1 and MDCKII-parental cells (non-hTfR1 expressing), normalised to total cell protein. No detectable uptake of either compound by MDCKII-parental cells was observed, confirming that mAb uptake in hTfR1 -transfected cells was mediated by hTfR1 . Data represents the mean ±SD of triplicate measurements in (A)-(C).

[0056] Figure 2 - Transcytosis efficiency derived from the mean amount of IgG transcytosed vs. the mean amount of IgG in the corresponding intracellular compartment at the beginning (t=0) of the chase (expressed as a percentage). Data represents the mean ±SD of triplicate measurements.

[0057] Figure 3 - Mean (± SD) by dose groups serum concentration-time profiles of RG6035 following a single IV administration of 7, 20, 70, 200, 700 and 2000 mg, semi-log scale.

[0058] Figure 4 - (A) Fold change in blood in B-cell frequencies over time per participant in Cohorts 1-6 (Part 1 study). (B) Fold change in blood in B-cell frequencies over time in each SAD IV Part 1 Cohorts 1-6 (mean + SD). (C) Fold change in blood in B-cell frequencies over time in each SAD IV Part 1 Cohorts 1-7 (mean + SD).

[0059] Figure 5 - (A) Fold change in CSF B-cells frequencies over time per participant in Cohorts 5 and 6 (Part 1 study). (B) Fold change in CSF B-cells frequencies over time per participant in Cohorts 5, 6 and 7 (Part 1 study).

[0060] Figure 6 - Individual serum concentration-time profiles of RG6035 following a single SC administration of 70 mg in linear and semi-log scale.

[0061] Figure 7 - (A) Individual serum concentration-time profiles of RG6035 following a single SC (with initial plotted RG6035 concentrations of around 1 ug / mL) and IV (with initial plotted RG6035 concentrations ofaround 10-50 ug / mL) administration of 70 mg in semi-log scale. (B) Mean serum concentration-time profiles of RG6035 following a single SC (with initial RG6035 concentration of less than 1 ug / mL) and IV (with initial RG6035 concentration of around 10-50 ug / mL)) administration of 70 mg in semi-log scale.

[0062] Figure 8 - Predicted typical concentration-time profiles of RG6035 following a single SC administration of 70 mg in semi-log scale, assuming different scenarios for rate and extent of absorption with 35% (red), 50% (green), 73% (light blue) or 93% bioavailability, with superimposed medians of observed data.

[0063] Figure 9 - Individual profiles of B-cells fold change from baseline versus time after single SC (N=6) and IV (N=9) dose administration of 70 mg.

[0064] Figure 10 - Individual CSF concentration-time profiles of RG6035 after single IV dose of either 700 mg or 2000 mg (Cohorts 5 and 6, Part 1).

[0065] Figure 11 - Individual serum concentration-time profiles of RG6035 following multiple SC administration of 70 mg in linear and semi-log scale (N=6).

[0066] Figure 12 - Predicted typical concentration-time profiles of RG6035 following multiple SC administration of 70 mg, assuming different scenarios for rate and extent of absorption with 35% (red), 50% (green), or 73% (light blue) bioavailability, with superimposed medians and inter-quartile range of observed data (broken line).

[0067] Figure 13 - (A) Individual profiles of B-cells in blood presented as fold change from baseline versus time after SC administration of multiple doses of 70mg RG6035 (N=6). (B) Percent B-Cells in CSF presented as fold change from baseline (screening) and post-treatment after SC administration of multiple doses of 70mg RG6035 (N=6).

[0068] Figure 14 - Predicted RG6035 serum concentrations and CSF B-cell depletion over time after 4 SC doses of 70 mg, 200 mg and 700 mg RG6035, respectively.

[0069] Figure 15 - BS-CD20 - Schematic representation of an anti-CD20 x anti-TfR1 bispecific antibody in “Roche Brainshuttle™” format.

[0070] Figure 16 - Mean (± SD) by dose groups serum concentration-time profiles of RG6035 following a single IV administration of 7, 20, 70, 200, 700, 2000, and 4000 mg, semi-log scale.

[0071] Figure 17 - Mean (± SD) by dose groups serum concentration-time profiles of RG6035 following multiple SC administration of 70 mg, 200 mg and 700 mg, semi-log scale.

[0072] Figure 18 - (A) Mean (± standard error) fold change in blood B cells (in frequencies) from baseline versus time by cohort, after multiple SC administration of RG6035. Multiple SC administration (at Day 1 , Day 8, Day 15 and Day 22) of 70 mg (N=7), 200 mg (N=8) and 700 mg (N=8). (B) Individual profiles of B cells inCSF following multiple SC administration of 70 mg (N=3 / 7), 200 mg (N=3 / 8), and 700 mg (N=1 / 7) RG6035 presented as fold change in frequencies from baseline over time.

[0073] DETAILED DESCRIPTION

[0074] The present invention provides for a treatment regimen involving the administration of RG6035 using a dose or dosing regimen that is surprisingly effective at providing a rapid depletion of B-cells in the CNS of patients with MS, even after a single dose of RG6035. A measure of the number of B-cells in the CSF may be used as a proxy for the number of B-cells in the CNS. Hence, depletion of B-cells in the CNS may be detected by measuring a depletion of B-cells in the CSF. The administration of RG6035, even after a high induction dose, surprisingly does not induce the production of anti-drug antibodies at least until around day 50 after first administration and is well tolerated, with minimal adverse events. Moreover, the RG6035 is efficiently transported across the blood brain barrier, triggering its therapeutic effects in the CNS (and may be transported across the blood brain barrier more efficiently than other compounds). Still further, RG6035 performs surprisingly better than other MS treatments, including those that deplete B-cells. For example, RG6035 may provide a more effective treatment and / or more rapid response in a patient compared to ocrelizumab.

[0075] The present invention also provides RG6035 for use in the treatment of MS, wherein the treatment comprises administering the RG6035 at a first dose, which is an induction dose, followed by one or more subsequent maintenance doses. Based on the clinical data provided herein, the present invention provides for a treatment regime that is well tolerated and causes a very rapid depletion of B-cells in the CNS. RG6035 does not cause any profound or sustained B-cell depletion in the blood.

[0076] DEFINITIONS

[0077] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular, and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0078] General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0079] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)and is referred to as “numbering according to Kabat” herein. Specifically, the Kabat numbering system (see pages 647-660) of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) is used for the light chain constant domain CL of kappa and lambda isotype, and the Kabat EU index numbering system (see pages 661-723) is used for the constant heavy chain domains (CH1 , Hinge, CH2 and CH3, which is herein further clarified by referring to “numbering according to Kabat EU index” in this case).

[0080] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0081] Unless otherwise defined herein the term “comprising of’ shall include the term “consisting of’.

[0082] The term “about” as used herein in connection with a specific value (e.g. temperature, concentration, time and others) shall refer to a variation of + / - 1 % of the specific value that the term “about” refers to.

[0083] The "blood-brain-barrier" or "BBB" refers to the physiological barrier between the peripheral blood circulation and the brain and spinal cord that is formed by tight junctions within the brain capillary endothelial plasma membranes, creating a tight barrier that restricts the transport of molecules into the brain, even very small molecules such as urea (60 Daltons). The BBB within the brain, the blood-spinal-cord-barrier within the spinal cord, and the blood-retinal-barrier within the retina are contiguous capillary barriers within the CNS, and are herein collectively referred to as the blood-brain-barrier or BBB. The BBB also encompasses the blood-CSF-barrier (choroid plexus) where the barrier is comprised of ependymal cells rather than capillary endothelial cells.

[0084] The terms “anti-human CD20 antibody” and “antibody specifically binding to human CD20” refer to an antibody that is capable of binding human CD20 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD20. Notably, the anti-CD20 antigen binding portion of an anti-CD20 antibody binds to CD20 with a higher affinity that it binds to any other antigen.

[0085] The “CD20” antigen is an approx. 35 kDa, non-glycosylated phosphoprotein found on the surface of greater than 90 % of B-cells from peripheral blood or lymphoid organs. CD20 is expressed during early pre-B-cell development and remains until plasma cell differentiation. CD20 is present on both normal B-cells as well as malignant B-cells. Other names for CD20 in the literature include “B-lymphocyte-restricted antigen” and “Bp35” and “MS4A1” (Comi et al., 2021). The CD20 antigen is described in Clark et al. Proc. Natl. Acad. Sci USA 82 (1985) 1766, for example. See also SEQ ID NO: 06.

[0086] The “CD19” antigen is an approximately 95 kDa transmembrane glycoprotein found on the surface of B-cells. CD19 is expressed on a broader range of B-cells than CD20, capturing B-cell maturation states frompro-B-cell to plasmablasts (Pieper et al., 2013). CD19 positivity may be used to identify total B-cells of interest, and may be measured as surrogates for CD20+ B-cells.

[0087] An “autoimmune disease” herein is a non-malignant disease or disorder arising from and directed against an individual's own tissues. Examples of autoimmune diseases or disorders include, but are not limited to, multiple sclerosis.

[0088] An “antagonist” is a molecule that upon binding to a B-cell surface marker destroys, kills or depletes B-cells in a mammal and / or interferes with one or more B-cell functions, e.g. by reducing or preventing a humoral response elicited by the B-cell. The antagonist is able to deplete B-cells (i.e. reduce circulating B-cell numbers or levels) in a mammal treated therewith. Such depletion may be achieved via various mechanisms such antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC), inhibition of B-cell proliferation and / or induction of direct B-cell death (e.g. via apoptosis). In the CNS, B-cell depletion may occur via ADCC.

[0089] Antagonists which “induce apoptosis” are those which induce programmed cell death, e.g. of a B-cell, as determined by standard apoptosis assays, such as binding of Annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and / or formation of membrane vesicles (called apoptotic bodies).

[0090] The “cerebral spinal fluid” or “CSF” refers to the fluid that is contained within the ventricles of the brain and within meningeal tissue that surrounds the vertebrate brain and spinal cord.

[0091] A "blood-brain-barrier receptor" (BBBR) is an extracellular membrane-linked receptor protein expressed on brain endothelial cells that is capable of transporting molecules across the BBB or can be used to transport exogenous administered molecules. Examples of BBBR include the transferrin receptor 1 (TfR1).

[0092] The "transferrin receptor 1" ("TfR1") is a transmembrane glycoprotein (with a molecular weight of about 180,000 Da) composed of two disulphide-bonded sub-units (each of apparent molecular weight of about 90,000 Da) involved in iron uptake in vertebrates. In certain embodiments of all aspects and embodiments of the current invention, the TfR1 as mentioned herein is human TfR1 comprising the amino acid sequence as in Schneider et al. (Nature 311 (1984) 675-678), for example.

[0093] “Brainshuttle™” is a registered trademark. “Brainshuttle™”, “BS”, “BS module” or other terms used herein refers to a domain swapped Fab fragment that specifically binds to transferrin receptor 1 (TfR1). The Fab fragment may be conjugated to an antibody to increase transport of the antibody across the blood brain barrier. The Fab fragment may be conjugated to the antibody via a linker, for example a GlySer linker. BS-CD20 as used herein refers to a multispecific antibody that binds both the B-cell antigen CD20 via two Fab regions on the antibody and to TfR1 via an anti-TfR1 domian swapped Fab fragment conjugated via a linker to the C terminus of one of the heavy chains of the anti-CD20 antibody. The BS module therefore allow the conjugated anti-CD20 antibody to cross the blood brain barrier (BBB). RG6035 as such comprises a BS module.A "multispecific antibody" denotes an antibody having binding specificities for at least two different antigens. Exemplary multispecific antibodies may bind both a BBBR (for example TfR1) and a B-cell antigen (for example CD20). Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies) or combinations thereof (e.g. full length antibody plus additional scFv or Fab fragments). Engineered antibodies with two, three or more (e.g. four) functional antigen binding sites have also been reported (see, e.g., US 2002 / 0004587).

[0094] The term "antibody" herein is used to encompass various antibody structures, including but not limited to monoclonal antibodies and multispecific antibodies (e.g., bispecific antibodies) so long as they exhibit the desired antigen-binding activity.

[0095] The term "antibody-dependent cellular cytotoxicity (ADCC)" is a function mediated by Fc receptor binding and refers to lysis of target cells by an antibody in the presence of effector cells. ADCC can be measured by the treatment of a preparation of CD19 expressing erythroid cells (e.g. K562 cells expressing recombinant human CD19) with an antibody in question in the presence of effector cells such as freshly isolated peripheral blood mononuclear cells (PBMCs) or purified effector cells from buffy coats, like monocytes or natural killer (NK) cells. Target cells are labelled with Cr51 and subsequently incubated with the antibody in question. The labelled cells are incubated with effector cells and the supernatant is analysed for released Cr51. Controls include the incubation of the target endothelial cells with effector cells but without the antibody. The capacity of the antibody in question to induce the initial steps mediating ADCC is investigated by measuring their binding to Fey receptors expressing cells, such as cells, recombinantly expressing FcyRI and / or FcyRIIA or NK cells (expressing essentially FcyRIIIA).

[0096] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

[0097] The term “fragment antigen-binding fragment” or “Fab fragment” refers to the region of an antibody that binds to antigens. The Fab region of an antibody refers to the fragment antigen-binding region comprising a light chain and a portion of a heavy chain, wherein the light chain consists of a variable domain (VL) and a constant domain (CL), and the heavy chain consists of a variable domain (VH) and the first constant domain (CH1). The Fab region includes the complementarity-determining regions (CDRs) responsible for antigen binding but lacks the Fc region of the antibody. A Fab fragment may be “domain swapped” and as such may be a domain swapped Fab fragment. In such a Fab fragment, the CL domain and the CH1 domain are exchanged, such that the domain swapped Fab fragment comprises a first chain consisting of the VL domain and the CH1 domain, and a second chain consisting of the VH domain and the CL domain. The domain swapped Fab fragment may retain specific binding to an antigen, such as TfR1.

[0098] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may befurther divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, gamma, epsilon and miu, respectively. RG6035 is of the human lgG1 class.

[0099] The term "complement-dependent cytotoxicity (CDC)" refers to lysis of cells induced by an antibody in question in the presence of complement. CDC can be measured by the treatment of CD19 expressing human endothelial cells with an antibody in question in the presence of complement. The cells are labelled with calcein. CDC is found if the antibody in question induces lysis of 20 % or more of the target cells at a concentration of 30 pg / ml. Binding to the complement factor C1q can be measured in an ELISA. In such an assay, an ELISA plate is coated with concentration ranges of the antibody in question, to which purified human C1q or human serum is added. C1q binding is detected by an antibody directed against C1q followed by a peroxidase-labelled conjugate. Detection of binding (maximal binding Bmax) is measured as optical density at 405 nm (OD405) for peroxidase substrate ABTS® (2,2'-azino-di-[3-ethylbenzthiazoline-6-sulfonate) (Obermeier et al., 2008).

[0100] “Effector function” refers to those biological activities attributable to the Fc-region of an antibody, which vary with the antibody class. Examples of antibody effector functions include C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B-cell receptor); and B-cell activation.

[0101] Fc receptor binding dependent effector functions can be mediated by the interaction of the Fc-region of an antibody with Fc receptors (FcRs), which are specialized cell surface receptors on hematopoietic cells and their descendants. Fc receptors belong to the immunoglobulin superfamily, and have been shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g. tumour cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC) (see e.g. Van de Winkel, J.G. and Anderson, C.L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: Fc receptors for IgG antibodies are referred to as FcyR. Fc receptor binding is described e.g. in Ravetch, J.V. and Kinet, J.P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P.J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin. Med. 126 (1995) 330-341 ; and Gessner, J.E., et al., Ann. Hematol. 76 (1998) 231-248.

[0102] Mapping of the binding sites on human lgG1 for Fc receptors, the above mentioned mutation sites and methods for measuring binding to FcyRI and FcyRIIA are described in Shields, R.L., et al., J. Biol. Chem.

[0103] 276 (2001) 6591-6604.

[0104] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0105] The term “Fc-region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc-regions and variant Fc-regions. A human IgG heavy chain Fc-region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc-region may or may not be present.

[0106] An “Fc-region of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies. The Fc-region of RG6035 is of the human lgG1 subclass comprising the effector function eliminating mutations L234A, L235A and P329G (numbering according to EU index of Kabat).

[0107] The term “full-length antibody” is used herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc-region as defined herein. A "full-length antibody" is an antibody that comprises the antigen-binding variable regions VH and VL as well as a light chain constant domain (CL) and heavy chain constant domains, CH1 , CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variants thereof. In more detail, a full-length antibody comprises two antibody light chains (each comprising a variable domain and a constant domain) and two antibody heavy chains (each comprising a variable domain, a hinge region and three constant domains including the CH2 and CH3 domains). The C-terminal amino acid residues K or GK may be present or not independently of each other in the two antibody heavy chains of a full-length antibody. A full-length antibody may have additional components or regions conjugated to it (for example an additional Fab region, such as a BS module).

[0108] CD20 mAbs may be grouped into two groups based on their mechanisms of action. “Type I CD20 antibodies” refer to CD20 mAbs that predominantly act through complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). For example, type I anti-CD20 antibody binding leads to CD20 clustering, by bridging two CD20 molecules due to the binding of the two Fabs of the anti-CD20 antibody to one CD20 molecule each forming a CD20 dimer, which in turn induces good complement activation. Meanwhile, “type II CD20 antibodies” refer to CD20 mAbs that predominantly act through direct cellular cytotoxicity mechanisms, such as programmed cell death (PCD), and ADCC mechanisms.

[0109] The term “anti-drug antibodies” or “ADAs” refers to an antibody that binds to a drug. ADAs can alter a drug’s pharmacokinetic and pharmacodynamic properties, reducing drug efficacy. In more severe cases, ADAs can neutralise the drug’s therapeutic effects or cause severe adverse events to the patient (Vaisman-Mentesh et al., 2020). ADAs are generated by a T-cell dependent or independent B-cell activation pathway. In the T-cell dependent pathway, mAbs act as antigens and are internalised by antigen presenting cells (APCs), processed, and presented to T cells via the cognate interaction between the MHC class II molecules and T-cell receptor. Depending on the cytokine environment, these stimulated T cells may be activated, and in turn, following their cognate interactions with B cells, induces the proliferation of plasma cells that secrete ADAs. In the T cell independent pathway mAbs with multiple epitopes can crosslink B-cell receptors and stimulate B-cells to differentiate into plasma cells to produce ADAs.

[0110] The term “adverse effects”, “adverse events” or “AEs”, according to the E2A ICH guideline for Good Clinical Practice, refers to any untoward medical occurrence in a participant or clinical investigation participant administered a pharmaceutical product and which does not necessarily have to have a causal relationship with this treatment. An AE can therefore be any unfavourable and unintended sign (including an abnormallaboratory finding), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. “Serious adverse events” or “SAEs” are defined as any untoward medical occurrence that at any dose: results in death; is life-threatening; requires inpatient hospitalisation or prolongation of existing hospitalisation; results in persistent or significant disability or incapacity; is a gongenital anomaly or birth defect; or other significant events.

[0111] “CSF:serum ratio” or “CSF to serum exposure ratio” refers to the area under the concentration-time curve [AUC] for measuring and comparing drug uptake into brain vascular endothelial cells and parenchyma from the blood serum. A positive CSF:serum ratio relates to a greater penetrative ability of the drug through the BBB and uptake into the CSF from the blood.

[0112] “Area under the curve” or “AUC” refers to the area under the plasma concentration versus time curve, and is a pharmacokinetic measure that indicates the exposure to a drug, such as RG6035. AUCo-i68h refers to the area under the serum concentration-time curve from 0 to 168 hours; AUCinf refers to area under the serum concentration-time curve from time 0 to infinity; AUCiast refers to area under the concentration-time curve up to last measurable concentration.

[0113] “Az” refers to the terminal rate constant calculated by linear regression of the log transformed terminal part of the concentration-time curve.

[0114] “Cmax” refers to the maximum serum concentration observed for a drug, such as RG6035, that has been administered to a subject. “Tmax” refers to time to maximum observed concentration of a drug, such as RG6035, that has been administered to a subject.

[0115] “CNS-compartmentalised B-cells” are associated with grey matter pathology and clinical progression in MS. “Meningeal B-cells” are located in the meninges of the brain. It is thought that efficient depletion of CNS-compartmentalised and meningeal B cells, in addition to peripheral B cells, might lead to further reduction of progressive CNS tissue damage in MS, and with that, further reduce the risk of disability progression in those who suffer with MS.

[0116] “B-cell depletion” refers to a reduction in B-cell number or concentration via the indirect or direct killing of B-cells. As described herein, B-cell depletion may be induced by anti-CD20 antibodies through various mechanisms.

[0117] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which one or more exogenous nucleic acid(s) have been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.A “humanised” antibody refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions and amino acid residues from human frameworks.

[0118] A “humanised form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanisation.

[0119] The term “hypervariable region” or “HVR”, as used herein, refers to each of the regions of an antibody variable domain comprising the amino acid residue stretches which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and / or form structurally defined loops (“hypervariable loops”), and / or contain the antigen-contacting residues (“antigen contacts”). Generally, antibodies comprise six HVRs; three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3).

[0120] HVRs include

[0121] a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, A.M., J. Mol. Biol. 196 (1987) 901-917); b) CDRs occurring at amino acid residues 24-34 ( L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91- 3242.);

[0122] c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262 (1996) 732-745); and d) combinations of (a), (b), and / or (c), including amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0123] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0124] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain preferred embodiments of all aspects and embodiments according to the current invention, the individual or subject is a human.

[0125] An "isolated" antibody is one, which has been separated from a component of its natural environment. In some embodiments of all aspects and embodiments according to the current invention, an antibody is purified to greater than 95 % or 99 % purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC). For review of methods for assessment of antibody purity, see, e.g., Flatman, S., et al., J. Chrom. B 848 (2007) 79-87.

[0126] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0127] “Isolated nucleic acid encoding an anti-human CD20 / human transferrin receptor antibody” refers to one or more nucleic acid molecules encoding the antibody heavy and light chains of the antibody, including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

[0128] The term "monoclonal antibody" or “mAb” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0129] "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1 , CH2, and CH3), whereby between the first and the second constant domain a hinge region is located. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (A), based on the amino acid sequence of its constant domain.

[0130] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0131] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments of all aspects and embodiments according to the current invention, RG6035 is used to delay development of a disease or to slow the progression of a disease, especially of multiple sclerosis.

[0132] The term “variable region” or “variable domain” refers to the domain of an antibody’s heavy or light chain that is involved in binding of the antibody to the antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T.J. et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91).

[0133] The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0134] Day 1 refers to the first day of the treatment regimen, i.e. the day that the first administration of the drug, such as RG6035, is given to the subject. All subsequent time points for patient monitoring or the administration of subsequent maintenance doses are calculated from this day.

[0135] “Q1W” refers to the administration of a drug, such as RG6035, once every week. “Q2W” refers to the administration of a drug, such as RG6035, once every 2 weeks. “Q3W” refers to the administration of a drug, such as RG6035, once every three weeks. “Q4W” refers to the administration of a drug, such as RG6035, once every 4 weeks.

[0136] “TI / 2“ refers to the apparent terminal half-life of a drug, such as RG6035, computed as ln(2) / Az.

[0137] RG6035 construct

[0138] RG6035 is a fusion between obinutuzumab and the Brainshuttle™ module, wherein the Fc-region of obinutuzumab was made Fc-region-effector-function-silent by introducing the P329G / L234A / L235A mutation (PGLALA mutation). This improves the safety whilst retaining B-cell depleting properties (Herter et al., 2018). Importantly, obinutuzumab containing the PGLALA mutation is still able to deplete B-cells in whole blood and to mediate anti-tumour efficacy in xenograft models in contrast to Fc-region-effector-function-silenced version of other anti-CD20 antibodies that lost B-cell depletion activity in absence of Fc-region-effector-functions. RG6035 is therefore an anti-CD20 antibody fused via the C-terminus of one ofthe heavy chains to a domain swapped Fab fragment that specifically binds to TfR1. The structure of RG6035 is depicted in Figure 15.

[0139] Thus, RG6035 has an advantageous safety profile compared with an Fc-region-effector-function-competent construct, while maintaining the ability to cross the BBB and to deplete B-cells.

[0140] RG6035 is based on a human immunoglobulin G1 isotype, containing two different heavy chains (“HC1”, SEQ ID NO: 2, and “HC2”, SEQ ID NO: 5) and two different light chains (“LC1”, SEQ ID NO: 3 and “LC2”, SEQ ID NO: 1). There are two copies of LC2 (SEQ ID NO: 1) in the final RG6035 construct, thus there are five polypeptides in total.

[0141] RG6035 is comprised of one fragment antigen-binding (Fab, SEQ ID NO: 4 and SEQ ID NO: 3) binding human Transferrin Receptor (C-terminal of HC2 plus LC1) and two Fabs binding human CD20 (HC1 and N-terminal of HC2 plus 2 x LC2). HC2 contains the HC part of the TfR binding Fab as a C-terminal fusion via an G2(S)(G4(S))x3 linker (GGSGGGGSGGGGSGGGGS (SEQ ID NO: 7)).

[0142] Selective pairing of cognate heavy and light chains is promoted via charge-pair substitutions in the CD20 binding Fab located at the CH1-CL interface. CH1 and CL kappa domain are exchanged in the TfR binding Fab to promote correct pairing. The anti-CD20 Fab contains two charge substitutions in the heavy chain HC1 and HC2 (K147E and K213E; Eu numbering, Edelman et al. 1969) and two substitutions in the light chain (E126R and Q127K; Eu numbering, Edelman et al. 1969).

[0143] Heterodimerization of the two half-antibodies is driven by “knobs-into-holes” mutations of the CH3 domain of the Fc region. The anti-CD20 anti-TfR heavy chain (HC2) carrying the “knob” contains two mutation (T366W and S354C; Eu numbering), while the anti-CD20 only heavy chain with the “hole” (HC1) bears four mutations (T366S, L368A, Y407V and Y349C; Eu numbering).

[0144] RG6035 contains the proprietary “PG LALA” mutations (L234A, L235A, P329G, Eu numbering, Edelman et al. 1969) in the Fc part preventing binding to Fey receptors and activation of the complement cascade (C1q binding), consequently, preventing Fc-effector function without changing functional binding of the neonatal Fc receptor (FcRn).

[0145] In more detail, RG6035 is a trivalent, bispecific antibody comprising

[0146] a) one full-length antibody comprising two pairs each of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding sites formed by each of the pairs of the full-length heavy chain and the full-length light chain specifically bind to human CD20, and

[0147] b) one additional Fab that is fused to the C-terminus of one heavy chain of the full-length antibody of a), wherein the binding site of the additional Fab specifically binds to human transferrin receptor 1 ,wherein each of the full-length antibody light chains comprises in the constant light chain domain at position 123 the amino acid residue arginine (instead of the wild-type glutamic acid residue; E123R mutation) and at position 124 the amino acid residue lysine (instead of the wild-type glutamine residue; Q124K mutation) (numbering according to Kabat),

[0148] wherein each of the full-length antibody heavy chains comprises in the first constant heavy chain domain at position 147 a glutamic acid residue (instead of the wild-type lysine residue; K147E mutation) and at position 213 a glutamic acid residue (instead of the wild-type lysine amino acid residue; K213E mutation) (numbering according to Kabat),

[0149] wherein the additional Fab specifically binding to human transferrin receptor 1 comprises a domain crossover such that the constant light chain domain and the constant heavy chain domain 1 are replaced by each other.

[0150] Thus, RG6035 is composed of four polypeptides that have the amino acid sequence of SEQ ID NO: 01 , SEQ ID NO: 02, SEQ ID NO: 03 and SEQ ID NO: 05.

[0151] In one embodiment of all aspects and embodiments according to the current invention, RG6035 is a trivalent, bispecific antibody comprising two polypeptides that comprise the amino acid sequence of SEQ ID NO: 01 , one polypeptide that comprises the amino acid sequence of SEQ ID NO: 02, optionally with additional N-terminal glutamine (Q) or pyroglutamic acid (pE) residue or / and optionally with additional C-terminal lysine (K) amino acid residue, one polypeptide that comprises the amino acid sequence of SEQ ID NO: 03, and one polypeptide that comprises the amino acid sequence of SEQ ID NO: 04, optionally with additional N-terminal glutamine (Q) or pyroglutamic acid (pE) residue.

[0152] In one preferred embodiment of all aspects and embodiments according to the current invention, RG6035 is a trivalent, bispecific antibody comprising two polypeptides that comprise the amino acid sequence of SEQ ID NO: 01 , one polypeptide that comprises the amino acid sequence of SEQ ID NO: 02, optionally with additional N-terminal glutamine (Q) or pyroglutamic acid (pE) residue and / or optionally with additional C-terminal lysine (K) amino acid residue, one polypeptide that comprises the amino acid sequence of SEQ ID NO: 03, and one polypeptide that comprises the amino acid sequence of SEQ ID NO: 05, optionally with additional N-terminal glutamine (Q) or pyroglutamic acid (pE) residue.

[0153] In one embodiment of all aspects and embodiments according to the current invention, RG6035 is a trivalent, bispecific antibody comprising two polypeptides that each comprise the amino acid sequence of SEQ ID NO: 01 , one polypeptide that comprises the amino acid sequence of SEQ ID NO: 02, one polypeptide that comprises the amino acid sequence of SEQ ID NO: 03, and one polypeptide that comprises the amino acid sequence of SEQ ID NO: 05.

[0154] In one embodiment of all aspects and embodiments according to the current invention, RG6035 is a trivalent, bispecific antibody comprising two polypeptides that each comprise an amino acid sequenceconsisting of the sequence of SEQ ID NO: 01 , one polypeptide that comprises an amino acid sequence consisting of the sequence of SEQ ID NO: 02, optionally with an additional N-terminal glutamine (Q) or pyroglutamic acid (pE) residue and / or optionally with an additional C-terminal lysine (K) amino acid residue, one polypeptide that comprises an amino acid sequence consisting of the sequence of SEQ ID NO: 03, and one polypeptide that comprises an amino acid sequence consisting of the sequence of SEQ ID NO: 05, optionally with additional N-terminal glutamine (Q) or pyroglutamic acid (pE) residue.

[0155] RG6035 specifically binds to CD20 and to TfR1. More specifically, RG6035 specifically binds to human and cyno CD20, and to human and cyno TfR1.

[0156] SEQ ID NO: 01 (RG6035 light chain 2) has the amino acid sequence:

[0157] DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC.

[0158] SEQ ID NO: 01 is a light chain comprising a variable light region that specifically binds to CD20, plus a CL domain. RG6035 comprises two polypeptides that comprise the sequence of SEQ ID NO: 1.

[0159] SEQ ID NO: 02 (RG6035 heavy chain 1) has the amino acid sequence:

[0160] VQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINVWRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPR EPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0161] SEQ ID NO: 02 is a heavy chain comprising a variable heavy region that specifically binds to CD20, plus the CH1 , CH2 and CH3 domains.

[0162] SEQ ID NO: 03 (RG6035 light chain 1) has the amino acid sequence:

[0163] AIQLTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQNYASSNVDNTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSC.

[0164] SEQ ID NO: 3 comprises a variable light region that specifically binds to TfR1 , plus a CH1 domain (part of the domain swapped Fab)SEQ ID NO: 04 (RG6035 Fab fragment) has the amino acid sequence:

[0165] SMQESGPGLVKPSQTLSLTCTVSGFSLSSYAMSWIRQHPGKGLEWIGYIWSGGSTDYASWAKSRVTISK TSTTVSLKLSSVTAADTAVYYCARRYGTSYPDYGDASGFDPWGQGTLVTVSSASVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC.

[0166] SEQ ID NO: 4 comprises a variable heavy region that specifically binds to TfR1 , plus a CL domain (part of the domain swapped Fab).

[0167] SEQ ID NO: 05 (RG6035 heavy chain 2) has the amino acid sequence:

[0168] VQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINVWRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRV TITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPR EPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSGGGGSGGGGSGGGGSQSMQESGPGLVKPS QTLSLTCTVSGFSLSSYAMSWIRQHPGKGLEWIGYIWSGGSTDYASWAKSRVTISKTSTTVSLKLSSVTA ADTAVYYCARRYGTSYPDYGDASGFDPWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASWCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.

[0169] SEQ ID NO: 5 is a heavy chain comprising a variable heavy region that specifically binds to CD20, plus the CH1 , CH2 and CH3 domains, plus a GlySer linker fusing the heavy chain at its C terminus to the sequence of SEQ ID NO: 4. As such, SEQ ID NO: 5 comprises a heavy chain comprising a variable heavy region that specifically binds to CD20, plus the CH1 , CH2 and CH3 domains, plus a GlySer linker, plus a variable heavy region that specifically binds to TfR1 , plus a CL domain.

[0170] SEQ ID NO: 06 (human CD20) has the amino acid sequence:

[0171] MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGL LMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKIS HFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWK RTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPI ENDSSP

[0172] In the event of any discrepancy between the sequences provided in the sequence listing and the sequences provided in the description, the sequences in the description should prevail.In one embodiment of all aspects and embodiments of the current invention, RG6035 is a monoclonal trivalent, bispecific antibody that specifically binds to CD20 and TfR1.

[0173] THERAPEUTICALLY EFFECTIVE AMOUNT

[0174] RG6035 was selected to enter a clinical study, BP42230 (NCT05704361), which was the first-in-human clinical study for patients with MS.

[0175] Aspects of the present invention come as a result of the surprising outcomes of the clinical study. More specifically, the present invention provides for a treatment regimen involving the administration of RG6035 using a dose or dosing regimen at a dose of 700 mg, 2000 mg, or 4000 mg that is surprisingly effective at providing a rapid and sustained depletion of B-cells in the CNS of patients with MS, even after a single dose of RG6035. What was also surprising was that even after a high induction dose of RG6035, anti-drug antibodies to RG6035 were not detected in treated subjects for at least about 50 days. Overall, RG6035 was surprisingly well tolerated, with minimal adverse events.

[0176] Aspects of the present invention therefore comprise administering to a subject a composition comprising a therapeutically effective amount of RG6035. Preferably, the subject is a human. As used herein, the term a “therapeutically effective amount” refers to an amount of RG6035 sufficient to produce a desired therapeutic effect of rapid depletion of B-cells in the CNS of a subject, whilst minimising adverse events, and simultaneously addressing issues such as patient compliance and convenience. Depletion of B-cells in the CNS may be measured by measuring the B-cells in the CSF. Measurement of B-cells may comprise determine the number or concentration of CD19 positive cells in a sample obtained from a subject (for example a CSF sample).

[0177] In certain embodiments, RG6035 is administered to the subject at a dose of 700 mg, 2000 mg, or 4000 mg. In some embodiments, RG6035 is administered to the subject at a dose of 700 mg. In some embodiments, RG6035 is administered to the subject at a dose of 4000 mg. In preferred embodiments, RG6035 is administered to the subject at a (first) dose of 2000 mg.

[0178] In certain embodiments, RG6035 is administered intravenously to the subject at a dose of 700 mg, 2000 mg, or 4000 mg. In some embodiments, RG6035 is administered intravenously to the subject at a dose of 700 mg. In some embodiments, RG6035 is administered intravenously to the subject at a dose of 4000 mg. In preferred embodiments, RG6035 is administered intravenously to the subject at a (first) dose of 2000 mg.

[0179] In some embodiments, the 700 mg, 2000 mg, or 4000 mg dose of RG6035 is administered on day 1. In some embodiments, the 700 mg dose of RG6035 is administered on day 1. In some embodiments, the 2000 mg dose of RG6035 is administered on day 1. In some embodiments, the 4000 mg dose of RG6035 is administered on day 1 .In some embodiments, the 700 mg, 2000 mg, or 4000 mg dose of RG6035 is administered intravenously on day 1. In some embodiments, the 700 mg dose of RG6035 is administered intravenously on day 1. In some embodiments, the 2000 mg dose of RG6035 is administered intravenously on day 1. In some embodiments, the 4000 mg dose of RG6035 is administered intravenously on day 1.

[0180] DEPLETION OF B-CELLS IN THE CNS FOLLOWING A SINGLE DOSE OF RG6035

[0181] According to the present invention, it was surprising to find that just a single dose of RG6035 at 700 mg, 2000 mg, or 4000 mg is sufficient to deplete B-cells in the CNS of the subject. B-cell depletion in the CNS in the subject is determined compared to the B-cell number or concentration in the CNS prior to the administration of the dose of RG6035. This may be determined by measuring the number or concentration of B-cells in the CSF. Preferably, the depletion of B cells in the CNS or CSF is an at least 50% depletion compared to the number or concentration of B cells prior to administration of RG6035, for example when the induction dose is 700 mg. In another preferred embodiment, the depletion of B cells in the CNS or CSF is an at least 80% depletion compared to the number or concentration of B cells prior to administration of RG6035, for example when the induction dose is 2000 mg. In another embodiment, the depletion of B cells in the CNS or CSF is an at least 80% depletion compared to the number or concentration of B cells prior to administration of RG6035, for example when the induction dose is 4000 mg.

[0182] In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0183] In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 50%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 55%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 60%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 65%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 70%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 75%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 80%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 85%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 90%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 95%. Preferably, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 50% when the induction dose is 700 mg. Preferably, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 80% when the induction dose is 2000 mg. Preferably, the dose of RG6035 is effective to deplete B-cells in the CNS of the subject by at least 80% when the induction dose is 4000 mg.

[0184] In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 50%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 55%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 60%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 65%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 70%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 75%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 80%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 85%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 90%. In some embodiments, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 95%. Preferably, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 50% when the induction dose is 700 mg. Preferably, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 80% when the induction dose is 2000 mg. Preferably, the dose of RG6035 is effective to deplete B-cells in the CSF of the subject by at least 80% when the induction dose is 4000 mg.

[0185] In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CSF is achieved within 14 days following administration of the 700 mg dose of RG6035. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CSF is achieved within 14 days following administration of the 2000 mg dose of RG6035. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CSF is achieved within 14 days following administration of the 4000 mg dose of RG6035.

[0186] In some embodiments, at least 50% depletion of B-cells in the CSF is achieved within 14 days, for example following administration of a 700 mg intravenous dose of RG6035. In some embodiments, at least 80% depletion of B-cells in the CSF is achieved within 14 days, for example following administration of a 2000 mg intravenous dose of RG6035. In some embodiments, at least 80% depletion of B-cells in the CSF is achieved within 14 days, for example following administration of a 4000 mg intravenous dose of RG6035.

[0187] In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CNS is achieved within 14 days following administration of the 700 mg dose of RG6035. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CNS is achieved within 14 days following administration of the 2000 mg dose of RG6035. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CNS is achieved within 14 days following administration of the 4000 mg dose of RG6035.

[0188] In some embodiments, at least 50% depletion of B-cells in the CNS is achieved within 14 days following administration of a 700 mg intravenous dose of RG6035. In some embodiments, at least 80% depletion of B-cells in the CNS is achieved within 14 days following administration of the 2000 mg intravenous dose of RG6035. In some embodiments, at least 80% depletion of B-cells in the CNS is achieved within 14 days following administration of the 4000 mg intravenous dose of RG6035.In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CSF is achieved within 7 days following administration of the 700 mg dose of RG6035. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CSF is achieved within 7 days following administration of the 2000 mg dose of RG6035. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CSF is achieved within 7 days following administration of the 4000 mg dose of RG6035.

[0189] In some embodiments, at least 50% depletion of B-cells in the CSF is achieved within 8 days following administration of a 700 mg intravenous dose of RG6035. In some embodiments, at least 80% depletion of B-cells in the CSF is achieved within 8 days following administration of a 2000 mg intravenous dose of RG6035. In some embodiments, at least 80% depletion of B-cells in the CSF is achieved within 8 days following administration of a 4000 mg intravenous dose of RG6035.

[0190] In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CNS is achieved within 8 days following administration of the 700 mg dose of RG6035. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CNS is achieved within 8 days following administration of the 2000 mg dose of RG6035. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CNS is achieved within 8 days following administration of the 4000 mg dose of RG6035.

[0191] In some embodiments, at least 50% depletion of B-cells in the CNS is achieved within 8 days following administration of a 700 mg intravenous dose of RG6035. In some embodiments, at least 80% depletion of B-cells in the CNS is achieved within 8 days following administration of a 2000 mg intravenous dose of RG6035. In some embodiments, at least 80% depletion of B-cells in the CNS is achieved within 8 days following administration of a 4000 mg intravenous dose of RG6035.

[0192] In preferred embodiments, depletion of B-cells in the CNS is achieved within 14 days of the first administration of RG6035.

[0193] In some embodiments, the depletion of B-cells in the CSF or the CNS may be a sustained depletion. In such embodiments, the depletion of B-cells may be sustained below a certain percentage compared to the number or concentration of B-cells prior to administration of RG6035. For example, at least 50% depletion of B-cells (e.g. for a 700 mg does) or at least 80% depletion of B-cells (e.g. for a 2000 mg or 4000 mg dose) may be achieved within a certain time period, which is then sustained. In some embodiments, the depletion of B-cells in the CNS is maintained for at least 14, at least 21 or at least 50 days. In some embodiments, the depletion of B-cells in the CSF is sustained for at least 14, at least 21 or at least 50 days. The number of days of sustained depletion in the CSF or CNS generally refers to the number of days since the first administration of RG6035. Rapid and sustained depletion of the B-cells in the CNS or CSF may surprisingly be achieved after a single intravenous dose of RG6035.In some embodiments, the depletion of B-cells in the CSF is sustained for at least 14 days following administration of a 700 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CSF is sustained for at least 14 days following administration of a 2000 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CSF is sustained for at least 14 days following administration of a 4000 mg intravenous dose of RG6035.

[0194] In some embodiments, the depletion of B-cells in the CSF is sustained for at least 21 days following administration of a 700 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CSF is sustained for at least 21 days following administration of a 2000 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CSF is sustained for at least 21 days following administration of a 4000 mg intravenous dose of RG6035.

[0195] In some embodiments, the depletion of B-cells in the CSF is sustained for at least 50 days following administration of a 700 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CSF is sustained for at least 50 days following administration of a 2000 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CSF is sustained for at least 50 days following administration of a 4000 mg intravenous dose of RG6035.

[0196] In some embodiments, the depletion of B-cells in the CNS is sustained for at least 14 days following administration of a 700 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CNS is sustained for at least 14 days following administration of a 2000 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CNS is sustained for at least 14 days following administration of a 4000 mg intravenous dose of RG6035.

[0197] In some embodiments, the depletion of B-cells in the CNS is sustained for at least 21 days following administration of a 700 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CNS is sustained for at least 21 days following administration of a 2000 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CNS is sustained for at least 21 days following administration of a 4000 mg intravenous dose of RG6035.

[0198] In some embodiments, the depletion of B-cells in the CNS is sustained for at least 50 days following administration of a 700 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CNS is sustained for at least 50 days following administration of a 2000 mg intravenous dose of RG6035. In some embodiments, the depletion of B-cells in the CNS is sustained for at least 50 days following administration of a 4000 mg intravenous dose of RG6035.

[0199] In some embodiments, at least 50% depletion of B-cells in the CNS is achieved within 8 days following administration of a 700 mg intravenous dose of RG6035, and the at least 50% depletion of B-cells in the CNS is sustained for at least 14 days following the administration of RG6035. In some embodiments, at least 50% depletion of B-cells in the CNS is achieved within 8 days following administration of a 700 mg intravenous dose of RG6035, and the at least 50% depletion of B-cells in the CNS is sustained for at least 21 days following the administration of RG6035. In some embodiments, at least 50% depletion of B-cellsin the CNS is achieved within 8 days following administration of a 700 mg intravenous dose of RG6035, and the at least 50% depletion of B-cells in the CNS is sustained for at least 50 days following the administration of RG6035.

[0200] In some embodiments, at least 80% depletion of B-cells in the CNS is achieved within 8 days following administration of a 2000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CNS is sustained for at least 14 days following the administration of RG6035. In some embodiments, at least 80% depletion of B-cells in the CNS is achieved within 8 days following administration of a 2000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CNS is sustained for at least 21 days following the administration of RG6035. In some embodiments, at least 80% depletion of B-cells in the CNS is achieved within 8 days following administration of a 2000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CNS is sustained for at least 50 days following the administration of RG6035.

[0201] In some embodiments, at least 80% depletion of B-cells in the CNS is achieved within 8 days following administration of a 4000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CNS is sustained for at least 14 days following the administration of RG6035. In some embodiments, at least 80% depletion of B-cells in the CNS is achieved within 8 days following administration of a 4000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CNS is sustained for at least 21 days following the administration of RG6035. In some embodiments, at least 80% depletion of B-cells in the CNS is achieved within 8 days following administration of a 4000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CNS is sustained for at least 50 days following the administration of RG6035.

[0202] In some embodiments, at least 50% depletion of B-cells in the CSF is achieved within 8 days following administration of a 700 mg intravenous dose of RG6035, and the at least 50% depletion of B-cells in the CSF is sustained for at least 14 days following the administration of RG6035. In some embodiments, at least 50% depletion of B-cells in the CSF is achieved within 8 days following administration of a 700 mg intravenous dose of RG6035, and the at least 50% depletion of B-cells in the CSF is sustained for at least 21 days following the administration of RG6035. In some embodiments, at least 50% depletion of B-cells in the CSF is achieved within 8 days following administration of a 700 mg intravenous dose of RG6035, and the at least 50% depletion of B-cells in the CSF is sustained for at least 50 days following the administration of RG6035.

[0203] In some embodiments, at least 80% depletion of B-cells in the CSF is achieved within 8 days following administration of a 2000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CSF is sustained for at least 14 days following the administration of RG6035. In some embodiments, at least 80% depletion of B-cells in the CSF is achieved within 8 days following administration of a 2000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CSF is sustained for at least 21 days following the administration of RG6035. In some embodiments, at least 80% depletion of B-cells in the CSF is achieved within 8 days following administration of a 2000 mg intravenous dose of RG6035,and the at least 80% depletion of B-cells in the CSF is sustained for at least 50 days following the administration of RG6035.

[0204] In some embodiments, at least 80% depletion of B-cells in the CSF is achieved within 8 days following administration of a 4000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CSF is sustained for at least 14 days following the administration of RG6035. In some embodiments, at least 80% depletion of B-cells in the CSF is achieved within 8 days following administration of a 4000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CSF is sustained for at least 21 days following the administration of RG6035. In some embodiments, at least 80% depletion of B-cells in the CSF is achieved within 8 days following administration of a 4000 mg intravenous dose of RG6035, and the at least 80% depletion of B-cells in the CSF is sustained for at least 50 days following the administration of RG6035.

[0205] The sustaining of the B-cell depletion discussed above may be achieved with a single intravenous dose. However further maintenance of the B-cell depletion may be achieved via one or more maintained doses, discussed below.

[0206] CSF to serum ratio

[0207] As discussed, many existing B-cell depleting monoclonal antibody (mAb) therapies approved for treating MS are unable to actively cross the BBB and address the disease in the CNS. Thus, one of the key hurdles of the development of novel B-cell depletion monoclonal antibody therapies remains their delivery to the CNS.

[0208] Uptake of a mAb to the CNS can be assessed by the ratio of mAb levels in the blood serum and the levels in the CSF. As reported herein, RG6035 is efficiently transported across the BBB and its therapeutic effects in the CNS. Delivery of RG6035 to a patient intravenously or subcutaneously results in a surprisingly high uptake of RG6035 into the CNS compared to other Brainshuttle™ or non-Brainshuttled mAbs, such as ocrelizumab.

[0209] In some embodiments, the CSF to serum ratio of RG6035 following the administration of the induction dose of RG6035 is at least 0.5% or greater. In some embodiments of the invention, the CSF to serum ratio of RG6035 following the administration of the induction dose of RG6035 is 1 % or greater. Despite there being a higher overall concentration of RG6035 in the blood compared to the CSF (or CNS, as measured the concentration of RG6035 in the CSF), RG6035 is surprisingly effective at depleting B-cells in the CNS or CSF.

[0210] DEPLETION OF B-CELLS IN THE BLOOD FOLLOWING A SINGLE DOSE OF RG6035

[0211] RG6035 was also found to deplete B-cells in the blood from a baseline value, which was calculated as the mean of screening and pre-dose values. An initial drop in B-cells in blood was observed on Day 2, with no predominant dose-dependent effect, and no profound and sustained B-cell depletion was recorded in theblood after single intravenous dosing of RG6035, up to doses of 4000 mg. Further detail of the impact of RG6035 on B-cell depletion in the blood is described in Examples 3-5 and (Figure 4A, 4B, and 4C, Figure 9, Figure 13A, Figure 18A). Thus, whilst a single intravenous dose of RG6035 is sufficient to trigger a profound and sustained depletion in B-cells in the CSF or CNS, there is no profound and sustained depletion in B-cells in the blood.

[0212] MULTIPLE DOSING REGIME

[0213] In some aspects, the present invention relates to RG6035 for use in the treatment of MS in a subject, wherein the treatment comprises administering the RG6035 at a first dose, which is an induction dose, and is followed by one or more subsequent maintenance doses of RG6035. In these aspects, the one or more subsequent maintenance doses of RG6035 help sustain the rapid B-cell depletion in the CNS induced by the induction dose, whilst maintaining good tolerability of the treatment. In certain embodiments, the first induction dose of RG6035 is administered to the subject on day 1. Generally, the (first) 700, 2000, and 4000 mg doses discussed here are the induction dose, which is administered on day 1 , preferably by intravenous administration.

[0214] In some embodiments of the invention, the first dose of RG6035, which is an induction dose, can be considered as an induction therapy. The subsequent maintenance doses of RG6035 can be considered as a maintenance therapy. The present invention includes embodiments relating to a maintenance therapy, for example in a subjects who has previously been administered an induction therapy described herein.

[0215] “Induction therapy” according to the invention comprises the administration of the induction dose of RG6035, as defined above. In some embodiments, the induction therapy is administered to the subject intravenously.

[0216] A “maintenance cycle”, “maintenance therapy” or “maintenance treatment” or similar according to the invention comprises the administration of subsequent, maintenance doses of RG6035 to the subject, as defined above. Each subsequent maintenance dose of RG6035 may be administered at a frequency suitable to maintain the B-cell depletion in the subject at a level that is therapeutically effective. In some embodiments, administration of each maintenance dose of RG6035 may further deplete B-cells in the CNS or CSF of the subject. A maintenance therapy may be provided to a subject who has previously been administered an induction therapy.

[0217] In some embodiments of the invention, the 700 mg, 2000 mg, or 4000 mg induction dose of RG6035 is administered intravenously to the subject on day 1. Preferably, the induction dose of RG6035 is a 2000 mg dose that is administered intravenously to the subject on day 1.

[0218] Maintenance dose of RG6035 may preferably be administered subcutaneously. Each maintenance cycle or therapy comprises administration of a maintenance dose of RG6035 at 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg. Generally, each maintenance dose in a given maintenance therapy will be of the same amount of RG6035. Preferably, the one or more subsequent maintenance doses of RG6035 are each 300mg. Preferably, the one or more subsequent maintenance doses of RG6035 are each 100 mg. Each RG6035 maintenance dose may be administered after there is a depletion of B-cells in the CSF of the subject achieved by the administration of the first induction dose of RG6035. In some embodiments, each subsequent maintenance dose of RG6035 is administered at a frequency to maintain B-cell depletion in the subject at a level that is therapeutically effective.

[0219] In some embodiments, the administration of one or more subsequent maintenance doses maintains an at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CSF of the subject compared to the number or concentration of B-cells in the CSF before the administration of the induction dose of RG6035. In some embodiments, the administration of one or more subsequent maintenance doses maintains an at least 50% depletion of B-cells in the CSF of the subject compared to the number or concentration of B-cells in the CSF before the administration of the induction dose of RG6035, wherein the induction dose was 700 mg. In some embodiments, the administration of one or more subsequent maintenance doses maintains an at least 80% depletion of B-cells in the CSF of the subject compared to the number or concentration of B-cells in the CSF before the administration of the induction dose of RG6035, wherein the induction dose was 2000 mg. In some embodiments, the administration of one or more subsequent maintenance doses maintains an at least 80% depletion of B-cells in the CSF of the subject compared to the number or concentration of B-cells in the CSF before the administration of the induction dose of RG6035, wherein the induction dose was 4000 mg.

[0220] In some embodiments, the administration of one or more subsequent maintenance doses maintains an at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% depletion of B-cells in the CNS of the subject compared to the number or concentration of B-cells number in the CNS before the administration of the induction dose of RG6035. In some embodiments, the administration of one or more subsequent maintenance doses maintains an at least 50% depletion of B-cells in the CNS of the subject compared to the number or concentration of B-cells in the CNS before the administration of the induction dose of RG6035, wherein the induction dose was 700 mg. In some embodiments, the administration of one or more subsequent maintenance doses maintains an at least 80% depletion of B-cells in the CNS of the subject compared to the number or concentration of B-cells in the CNS before the administration of the induction dose of RG6035, wherein the induction dose was 2000 mg. In some embodiments, the administration of one or more subsequent maintenance doses maintains an at least 80% depletion of B-cells in the CNS of the subject compared to the number or concentration of B-cells in the CNS before the administration of the induction dose of RG6035, wherein the induction dose was 4000 mg.

[0221] In some embodiments, each maintenance dose maintains B-cell depletion in the CSF for 7 days. In some embodiments, each maintenance dose maintains B-cell depletion in the CSF for at least 14 days. In some embodiments, each maintenance dose maintains B-cell depletion in the CSF for at least 21 days. In some embodiments, each maintenance dose maintains B-cell depletion in the CSF for at least 28 days. In some embodiments, each maintenance dose maintains B-cell depletion in the CSF for more than 28 days.

[0222] In some embodiments, each maintenance dose maintains B-cell depletion in the CNS for 7 days. In some embodiments, each maintenance dose maintains B-cell depletion in the CNS for at least 14 days. In someembodiments, each maintenance dose maintains B-cell depletion in the CNS for at least 21 days. In some embodiments, each maintenance dose maintains B-cell depletion in the CNS for at least 28 days. In some embodiments, each maintenance dose maintains B-cell depletion in the CNS for more than 28 days.

[0223] Preferably the invention comprises multiple maintenance doses, e.g. each maintenance dose is repeated multiple times, i.e. is carried out two, three, four or more times. In some embodiments, the one or more subsequent maintenance doses of RG6035 are Q1W, Q2W, Q3W, or Q4W. Preferably, the dosing schedule for the maintenance doses is Q2W. Each maintenance dose may maintain the B-cell depletion in the CNS or CSF until the next maintenance dose.

[0224] In some embodiments, the maintenance doses may be continued for at least 3 cycles. In some embodiments, the maintenance dose may be continued for at least 10 cycles. In a preferred embodiment, the maintenance dose is a chronic dose.

[0225] References herein to “every 7 days thereafter”, “every 14 days thereafter”, “every 21 days thereafter”, or “every 28 days thereafter” and the like mean the maintenance dose is continued on a regular cycle until the treatment is discontinued.

[0226] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 100 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 100 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 100 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 100 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0227] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 150 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 150 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 150 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 150 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0228] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 300 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 300 mg once every 2 weeks starting on day 15 andevery 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 300 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 300 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0229] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 600 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 600 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 600 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 600 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0230] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 700 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 700 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 700 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 700 mg on day 1 , followed by maintenance doses of 700 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0231] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0232] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every 3 weeksstarting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0233] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every week starting on day 8 and every 7 days thereafter. In a particularly preferred embodiment, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0234] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 600 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 600 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 600 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 600 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0235] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 700 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 700 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 700 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 700 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0236] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 100 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 100 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 100 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprisesadministering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 100 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0237] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 150 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 150 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 150 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 150 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0238] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 300 mg once every week starting on day 8 and every 7 days thereafter. In a particularly preferred embodiment, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 300 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 300 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 300 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0239] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 600 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 600 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 600 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 600 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0240] In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 700 mg once every week starting on day 8 and every 7 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 700 mg once every 2 weeks starting on day 15 and every 14 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 700 mg once every 3 weeks starting on day 22 and every 21 days thereafter. In some embodiments, the treatment comprises administering the RG6035 at an induction dose of 4000 mg on day 1 , followed by maintenance doses of 700 mg once every 4 weeks starting on day 29 and every 28 days thereafter.MAINTENANCE DOSE AS THE SOLE DOSING REGIMEN

[0241] The present invention also provides treatment comprising the maintenance doses, without a prior induction dose.

[0242] Accordingly, in some embodiments there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg.

[0243] The dose of RG6035 may be a subcutaneous Q1W, Q2W, Q3W, or Q4W dose. Preferably, the dose of RG6035 may be a subcutaneous Q2W dose.

[0244] In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q1W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q2W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q3W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q4W.

[0245] In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 150 mg Q1W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 150 mg Q2W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 150 mg Q3W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 150 mg Q4W.

[0246] In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 300 mg Q1W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 300 mg Q2W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 300 mg Q3W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 300 mg Q4W.

[0247] In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 600 mg Q1W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 600 mg Q2W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of600 mg Q3W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 600 mg Q4W.

[0248] In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg Q1W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg Q2W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg Q3W. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg Q4W.

[0249] In some embodiments, the RG6035 may be administered for at least 3 cycles. In some embodiments, the RG6035 may be administered for at least 10 cycles. In a preferred embodiment, the RG6035 may be administered chronically.

[0250] The above doses may preferably be administered subcutaneously.

[0251] MAINTENANCE DOSE AS THE SOLE DOSING REGIMEN IN A SUBJECT WHO HAS PREVIOUSLY BEEN ADMINISTERED WITH OCRELIZUMAB

[0252] The present invention also provides treatment comprising the maintenance doses, without a prior induction dose of RG6035, but with a prior treatment with ocrelizumab.

[0253] Without wishing to be found by theory, it is noted that a prior treatment with ocrelizumab can induce peripheral B-cell depletion. As such, a prior treatment with ocrelizumab can be used as an alternative to the described induction doses, to induce peripheral B-cell depletion before subsequent administration of RG6035. In some embodiments, a prior treatment with ocrelizumab induces peripheral B-cell depletion before subsequent administration of RG6035. In these embodiments, subsequent administration of maintenance doses of RG6035 induces B-cell depletion in the CNS and the CSF.

[0254] Accordingly, in some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis in a subject who has previously been administered ocrelizumab. In some embodiments, ocrelizumab may have been administered intravenously. In some embodiments, ocrelizumab may have been administered subcutaneously. In some embodiments, ocrelizumab may have been administered intravenously and / or subcutaneously.

[0255] In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis in a subject who has previously been administered ocrelizumab, wherein the subject is refractory to an anti-CD20 therapy, e.g. ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis in a subject who has previously been administered ocrelizumab, wherein the subject is experiencing clinical progression after an anti-CD20 therapy, e.g. ocrelizumab. In some embodiments, theRG6035 is for use in the treatment of multiple sclerosis in a subject who has previously been administered ocrelizumab and is experiencing relapse.

[0256] In some embodiments there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg, in a patient that has been previously administered ocrelizumab.

[0257] The dose of RG6035 may be a subcutaneous Q1W, Q2W, Q3W, or Q4W dose. Preferably, the dose of RG6035 may be a subcutaneous Q2W dose.

[0258] In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q1 W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q2W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q3W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q4W, in a patient that has been previously administered ocrelizumab.

[0259] In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 150 mg Q1 W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 150 mg Q2W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 150 mg Q3W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 150 mg Q4W, in a patient that has been previously administered ocrelizumab.

[0260] In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 300 mg Q1 W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 300 mg Q2W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 300 mg Q3W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 300 mg Q4W, in a patient that has been previously administered ocrelizumab.In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 600 mg Q1 W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 600 mg Q2W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 600 mg Q3W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 600 mg Q4W, in a patient that has been previously administered ocrelizumab.

[0261] In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg Q1 W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg Q2W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg Q3W, in a patient that has been previously administered ocrelizumab. In some embodiments, there is provided RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg Q4W, in a patient that has been previously administered ocrelizumab.

[0262] In some embodiments, the RG6035 may be administered for at least 3 cycles, in a patient that has been previously administered ocrelizumab. In some embodiments, the RG6035 may be administered for at least 10 cycles, in a patient that has been previously administered ocrelizumab. In a preferred embodiment, the RG6035 may be administered chronically, in a patient that has been previously administered ocrelizumab.

[0263] The above doses of RG6035 may preferably be administered subcutaneously.

[0264] ANTIDRUG ANTIBODIES (ADAS)

[0265] The development of anti-drug antibodies (ADAs) in most animals treated with RG6035 was observed after repeated dosing but did not significantly affect the exposure. The incidence of ADAs in non-human primates (NHPs) is not considered predictive for humans.

[0266] It was therefore surprising to find that the dosing regime according to the present invention, is associated with trends of a lower prevalence and a later onset of ADAs following higher doses of RG6035. For example, after 700 mg IV, 2000 mg IV, or 4000 mg IV doses of RG6035, no ADAs were detected until day 57.

[0267] Accordingly, in some embodiments, no anti-drug antibodies to RG6035 are detected in the CSF following administration of the 700 mg, 2000 mg, 4000 mg dose of RG6035 until at least day 50.METHODS OF TREATMENT

[0268] The present invention provides methods of treatment of multiple sclerosis. The methods of treatment may employ any of the dosing regimens disclosed herein.

[0269] The present invention relates to a method for the treatment of multiple sclerosis in a subject comprising administering a therapeutically effective amount of RG6035 to the subject, wherein RG6035 is administered at a dose of 700 mg, 2000 mg, 4000 mg.

[0270] In some embodiments, the method of the present invention comprises administering a therapeutically effective amount of RG6035 intravenously. In some embodiments, the method of the present invention comprises administering a therapeutically effective amount of RG6035 intravenously at a dose of 700 mg, 2000 mg, 4000 mg.

[0271] In some embodiments, the method for treating multiple sclerosis in a subject comprises administering to the subject:

[0272] (i) a first induction dose of RG6035 of 700 mg, 2000 mg, or 4000 mg; and

[0273] (ii) one or more subsequent maintenance doses of RG6035.

[0274] In preferred embodiments, the first induction dose of RG6035 is administered intravenously to the subject, and the one or more subsequent maintenance doses of RG6035 is administered subcutaneously to the subject.

[0275] Each maintenance dose of RG6035 may be administered at 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg. Preferably, the one or more subsequent maintenance doses of RG6035 are each 300 mg. Preferably, the one or more subsequent maintenance doses of RG6035 are each 100 mg. Each RG6035 maintenance dose may be administered after there is a depletion of B-cells in the CSF of the subject achieved by the administration of the first induction dose of RG6035. Preferably, each subsequent maintenance dose of RG6035 is administered at a frequency to maintain B-cell depletion in the CSF of the subject at a therapeutically effective level.

[0276] In some embodiments, each RG6035 maintenance dose is administered after there is a depletion of B-cells in the CNS of the subject achieved by the administration of the first induction dose of RG6035. Preferably, each subsequent maintenance dose of RG6035 is administered at a frequency to maintain B-cell depletion in the CNS of the subject at a therapeutically effective level.

[0277] In some embodiments, each RG6035 maintenance dose is administered after a CSF to serum ratio of 0.5% or greater, or, preferably, 1% or greater is achieved by the administration of the first maintenance dose of RG6035 to the subject.

[0278] Preferably the invention comprises multiple maintenance doses, e.g. each maintenance dose is repeated multiple times, i.e. is carried out two, three, four or more times. In some embodiments, the one or moresubsequent maintenance doses of RG6035 are Q1W, Q2W, Q3W, or Q4W. In preferred embodiments, the one or more subsequent maintenance doses are Q2W.

[0279] The present invention also provides a method for the treatment of multiple sclerosis in a subject comprising administering a therapeutically effective amount of RG6035 to the subject, wherein RG6035 is administered at a dose of 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg. The dose of RG6035 may be Q1 W, Q2W, Q3W, or Q4W. In a preferred embodiment, the dose may be 300 mg Q2W. In a preferred embodiment, the dose may be 100 mg Q2W.

[0280] In some embodiments, there is provided RG6035 for use in a method of depleting B-cells in the CNS of a multiple sclerosis patient. In some embodiments, there is provided RG6035 for use in the selective depletion of B-cells in the CNS of a multiple sclerosis patient. In some embodiments, there is provided RG6035 for use in a method of depleting B-cells in the CSF of a multiple sclerosis patient. In some embodiments, there is provided RG6035 for use in the selective depletion of B-cells in the CSF of a multiple sclerosis patient. The compositions and pharmaceutical compositions disclosed herein may also be provided for such purposes.

[0281] In some embodiments, there is provided a method of depleting B-cells in the CNS of a multiple sclerosis patient. In some embodiments, there is provided a method of selectively depleting B-cells in the CNS of a multiple sclerosis patient. In some embodiments, there is provided a method depleting B-cells in the CSF of a multiple sclerosis patient. In some embodiments, there is provided a method of selectively depleting B-cells in the CSF of a multiple sclerosis patient. The compositions and pharmaceutical compositions disclosed herein may also be provided for such purposes.

[0282] Selective B-cell depletion may refer to the preferential depletion of B-cells in the CNS or CSF compared to B-cells elsewhere in the body, for example B-cells in the blood.

[0283] ROUTES OF ADMINISTRATION

[0284] An antibody as reported herein (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. Subcutaneous administration of a pharmaceutical formulation may be more convenient to the patient than an administration by intravenous infusion.

[0285] In addition, the bispecific antibody may suitably be administered by pulse infusion, e.g., with declining doses of the bispecific antibody. The dosing can be given by injections, intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.Antibodies as reported herein would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0286] In the present invention, the induction dose may be administered intravenously, and any one or more maintenance doses may be administered subcutaneously.

[0287] PHARMACEUTICAL FORMULATIONS

[0288] Pharmaceutical formulations of a bispecific anti-human CD20 / human transferrin receptor antibody as described herein are prepared by mixing such antibody having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone); amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rhuPH20, are described in US 2005 / 0260186 and US 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0289] Exemplary lyophilized antibody formulations are described in US 6,267,958. Aqueous antibody formulations include those described in US 6,171 ,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer. Exemplary anti-CD20 antibody formulations are described in WO98 / 56418, expressly incorporated herein by reference.

[0290] Lyophilized formulations adapted for subcutaneous administration are described in WO 97 / 04801. Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the mammal to be treated herein.

[0291] The formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0292] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed.) (1980).

[0293] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (US 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[0294] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. In one embodiment the formulation is isotonic.

[0295] RG6035 may be formulated for intravenous administration, for example for an induction dose. RG6035 may be formulated for subcutaneous administration, for example for one or more maintenance doses.

[0296] The present invention provides compositions, in particular pharmaceutical compositions, that are suitable for use in the medical uses and methods of treatment disclosed herein. The compositions or pharmaceutical compositions may comprise a fixed dose of RG6035.

[0297] For example, the present invention provides a pharmaceutical composition comprising RG6035 and one or more pharmaceutically acceptable excipients, wherein the RG6035 is present in an amount of 700 mg, 2000 mg, 4000 mg. Such pharmaceutical compositions may be for intravenous administration. Hence, thepharmaceutical composition may be an intravenous pharmaceutical composition. In some embodiments, the pharmaceutical composition is for use as a medicament, wherein the pharmaceutical composition is administered intravenously. The pharmaceutical composition may be a unit dose composition.

[0298] For example, the present invention also provides a pharmaceutical composition for subcutaneous administration comprising RG6035 and one or more pharmaceutically acceptable excipients, wherein the RG6035 is present in an amount of 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg. Such pharmaceutical compositions may be for subcutaneous administration. Hence, the pharmaceutical composition may be a subcutaneous pharmaceutical composition. In some embodiments, the pharmaceutical composition is for use as a medicament, wherein the pharmaceutical composition is administered subcutaneously. The pharmaceutical composition may be a unit dose composition.

[0299] The pharmaceutical compositions for subcutaneous administration may be provided as a kit comprising multiple unit doses of RG6035, wherein the multiple unit doses of RG6035 are each 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg. The kit may further comprise instructions for use, in particular instructions for the subcutaneous administration of a unit dose of RG6035 for the purpose of treating multiple sclerosis. In a preferred embodiment, the kit may comprise multiple unit doses of RG6035, wherein the multiple unit doses of RG6035 are each 300 mg. In a preferred embodiment, the kit may comprise multiple unit doses of RG6035, wherein the multiple unit doses of RG6035 are each 100 mg.

[0300] COMBINATION WITH OCRELIZUMAB

[0301] Direct targeting of CNS-compartmentalised B-cells in addition to B-cells in the peripheral immune system by RG6035 has the potential to improve patient response rates as compared to current standard of care anti-CD20 mAb MS therapies that systemically target the peripheral immune system.

[0302] In some embodiments, however, peripheral B-cells could also be targeted by administration of another B-cell-depleting mAb, such as ocrelizumab.

[0303] Ocrelizumab is a recombinant humanized B-cell-depleting mAb that selectively targets CD20-expressing B-cells involved in the pathological inflammatory mechanisms associated with relapses and relapse associated worsening. To date, only ocrelizumab is indicated for primary progressive MS (PPMS) (Klein et al. 2013). Since ocrelizumab is associated with poor transcytosis of the BBB, B-cell depletion from CSF after treatment with ocrelizumab is assumed to be the consequence of a reduced influx of B-cells into the CSF due to the depletion in blood (Cross et al., 2019).

[0304] In some embodiments of the invention, RG6035 is for administration in combination with ocrelizumab. In some embodiments, RG6035 is for subsequent, sequential or separate administration with ocrelizumab.

[0305] In some embodiments, the methods of treatment comprises administering one or more doses of ocrelizumab to a patient who has received or who will receive one or more doses of RG6035.In some embodiments, there is provided the combination of RG6035 and ocrelizumab. The RG6035 may be for administration according to a dosing regimen disclosed herein. In some embodiments there is provided ocrelizumab for use in treatment multiple sclerosis, wherein the ocrelizumab is for combinatorial treatment with RG6035, for example according to a dosing regimen disclosed herein. In some embodiments there is provided RG6035 for use in treatment multiple sclerosis, wherein the ocrelizumab is for combinatorial treatment with ocrelizumab, wherein the RG6035 may be for administration according to a dosing regimen disclosed herein.

[0306] In some embodiments, the ocrelizumab is administered before the first administration of RG6035. In some embodiments, the ocrelizumab is administered before the first maintenance dose of RG6035. In some embodiments, ocrelizumab is administered intravenously. In some embodiments, ocrelizumab is administered subcutaneously. In some embodiments, ocrelizumab is administered intravenously and / or subcutaneously.

[0307] The RG6035 and ocrelizumab may be provided as a kit.

[0308] The invention will now be further described by way of the following Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention, with reference to the Figures.

[0309] EXAMPLES

[0310] Example 1 - In vitro and ex vivo binding of RG6035 to huTfR and CD20

[0311] Antibody and bispecific antibody production

[0312] Brainshuttle™ (BS) constructs were engineered by fusing an anti-huTfR1 cross Fab (human; clone 1026) to the C-terminus of a heavy chain of obinutuzumab or obinutuzumab-PGLALA variant (Klein et al., 2016; Schlothauer et al., 2016). Knobs-into-holes technology (Merchant et al., 1998) was used to favour heterodimeric pairing of a heavy chain carrying the BS module with a non-fused heavy chain. CrossMab technology (Klein et al., 2016; Schaefer et al., 2011) was used to avoid mispairing of the different light chains on the antibody complex. In the same way, the huTfRI positive control reference binder, BS-DP47, was generated, except that the fusion partner was DP47 (a non-target binding germline human IgG used as a blood tracer for brain contamination), instead of anti-CD20. To generate the mouse TfR1 surrogate BS, an anti-muTfR1 single chain Fab (rat; clone 8D3) was fused to the C-terminus of the heavy chain of obinutuzumab or obinutuzumab-PGLALA variant, also utilizing knobs-into-holes technology. A glycineserine peptide linker was used to construct the single-chain Fab fragment.

[0313] Genes for the different chains of the different antibodies were cloned into expression cassettes, which harbour regulatory elements for the expression of the genes comprising CMV promoter, bGH poly A signal, and hGH transcriptional terminator. The expression cassettes were cloned into expression vectors. Expi293TM or ExpiCHO-STM cells (ThermoFisher Scientific; A14527 and A29127, respectively) were co-transfected with the appropriate vector combination according to the manufacturer’s protocol and antibodies were purified from cell culture supernatants as previously described (Niewoehner et al., 2014).

[0314]

[0315] Human B-cell lymphoma cells were maintained in RPMI1640 medium (ThermoFisher, 11875093) with 10 % fetal calf serum (FCS; Gibco 16140) and 1 % (v / v) 2 mM N-acetyl-L-alanyl-L-glutamine (GlutaMAXTM; ThermoFisher, 35050061) at 0.3 to 0.9*1 E6 cells / mL at 37 °C in a 5 % CO2 humidified incubator. Harvested cells (98.2 % viability) were pelleted by centrifugation (4 min., 400xg) and re-suspended in Fluorescence- Activated Cell Sorting (FACS) buffer (phosphate buffered saline (PBS) with 2 % (v / v) FCS, 5 nM EDTA, and 0.25 % sodium azide) at about 0.6*1 E6 viable cells / mL and seeded into 96-U-bottom plates at 1*1 E5 cells per well. Test antibodies were added to cells with final concentrations ranging from 1000 nM to 0.0128 nM (1 :5 dilution steps, in triplicate) for 30 min at 4 °C. Cells were then washed with FACS buffer and incubated with a secondary antibody (Jackson ImmunoResearch, 109-096-098; FITC F(ab)’2 anti-human Fcg specific; 1 :20) for 30 min at 4 °C. Cells were then washed twice with FACS buffer and fixed using 2 % paraformaldehyde in FACS buffer before proceeding with FACS acquisition (BD FACS Cantoll flow cytometer). FACS gating was performed using FACS Diva Software and the median fluorescence intensities were determined. EC50 values were calculated based on sigmoidal dose-response (variable slope) analysis using GraphPad Prism. No further statistical analysis was performed.

[0316] Cell death induction

[0317] Human B-cell lymphoma cells were incubated with test antibodies (0.0128 nM to 1000 nM) for 24 hours before measuring cell death by assessing Annexin V (annV) expression and propidium iodide (PI) uptake.

[0318] Human B-cell lymphoma cells were seeded in a 96-U-bottom plate at 1 *1 E5 cells per well. Test antibodies (0.0128 nM to 1000 nM in triplicate) were incubated with cells at 37 °C for 24 hours. Afterwards, cells were resuspended and centrifuged (4 minutes at 400xg), then washed with annV Binding Buffer (BB; 10 mM HEPES / NaOH pH 7.4, 140 mM NaCI, 2.5 mM CaCI2). Final cell pellets were resuspended in annV-FLUOS (Roche Diagnostics GmbH, Mannheim, Germany; 11828681001 ; 1 :100 in annV BB) before a 15-minute incubation at room temperature with protection from light. Cells were then washed with annV BB before the addition of annV BB containing PI (Merck, P4864; 1 :4000). Signal acquisition was performed immediately upon addition of PI using a BD FACS Cantoll flow cytometer. FACS gating was performed using BD FACS Diva Software to determine the percentages of annV / PI double positive cells. No further statistical analysis was performed.

[0319] TfR-mediated cell internalization and transcvtosis

[0320] Parental MDCKII cells were cultured in high-glucose DMEM (ThermoFisher, 41965062) supplemented with 10 % (v / v) FCS (Sigma-Aldrich, F4135) at 37 °C in a 5 % CO2 humidified incubator. Cells were seeded into 24-well plates at 7.5*1 E4 cells per well and transiently transfected 24 hours post seeding. For MDCKII transient transfection, vector DNA mix was prepared in Opti-MEMTM reduced serum media (ThermoFisher,31985070) at a concentration of 0.02 pg / pL, giving 0.5 pg DNA per 25 pL volume transferred per well (i.e. , the total amount of DNA per 24-well plate well for transfection). Lipofectamine™ 2000 reagent (ThermoFisher, 11668027) was also diluted in Opti-MEM reduced serum media, to a concentration giving 1.5 pL Lipofectamine™ per 25 pL transferred per well. The separately prepared diluted DNA and Lipofectamine™ reagent were combined in a 1 :1 ratio and incubated at room temperature for 10 minutes to allow formation of DNA / Lipofectamine™ complexes. After incubation, transfection complexes were carefully transferred into each well. Parallel mock transfections were included, where the same procedure was conducted for transfection but without the vector DNA.

[0321] MDCKII cells were transiently transfected with huTfR 24 hours prior to the transcytosis assays. huTfR-mediated internalization (uptake) and transcytosis (pulse-chase) assays were performed 24-hours post transfection; each treatment condition was evaluated in triplicate. The assay buffer (AB) used consisted of Hanks balance salt solution (Mg++ / Ca++; ThermoFisher, 14065072) supplemented with 20 mM HEPES (ThermoFisher, #15630056), pH 7.4. All assay incubation steps were conducted at 37 °C, 5 % CO2 without shaking. Cells were rinsed twice with AB prior to initiation of the pulse phase (uptake / cell loading) with the addition 0.2 mL AB spiked with test antibodies per well. Antibodies were incubated with the cells for the indicated time points (30 or 45 minutes), depending on the experiment. At the end of the incubation, uptake was stopped with the immediate aspiration of spiked AB, followed by two rapid rinses with AB containing 0.1 % (v / v) bovine serum albumin (BSA), followed by a final wash with AB without BSA to remove any residual protein. The pulse step was performed in duplicate plates; one plate was immediately lysed, representing the intracellular compartment IgG content at the start of the chase phase; time 0, and the other plate evaluated across multiple time points. Following a final rinse step (as described above), cells were incubated with pre-warmed AB (37 °C) and left to initiate the chase. Samples of the AB (extracellular compartment) were then collected at the end of each time point (5, 10, and 30 minutes). At the end of the final time point, cells were rinsed three times as described above for the pulse phase, before being lysed and assayed for intracellular IgG content.

[0322] To measure intracellular IgG content, cells were lysed in RIPA Lysis and Extraction Buffer (ThermoFisher Scientific, 89900) containing protease inhibitors (Sigma Aldrich, 11697498001) and incubated for 30 minutes at 4 °C (under frequent agitation). Solubilized cell lysates were transferred to 96-well LoBind deep well plates and stored at 4 °C (or -80 °C for longer periods) until ready for analysis. Samples from the chase phase were immediately diluted with assay buffer in 96-well LoBind deep well plates. Total cell protein content in cell lysates were determined using the PierceTM BCA Protein assay (ThermoFisher, 23225), following the manufacturers’ protocol. IgG content was quantitatively evaluated using generic IgG ELISA with chemiluminescence-based detection.

[0323] B-cell depletion in human CSF

[0324] Human PBMCs and human CSF were used as a matrix for B-cell depletion assays. PBMCs from five healthy donors and CSF from 12 individual donors (female and male) across all age groups were obtained from Discovery Life Sciences, Huntsville, USA. CSF samples were thawed once, pooled, aliquoted, and stored at -80 °C until needed. PBMCs were thawed in RPMI (with 10 % (v / v) FCS, 1 %penicillin / streptomycin (PS), and 1 % non-essential amino acid (NEAA)) and equilibrated at 37 °C, 5 % CO2 for at least 2 hours. Approximately 2*1 E6 cells / mL (2*1 E5 per well) were seeded in a 96-well plate. After 2 hours of incubation, the plate was centrifuged at 300xg for 5 minutes at room temperature. Cells were then resuspended in 100 pL human CSF.

[0325] Treatment antibodies were diluted and added to PBMCs in CSF in the range of 0.002 nM to 100 nM for 22 hours. Cells were then pelleted and processed for FACS staining with the following antibodies: anti-CD45 antibody (APC, F20; BD Biosciences, 560973), anti-CD19 antibody (PE, F20; BioLegend, 982402), and anti-CD3 antibody (PE / Cy7, F20; BioLegend, 300419). Cell viability was measured with the LIVE / DEADTM Fixable Aqua Dead Cell Stain Kit (ThermoFisher, L34957), Zombie Aqua™ Fixable Viability Kit (BioLegend, 423101), and F1000 BioLegend. Samples were measured with a BD Biosciences FACSLyric™ flow cytometer using FACS Suite software in a volume-based acquisition. FACS raw data was analyzed using FlowJo software. B-cell depletion was calculated using the B-cell / T-cell ratio of untreated control versus the B-cell / T-cell ratio of the sample. Statistical evaluations were obtained using GraphPad Prism. Statistical significance of the difference in maximal CD19+-B-cell depletion between the different antibodies was calculated using a one-way analysis of variance (multiple comparisons).

[0326] B-cell depletion in human tonsil-derived cells

[0327] Freshly resected tonsil tissue was obtained from adult donors undergoing routine tonsillectomy at Hirslanden Clinic Muenchenstein Birshof, Switzerland. Written patient consent was obtained for each patient-derived sample. Upon collection, tissue was stored in 4 °C in Hanks balanced salt solution (Mg++ / Ca++; ThermoFisher, 14065-072). Subsequently, tonsil tissue underwent mechanical and enzymatic digestion to obtain a cell suspension, which was directly used for B-cell depletion assays. Concentration-dependent and B cell subset depletion was determined as described below.

[0328] Total B-cell depletion

[0329] B-cells were transferred to 96-well U bottom plates in cultivation medium (RPMI with 10 % FBS, 1 % PS, and 1 % NEAA at 2.5*1 E5 cells / well). Treatment antibodies were diluted and added in the range of 0.03 nM to 100 nM. Further experimental conditions follow B-cell depletion in human CSF (described above).

[0330] B-cell subset depletion

[0331] B-cells were transferred to 96-well U bottom plates in cultivation medium (1*1 E6 cells / well). Treatment antibodies were diluted and added at 1 nM to 100 nM and incubated for 8 hours at 37 °C, 5 % CO2. Cells were then pelleted and processed for FACS staining. Cell viability was measured with the LIVE / DEADTM and Zombie Aqua™ kits, the antibodies listed in Table 1 , and BD Horizon™ Brilliant Stain Buffer (BD Biosciences, 563794). Samples were analysed on a FACSymphony™ or FACS LSRFortessa™ using FACSDiva™ software (BD Biosciences). The phenotypic identification of B-cell subsets was performed based on the surface marker expression and staining strategy previously described (Clavarino et al., 2016).Table 1 : Antibodies used for B-cell depletion and human whole blood assay.

[0332]

[0333] Lymphocytes were identified based on size (forward scatter and granularity side scatter), as well as single-cell gating. Following a live-dead discrimination, live CD45+-lymphocytes were further gated into CD3--and CD3+-cells. CD3+-T-cells were later used to normalize for B-cell subset population depletion. CD19 positivity was used to identify total B-cells of interest. CD19+-B-cell subsets were further identified based on surface marker expression. Thereby, distinct expression of CD38 and CD10 on CD19+-B-cells served as initial discriminator of three major B-cell populations.

[0334] Plasma cells express the highest levels of CD38 and do not express CD10 (CD38+++ / CD10-), whereas CD38-low and CD10-CD19+-B-cells give rise to CD27+-memory and CD27--naive B-cells. CD38-intermediate (CD38++) and CD10+CD19+-B-cells can be further broken down to GC B-cells, transitional B-cells, and immature B-cells. During transition from immature bone marrow-derived B-cells to peripheral blood transitional cells, CD5 expression is upregulated. Accordingly, CD38++CD10+CD5+-cells have been classified as transitional B-cells. Furthermore, immature B-cells have been discriminated from GC B-cellsbased on their surface IgM expression. lgD-CD38++CD10+-cells were considered as GC B-cells and IgD-but lgM+CD38++CD10+-cells as immature B-cells.

[0335] Results

[0336] In CD20 binding assays, the Fc-region-effector-function-competent variant of RG6035 (Brainshuttle™-anti-CD20-WT) and RG6035 itself (Brainshuttle™-anti-CD20-PGLALA) demonstrated comparable CD20 binding in a concentration-dependent manner. The Brainshuttle™ constructs had a slightly higher affinity than the corresponding non-brain shuttle control antibodies, i.e. anti-CD20-WT and anti-CD20-PGLALA. Without being bound by this theory, it is assumed that this may be caused by additional binding of the Brainshuttle™ moiety to TfR1 expressed on the surface of B-cells.

[0337] Maximum B-cell depletion in human CSF spiked with human peripheral blood mononuclear cells (PBMCs) was similar for both RG6035 (Brainshuttle™-anti-CD20-PGLALA) and anti-CD20-PGLALA and there were no significant differences between the two constructs in terms of binding and B-cell depletion in ex vivo human tonsil-derived cells, confirming the ability of RG6035 to deplete B-cells stemming from lymphoid tissues.

[0338] In more detail, to assess B-cell death induction properties in cerebrospinal fluid (CSF), human peripheral blood mononuclear cells (PBMCs) were incubated with RG6035 and anti-CD20-PGLALA in CSF from healthy human donors. The average maximum depletion (± standard deviation [±SD]) after 22 hours incubation was 75.11 % ± 5.94 and 69.87 % ± 13.3 for RG6035 and anti-CD20-PGLALA, respectively. These results indicate strong direct B-cell depleting potency in the CSF cell culture matrix, confirming the susceptibility of B-cells in CSF as an example of central nervous system (CNS)-compartmentalized B-cells to the caspase-independent (non-apoptotic) B-cell depletion mechanism, mediated by RG6035 and anti-CD20-PGLALA (Klein et al., 2013).

[0339] This PBMC-CSF assay shows that the mechanism of action of RG6035 and anti-CD20-PGLALA is an Fc-region-effector-function-independent, direct B-cell killing mechanism leading to efficient B-cell depletion in the CSF. Furthermore, B-cell depletion does not require availability of the complement system.

[0340] RG6035 binding and B-cell depletion were also assessed in an ex vivo autologous B-cell depletion assay in human tonsil-derived cells to assess efficacy of direct cell death induction. Without being bound by this theory, it is assumed that B-cells from tonsil tissue may better reflect the response of B-cells in secondary lymphoid tissues compared with peripheral blood B-cells.

[0341] The assessment indicated that human tonsils contain a large proportion of B-cells and substantial heterogeneity of B-cell subpopulations, particularly naive B-cells, tissue resident memory B-cells, and immunologically active germinal centre (GC) B-cells.

[0342] It has been found that anti-CD20-PGLALA induced direct CD19+-B-cell death and depletion with a half-maximal effective concentration (EC50) of 0.523 ± 0.134 nM. The ability for direct cytotoxicity waspreserved in RG6035 (EC50 = 0.396 ± 0.177 nM). A cytotoxicity dose effect depending on the antibody concentration was observed 22 hours after the addition of both constructs to tonsil-derived cells and concentration dependent CD19+-B-cell depletion for three tonsil donors was shown. Maximum depletion was reached for both compounds in a concentration range between 25 nM and 100 nM (see Table 2). There was no significant difference in maximum B-cell depletion between the two constructs at any of the tested concentrations. RG6035 was able to reduce the amount of CD19+ human tonsil B-cells in this experiment by a maximum mean of 60 %.

[0343] Table 2: anti-CD20-PGLALA and RG6035 binding and B-cell depletion in an ex vivo autologous human tonsil-derived B-cell depletion assay.

[0344]

[0345] Three tonsil donors were used to determine the EC50 value for CD19+-B-cell depletion as well as B-cell subset depletion. It has been found that there was a different cellular depletion profile between the three donors. A comparative analysis showed a correlation between maximal B-cell subset depletion and surface CD20 expression, with the highest CD20 expression on transitional and the lowest CD20 expression on naive B-cells. The highest maximal depletion was observed on transitional and the lowest on naive B-cells. B-cells with intermediate CD20 expression levels showed also intermediate maximal B-cell depletion levels. Thus, the response to RG6035 correlates with the degree of CD20 surface expression.

[0346] Thus, RG6035 is able to induce direct cell death and deplete extravascular human B-cell subsets located in secondary lymphoid tissues.

[0347] Without being bound by this theory, it is assumed that an explanation for incomplete depletion of B-cells could be the differential expression of CD19 and CD20 on late-stage B-cells, specifically plasmablasts, which express low levels of CD20 and plasma cells, which express no CD20, while both cell types express CD19. Furthermore, a potential modulation of CD19 expression levels after CD20 antibody incubation could explain insufficient detection of maximal depletion (Jones et al., 2012).

[0348] Thus, the presence of a TfR1 binding moiety does not negatively interfere with the B-cell-depleting potency. No significant differences in EC50 and maximal depletion were observed between the two constructs, proving for a preserved direct B-cell death induction ability of RG6035 despite BS-mediated TfR1 cobinding.

[0349] RG6035 and BS-CD20-WT displayed a 30-fold higher intracellular uptake in Madin-Darby canine kidney II (MDCKII) cells transfected with huTfRI (MDCKII-huTfR) following a 30-minute loading (pulse) phase compared with huTfRI -negative non-transfected cells (MDCKII-parental). Following huTfRI -mediated uptake, comparable transcytosis activity profiles were observed for both BS constructs, with the amount ofIgG released into the extracellular compartment being identical. The non-target binding huTfRI positive control molecule, BS-DP47-PGLALA showed similar huTfRI -mediated uptake and transcytosis. Data are the mean of triplicates + / - SD. Following a loading (pulse) phase of 30 minutes, cells were extracted and analysed for intracellular IgG content using ELISA. The results are expressed as uptake of IgG normalized against the total cell protein in each well (ng IgG / mg). The results are shown in Figure 1.

[0350] The transcytosis efficiency is shown in Figure 2. The ng IgG transcytosed are compared with the ng IgG in the corresponding intracellular compartment at the beginning (t=0) of the chase (expressed as a %).

[0351] Thus, Fc-effector-function-silencing and the conjugation to a BS-module do not impair CD20 binding nor TfR1 -mediated transcytosis. The extent of direct B-cell death as a result of binding was comparable and concentration-dependent for these molecules.

[0352] In summary, it has been shown that RG6035 is well tolerated, with no indication of risk for IRRs, while retaining B-cell killing properties. It has been demonstrated that there is a TfR1 -dependent uptake of RG6035 into the brain in huCD20 transgenic mice. Taken together, RG6035 is able to and has advantageous properties to target and deplete CNS-located B-cells in MS more effectively, as compared to currently available therapies. It has been shown that for peripherally expressed targets, Fc-region-effector-function-silencing mitigates the risk of IRRs and effects on reticulocytes, resulting in an advantageous safety profile.

[0353] Example 2 - Preclinical development of RG6035 for treatment of MS

[0354] Nonclinical pharmacology

[0355] Pharmacological properties of RG6035 were characterised by in vitro studies using human and cynomolgus monkey derived B-cells, and in vivo in cynomolgus monkeys. Efficient total B-cell and B-cell subset depletion by RG6035 was demonstrated with human tonsil-derived immune cell suspension as well as human and cynomolgus monkeys whole blood cultures. Furthermore, it was shown that human cerebrospinal fluid (CSF) components do not impede the direct cell killing mechanism of RG6035.

[0356] In vivo, RG6035 induced a time-dependent reduction of B-cells not only in peripheral blood, but also in the spleen, tonsils, and mesenteric and mandibular lymph nodes in cynomolgus monkeys. The PD of peripheral B-cell depletion was characterized by four phases (initial rapid drop, rebound, sustained depletion, and return to baseline), and was analysed by PK / PD modelling based on available data in cynomolgus monkeys.

[0357] Pharmacokinetics and Drug Metabolism in cynomolgus monkeys

[0358] RG6035 PK was characterised in cynomolgus monkeys (the only cross-reactive species for CD20 and TfR1 binding) in serum, CSF, and brain tissue over the dose range of 0.3 mg / kg to 180 mg / kg. RG6035 showed a non-linear PK behaviour (attributed to target mediated drug disposition [TMDD]) after IV administration atdoses lower than 30 mg / kg and up to target saturation with an accelerated clearance (CL) up to 10 fold. RG6035 serum CL was overall linear over the dose range of 30 mg / kg to 180 mg / kg. The volume of distribution of RG6035 was low, ranging from 20 mL / kg to 80 mL / kg and typical for a therapeutic protein.

[0359] RG6035 uptake into brain vascular endothelial cells and parenchyma was confirmed with the CSF to serum exposure (area under the concentration-time curve [AUC]) ratio of the order of 1%, which corresponds to an approximately 25-fold higher brain AUC at steady state than a typical IgG. In brief, the greater the CSF to serum exposure ratio of RG6035, the greater the concentration of the drug within CNS niches, thus achieving higher exposure to pathological CNS-localised B-cells and improved treatment outcomes.

[0360] Toxicology and Safety Pharmacology

[0361] RG6035 was evaluated in a comprehensive package of nonclinical safety studies, including safety pharmacology endpoints, to support the FIH clinical study. Both the CD20- and TfR1 -binding moieties of RG6035 are cross-reactive to cynomolgus monkey only and no other commonly used species. Therefore, the cynomolgus monkey was selected as the relevant toxicity species.

[0362] The toxicity of RG6035 was studied for up to 4 weeks following once every 2 weeks (Q2W) IV (30 to 180 mg / kg), once a week (QW) IV (15 mg / kg) and QW subcutaneous (30 mg / kg) administrations.

[0363] Overall, treatment with RG6035 resulted in pharmacological changes, i.e., serum and peripheral B-cell depletion and decreased lymphocytes in lymphatic organs. Non adverse, dose dependent mild iron-restricted erythropoiesis with reduced haemoglobin synthesis rate and transient effects on platelet parameters were noted. With no adverse RG6035-related findings observed, RG6035 was well tolerated at all dose levels and regimens tested. The no observed-adverse effect level (NOAEL) was defined as 180 mg / kg Q2W in the dose range finding (DRF) study, and 150 mg / kg Q2W in the 4 week Good Laboratory Practice (GLP) study (i.e., the highest tested dose levels in both studies). No test item related adverse effects were noted for safety pharmacology endpoints for CNS, respiratory, and cardiovascular systems in the 4 week, GLP, repeat-dose toxicity study.

[0364] Further investigations demonstrated that RG6035 did not show an unexpected tissue staining pattern, induce haemolysis in human whole blood, precipitate in human plasma, or finally induce cytokine release in an in vitro human whole blood assay, indicating a low risk for infusion-related reactions (IRRs) upon first dosing in humans.

[0365] The nonclinical toxicity and safety pharmacology assessments therefore supported the initiation of the first-in-human clinical study in patients with MS.

[0366] Example 3 - depletion of B-cells in the CSF with ocrelizumab and RG6035

[0367] In the CNS, treatment with ocrelizumab standard dose (600 mg, IV infusion) results in low concentration of ocrelizumab in CNS (CSF plasma ratio of approximately 0.1-0.2%), but leads to a large and consistentreduction of CSF CD19+ B cell counts, observed from week 12 post-treatment (83.7%). It was also concluded that ocrelizumab standard dose has limited effect on other measures associated with B-cell intrathecal biology - IgG oligoclonal bands (OCBs), IgG Index, B cell related cytokines and chemokines -suggesting the maintenance of some B cell populations in the CNS.

[0368] In contrast, as demonstrated in Table 3 and in Figures 5 and 10, a single intravenous dose administration of 700 mg and 2000 mg RG6035 produced mean (± SD) CSF to serum ratios of 1.27% (± 0.29%) and 1.09% (± 0.42%), respectively. Thus, the CSF:Plasma ratios reported for RG6035 were significantly greater than those reported for the conventional mAb, ocrelizumab.

[0369] Table 3 - CSF drug concentration and CSF / serum ratio (%) following single IV dose of 700 mg and 2000 mg RG6035

[0370]

[0371]

[0372] BLQ = below limit of quantification.

[0373] These results therefore suggest that RG6035 may be a better mAb candidate for infiltrating the CNS and promoted depletion of CNS-compartmentalised B cells.

[0374] The following Examples 4-6 follow the published clinical study protocol BP42230 (NCT05704361). The results of the clinical study BP42230 are first described herein.

[0375] The clinical study BP42230 is the first study where RG6035 will be administered to humans. A nonrandomized, open-label, adaptive design was chosen to assess the safety, tolerability, and immunogenicity, as well as characterization of the pharmacokinetics (PK) and pharmacodynamics (PD) following administration of RG6035 to patients with MS.

[0376] The clinical study BP42230 is comprised of 3 parts:

[0377] Part 1 (Example 4) - single ascending doses (SAD) of RG6035, administered intravenously (IV) to patients with MS;

[0378] Part 2 (Example 5) - single ascending doses (SAD) of RG6035, administered subcutaneously (SC) to patients with MS;

[0379] Part 3 - (Example 6) - multiple ascending doses (MAD) of RG6035, administered subcutaneously (SC) to patients with MS.

[0380] The population of both RMS and PMS was selected in order to cover the entire MS spectrum.

[0381] Blood and CSF samples were taken to monitor and measure RG6035 concentrations and PD effects (CD19+ B cell depletion). CSF RG6035 concentrations were used to inform the PK / PD model and subsequent dosing in studies in patients. Exploratory assessment of ADA was also performed in CSF to help investigate the relationship between ADAs and safety / PK / PD.

[0382] Example 4 - Clinical study with RG6035 for treatment of MS - Part 1

[0383] Study Design for Part 1

[0384] Study BP42230 part 1 is a multiple-centre, non-randomized, open-label, adaptive study to investigate the safety, tolerability, immunogenicity, PK, and PD of single ascending IV doses of RG6035 in patients with MS.All participants in this study received a single IV dose of RG6035 in the morning of Day 1 .

[0385] 55 participants completed this study. Each cohort was split in two groups as follows: a sentinel participant dosed on Day 1 , and at least 5 participants dosed no earlier than 36 hours thereafter.

[0386] Treatment Groups for Part 1

[0387] Seven dose levels:

[0388] Cohort 1 - 7 mg RG6035 (starting dose)

[0389] Cohort 2 - 20 mg RG6035

[0390] Cohort 3 - 70 mg RG6035

[0391] Cohort 4 - 200 mg RG6035

[0392] Cohort 5 - 700 mg RG6035

[0393] Cohort 6 - 2000 mg RG6035

[0394] Cohort 7 - 4000 mg RG6035

[0395] Subsequent doses were selected in an adaptive manner during study conduct based on emerging data. Doses were repeated (or additional participants added), adjusted downwards, or intermediate doses may be investigated based on safety, tolerability, PK data, and / or PD data at each dose level, as well as on model-based prediction of anticipated maximum tolerated dose and / or PK and PD parameter(s). The maximum dose did not exceed 4000 mg, which is considered to the maximum feasible dose for IV administration.

[0396] A Dose-Decision Committee was established to provide dose recommendations for all cohorts of the study.

[0397] Objectives and endpoints

[0398]

[0399]

[0400] Dose-Escalation-Decision Criteria

[0401] The decision to escalate to the next dose level was made jointly by the Applicant’s study team, the Investigators, and any other person the Investigators or Clinical Pharmacologist considers necessary to assist with the decision (Dose-Decision Committee).

[0402] Dose decisions (to escalate, reduce, or repeat) between cohorts were made after careful and complex review of all available cumulative (i.e., including emergent new data from all previous cohorts) safety data (including adverse events [AEs], electrocardiograms [ECGs], vital signs, laboratory safety test results) collected up to at least Day 15 ( ±2 days) after receiving RG6035, and PK and PD data (whole blood panel of T-cells, B-cells, and natural killer cells) collected up to at least Day 8 ( ±1 day) after receiving RG6035.

[0403] Evaluable data from at least 6 participants of the previous dose level after single IV administration of RG6035 was required to make the decision to escalate to the next dose level.

[0404] General Study Stopping Rules Criteria

[0405] Dosing may have been stopped at any time during the study if one of the circumstances listed below occured:

[0406] • One RG6035-related serious AE (SAE)

[0407] • Severe (> Grade 3 per National Cancer Institute Common Terminology Criteria for Adverse Events [NCI CTCAE] v5 grading) non-serious RG6035 related AEs in 2 participants in the same cohort.

[0408] During the Parts 1-3 (Examples 3-5), the study stopping rules criteria were not reached.

[0409] Brain Magnetic Resonance Imaging

[0410] MRI was used to monitor brain lesions related to MS pathology and potentially other pathology findings. Screening brain MRI scans were obtained and checked for quality, only if no MRI scan taken within 3 months prior to Screening is available.

[0411] For eligibility, an MRI scan was taken at least 12 months (minus 2 weeks) prior to the screening MRI as a reference (activity / non-activity defined by MRI).For participants of Cohorts 5, 6 and 7, and later cohorts, as appropriate, results from the Screening MRI were assessed before CSF sampling to avoid exposing participants to an invasive procedure in case of exclusionary findings.

[0412] The evaluation of scans for incidental pathology not related to MS, such as PML, is a local responsibility that should be handled according to the local practice. Any clinically significant findings should be reported on the Adverse Event eCRF.

[0413] MRI assessments can include, but may not be limited to, T1 -weighted scans before and after injection of Gd contrast and T2-weighted scans before injection of Gd contrast.

[0414] Lumbar Puncture (Cohorts 5, 6, and 7, and Later Cohorts, as Appropriate)

[0415] Prothrombin time (I NR) and platelet count was performed within 7 days before the planned lumbar puncture and results must be available for review by site staff before the lumbar puncture can proceed. The participant must have an INR < 1.3 and a platelet count of > 100000 to minimize the risk of bleeding during the procedure.

[0416] Lumbar punctures were performed by a qualified physician who meets all local requirements and is proficient in the procedure to collect CSF from each participant. Two mandatory CSF samples were obtained; one between Day -7 to Day 1 (to obtain a predose or baseline sample) AND at one other timepoint (or at two timepoints if consent for an optional third sample is given) up to the Follow-up visit (this includes any unscheduled visit in case of relapse).

[0417] Lumbar puncture procedures and post-lumbar puncture care were performed in accordance with local practice. CSF sampling should be performed in the morning (between 8:00 a.m. and noon) to minimize potential diurnal variation of CSF parameters.

[0418] Atraumatic needles will be used for the lumbar puncture; the liquid will be sampled in a free flowing manner, which will take about 5 minutes.

[0419] CSF samples were collected from participants in Cohorts 5,6, and 7 (and later cohorts, as appropriate) and aliquoted for the following:

[0420] • Measurement of RG6035

[0421] • Biomarker analysis (Ocrelizumab Biomarker Outcome Evaluation [OBOE] panel; quantitative immunoglobulins, oligoclonal bands, and albumin; and exploratory biomarkers).

[0422] Exploratory assessment of ADAs were also performed, using the remaining PK CSF samples, in case of positive ADA results in serum.

[0423] Blood samples were also collected at the time of the CSF sample collection and aliquoted per timepoint for the measurement of serum PK; serum ADA; exploratory biomarkers; OBOE panel; immunoglobulins,oligoclonal bands, and albumin; TBNK,B cell subset and T-cell subset panels; and safety biomarkers only if required for safety reasons.

[0424] Pharmacokinetics

[0425] Blood and CSF samples for determination of serum and CSF concentrations of RG6035 and biotransformation products, as appropriate, were collected as described above. The date and time of each sample collection were recorded in the eCRF.

[0426] Blood samples were not taken from the same arm as IV drug administration.

[0427] Serum and CSF concentrations of RG6035 and biotransformation products, as appropriate, were measured by specific and validated methods. Samples may be used for exploratory biotransformation products identification. CSF PK data was not required for dose-escalation criteria.

[0428] A decision to stop PK sampling earlier or to collect more samples than currently proposed was based on the emergent PK profile of RG6035 from preceding dose groups.

[0429] Immunogenicity assessments

[0430] As RG6035 is a human antibody, there is a risk that ADA against RG6035 could develop, potentially reducing its efficacy and / or potentially resulting in a symptomatic hypersensitivity reaction, in particular immune-complex reactions.

[0431] Antibodies to RG6035 were evaluated in serum samples collected from all participants. The date and time of each sample was recorded in the electronic case report form (eCRF). ADA data was not required for dose-escalation decisions. Additional ADA samples were collected in participants if they experienced a Grade > 2 IRR and in participants with clinical signs of hypersensitivity reaction, in particular immune-complex reaction. In each case, for each collected ADA sample, a corresponding PK sample was collected at the same timepoint for the determination of the RG6035 concentration.

[0432] Validated screening, confirmatory, and titre assays were employed to detect ADAs against RG6035.

[0433] Pharmacodynamics and biomarkers analyses

[0434] Blood and CSF samples for PD tests and biomarker analyses were collected as described above.

[0435] The timing of PD sampling (blood and CSF) may have been changed based on emergent PD results in order to allow optimal characterization of the PD, and up to three additional PD blood samples may be taken per participant, after agreement with the Applicant’s Clinical Pharmacologist and the Investigator.Pharmacodynamics and biomarker samples

[0436] The samples may also be used for research purposes to identify biomarkers useful for predicting and monitoring response to RG6035, identifying biomarkers useful for predicting and monitoring RG6035 safety, assessing PD effects of RG6035, and investigating mechanisms of therapy resistance.

[0437] Biomarkers were presented as absolute value over time and / or percent change relative to baseline over time. Biomarker levels at baseline or overtime could be compared with efficacy or safety measurements to assess prognostic or predictive properties. Descriptive or summary statistics were used to describe biomarker assessments. Samples taken at screening and baseline before RG6035 dose could be averaged to better normalize postdose endpoints, taking into account normal variability.

[0438] The following samples for PD and biomarker research are required and will be collected from participants as detailed below.

[0439] CSF Samples (Only Cohorts 5 and 6 and Later Cohorts, as appropriate)

[0440] CSF samples were collected from participants in Cohorts 5 and 6 (and later cohorts, as appropriate) as described above. Two mandatory CSF samples were obtained; one between Day -7 to Day 1 (to obtain a predose or baseline sample) AND at one other timepoint (or at two timepoints if consent for an optional third sample is given) up to the Follow-up visit (this includes any unscheduled visit in case of relapse).

[0441] CSF samples were collected for the measurement of RG6035 and biomarker analyses.

[0442] Exploratory assessment of ADA was also performed, using the remaining PK CSF samples, in case of positive ADA results in serum.

[0443] The timing of CSF collection was selected based on emergent PD results in order to allow optimal characterization of the PD. Safety biomarkers may be analyzed at the discretion of the Investigator and the Sponsor in case of a safety concern.

[0444] CSF Samples (Cohort 7 only)

[0445] Per protocol, in Cohort 7 (Part 1) and in Part 3, two mandatory CSF samples were planned to be obtained (one at predose and one at postdose) with an optional third CSF sample (at postdose, if consent was given) to investigate RG6035 CSF concentrations and to assess the effect of RG6035 on CSF PD-related to changes in B-cell count (as a proxy for the brain). No information on PK was expected for the pre-dose sample, which was collected to assess the change in B-cell count.

[0446] CSF sampling was optimized to provide information on the time course of RG6035 concentrations in the CSF and on its PD effect with respect to changes on CSF B-cells. The proposed time points to collect postdose CSF samples in Cohort 7 were guided by preliminary PKPD model predictions for CSF PK and B cells at the scheduled protocol visits:• Three or more participants on Day 8 (±1 day) or on Day 11 (±1 day): Rationale: Best for measurable PK, and may capture maximum B-cell depletion.

[0447] • Three or more participants on Day 15 (±2 days) or on Day 22 (±3 days): Rationale: Expected measurable PK, and close to maximum expected B-cell depletion.

[0448] • Three or more participants on Day 57 (±12 days): Rationale: No information on PK was expected, could demonstrate sustained B-cell depletion (and B-cells could come back towards baseline).

[0449] In case of an optional second postdose sample, it could be collected combined on Day 15 / 22 and Day 57.

[0450] In Part 3, it was suggested to collect the CSF samples at the following visits:

[0451] • 3 participants: Day 29 (±2 days) or Day 36 (±2 days)*

[0452] • 3 participants: Day 57 (±12 days)*

[0453] • Optional second post-dose sample: combined Day 29 or Day 36 and Day 57

[0454] * sampling windows per protocol.

[0455] Blood Samples

[0456] Blood samples (including blood PD and biomarker samples) were collected as specified above for PD and biomarker analyses.

[0457] Pharmacokinetic Analyses

[0458] All PK analyses were carried out on the PK analysis population.

[0459] Serum and CSF Pharmacokinetic Parameters

[0460] The RG6035 serum and CSF concentrations were summarised descriptively by dose group.

[0461] PK parameters were read directly from the serum concentration-time profiles or calculated using standard non-compartmental methods.

[0462] Serum PK parameters of RG6035 include (if appropriate):

[0463] Tmax: Time to maximum observed concentration

[0464] Cmax: Maximum observed serum concentration

[0465] AUCo i68h: Area under the serum concentration-time curve from time 0 to 168 hours postdose AUCiast: Area under the serum concentration-time curve up to the last measurable concentration AUCint: AUC from time 0 to infinity

[0466] CL: Total body clearance calculated as Dose / AUC

[0467] Az: Terminal rate constant calculated by linear regression of the log transformed terminal part of the concentration-time curve

[0468] • T1 / 2: Apparent terminal half-life computed as ln(2) / Az

[0469] Statistical analyses

[0470] PK analysesPK analyses were carried out on the PK analysis population. PK concentration data (serum and CSF) and calculated PK parameters for RG6035 were displayed as appropriate. Individual listings, summary tables (including descriptive statistics: means, medians, ranges, standard deviations, and coefficients of variation), and graphs (including concentration versus time plots on linear and semilogarithmic scales) were displayed as appropriate. PK parameters were estimated using non compartmental methods.

[0471] Dose-normalized PK parameters of interest were explored graphically for dose linearity. In addition, analysis of variance models were used to test for dose linearity.

[0472] PK / PD relationships was explored using graphical analysis and if deemed appropriate exposure-response was modelled mathematically for selected PD and / or safety measurements.

[0473] Immunogenicity Analyses

[0474] The immunogenicity analyses were carried out on the immunogenicity analysis population.

[0475] The numbers and proportions of ADA positive participants and ADA negative participants at baseline (baseline prevalence) and after study drug administration (post-baseline incidence during both the treatment and follow-up periods) were summarized.

[0476] • Participants are considered to be ADA positive if they are ADA negative or have missing data at baseline but develop an ADA response following study drug administration (treatment induced ADA response), or if they are ADA positive at baseline and the titre of one or more post baseline samples is at least 4-fold (e.g., > 0.60-titer units) greater than the titre of the baseline sample (treatment- enhanced ADA response).

[0477] • Treatment-induced ADA responses were further categorized as either:

[0478] o Transient ADA response: defined as a) ADA negative or missing data at baseline and b) at least one post-treatment ADA-positive sample and c) only one ADA positive sample or the time between the first and last ADA-positive sample is less than 16 weeks and d) the last ADA sample is negative.

[0479] o Persistent ADA response: defined as a) ADA negative or missing data at baseline and b) post-treatment ADA-positive samples over 16 weeks or more or the last ADA timepoint is positive (Shankar et al 2014).

[0480] • Participants were considered to be ADA negative if they are ADA negative or have missing data at baseline and all post-baseline samples are negative, or if they are ADA positive at baseline but do not have any post baseline samples with a titre that is at least 4 fold greater than the titre of the baseline sample (treatment unaffected).

[0481] The relationship between ADA status and PK, safety, and biomarker endpoints was analysed and reported descriptively below.

[0482] Pharmacodynamic Analyses

[0483] PD data and parameter estimates were presented by listings and descriptive summary statistics (including means, medians, ranges, standard deviations, and coefficients of variation) and graphs as appropriate.Pharmacokinetic / Pharmacodynamic Relationships

[0484] PK / PD relationships were explored using graphical analysis or exploratory PK / PD modelling (e.g., nonlinear mixed effects modelling) may be performed in addition. Pharmacometric modelling and simulation based on study data were applied if considered appropriate and used for dose escalation / decision / CSF sampling time selections purposes, as appropriate.

[0485] Results

[0486] Safety and tolerability evaluations

[0487] Intravenous administration of 7 mg, 20 mg, 70 mg, 200 mg, 700 mg, 2000 mg, and 4000 mg RG6035 was safe and well tolerated in all the 55 participants. Three serious adverse events (SAE) were reported, all considered unrelated to the study drug. No Non-Serious Adverse Events of Special Interest or deaths were reported. There were no withdrawals due to an adverse event (AE).

[0488] 28 AEs have been reported for 10 (out of 12) participants in Cohort 5. One Grade 3 SAE of cellulitis was reported, unrelated to study drug and resolving after 3 days in hospital. All other AEs were non serious and Grade 1-2 in severity.

[0489] 32 AEs have been reported for 8 (out of 12) participants in Cohort 6. One Grade 1 SAE of Endometrial Polyp was reported, unrelated to study drug and still ongoing. All other AEs were non serious and Grade 1-2 in severity.

[0490] 10 AEs have been reported for 4 (out of 5) participants in Cohort 7. One participant experienced a Grade 3 SAE of ophthalmoplegia with onset on Study Day 97. The event was assessed to be unrelated to the study drug by the Investigator and recovered / resolved without treatment. No Grade 4 or 5 AEs were reported. No participant experienced an AE leading to treatment withdrawal and dose modification / interruption. One participant experienced a Grade 2 AE of IRR, which was assessed by the investigator as related to study treatment.

[0491] No apparent pattern of clinically significant abnormalities in the electrocardiogram (ECG), vital sign (VS) or laboratory safety parameters. However, a dose-dependent trend for an increase in soluble transferrin receptor was observed in Cohorts 5 and 6 with a trend of decrease in ferritin and increase in transferrin but with no clear dose dependency. In Cohort 7, the mean soluble transferrin receptor level showed an initial decline followed by a slow increase with a peak around Day 29 and returning to baseline at the follow-up visit. All participants were clinically unremarkable and most values were within normal range. A trend for an increased erythrocytes distribution width after Day 29-57 and a decreased reticulocyte corpuscular haemoglobin content from Day 5 to Day 57 was observed in Cohorts 5 and 6. In Cohort 7, there was a decrease in reticulocyte corpuscular haemoglobin content observed from Day 8 to 57 with gradual recovery back to baseline levels from Day 22 to Day 85.MRI scans were scheduled at screening and follow-up (Day 169) in Part 1 and at screening, Day 57 and follow-up (Day 197) in Part 3. At screening, up to 3 new / enlarging T2 lesions as compared to a reference scan are allowed per protocol. In Cohort 7, all 5 participants had an MRI scan at screening (equivalent to baseline in the outputs), of whom 1 participant reached the final follow-up MRI visit (Day 169). No participants had new / enlarging T2 lesions at screening. Only one participant (with results available at the follow-up visit) reported an abnormal MRI scan at this visit, which indicated new or enlarging T2 lesion(s).

[0492] Pharmacokinetics

[0493] Serum PK

[0494] Table 4 summarises the computed RG6035 serum PK parameters (non-compartment analysis [NCA], Phoenix WinNonlin 8.3.4). The serum concentration-time profiles of 7, 20, 70, 200, 700 and 2000 mg RG6035 are illustrated in Figure 3 (mean by dose group). The serum concentration-time profiles of 7, 20, 70, 200, 700, 2000, and 4000 mg RG6035 are illustrated in Figure 16 (mean by dose group).

[0495] Table 4 Mean Serum PK Parameters of RG6035 Following a Single IV Administration of 7, 20, 70, 200, 700, 2000, 4000 mg

[0496]

[0497] * Median (Min-Max) for Tmax ; GeoMean = Geometric mean; CV = coefficient of variation; Cmax = maximum observed serum concentration; Tmax = time to maximum observed serum concentration; N = number of participants. **N=4

[0498] CSF PK

[0499] Table 3, as presented above in Example 3, and Figure 10 presents the individual CSF concentrations of RG6035 following a single IV administration of 700 and 2000 mg. Table 3 also presents the individual CSF concentrations of RG6035 following a single IV administration of 4000 mg.

[0500] After a single dose administration of 700 mg and 2000 mg RG6035, the mean (± SD) CSF to serum ratio was 1.27% (± 0.29%) and 1.09% (± 0.42%) for a dose of 700 mg and 2000 mg, respectively. These data from cohorts 5 and 6 is presented in Figure 5A.After single dose IV administration of 700 mg, 2000 mg and 4000 mg RG6035, mean (± SD) CSF to serum concentration ratios were 1.27% (± 0.29%), 1.07% (± 0.40%) and 0.75% (± 0.35%), respectively. There was no apparent change in the ratio of CSF / serum concentrations over the investigated concentration and time range (Day 8-Day 57). These data from cohorts 5, 6, and 7 is presented in Figure 5B.

[0501] Pharmacodynamics

[0502] Whole blood panel of T-cells, B-cells and natural killer [NK] cells (TBNK panel) was analysed by flow cytometry.

[0503] Figure 4A shows individual profiles of B-cells in blood per participant in cohorts 1-6 presented as fold change in frequencies of cells from baseline. Figure 4B indicates mean fold change in B-cells from each cohort 1-6. Figure 4C indicates mean fold change in B-cells from cohorts 1-7. Baseline has been calculated as the mean of screening and pre-dose values.

[0504] An initial drop in B-cells in blood on Day 2 was observed in cohorts 1 -6 with no predominant dose dependent effect. In Cohort 5 all participants (n=10) show depletion at Day 2 (range -13% to -43%; mean -28%). In Cohort 6 only 6 / 9 participants with measurable readout at Day 2 showed B-cell depletion in blood and to different extent (overall range +15% to -43%; mean - 16%). The mean drop (-16%) in B-cells in blood on Day 2 in Cohort 6 is of a smaller magnitude as compared to cohorts 1 -5. Following single IV administration of 4000 mg RG6035, peripheral blood B-cell frequencies exceeded baseline levels at all time points. No significant changes were observed for monocytes, T cells, and NK cells.

[0505] In CSF CD19+ B-cells were assessed by flow cytometry using A872 panel. A minimum of 15 B-cell events need to be measured on flow cytometer to reliably report resulting frequencies of B-cells in the CSF sample. Figure 5A shows individual profiles of change in CSF B-cells frequencies over time in Cohorts 5 and 6, presented as fold change in frequencies from baseline over time. Figure 5B shows individual profiles of change in CSF B-cells frequencies overtime in Cohorts 5, 6, and 7, presented as fold change in frequencies from baseline overtime. Baseline has been calculated as the mean of screening and pre-dose values.

[0506] In Cohort 5, the CSF B-cell change from baseline ranges from +13% to -62% with a median of 57% depletion observed in 6 participants with measurable B-cells at early post-dose time points (Day 8-22) after a dose administration of 700 mg. For one participant (10033) in Cohort 5, two post-dose CSF data are available showing a CSF B-cell depletion of 59% and an increase in CSF B-cells of 30% on Day 11 and Day 131 , respectively. In Cohort 6, after a dose administration of 2000 mg, B-cell depletion ranges from -64% to -97% with a median of 88% depletion at early post-dose time points (Day 8-14). In one participant, a sustained depletion of up to 65% was observed at the second time point (Day 56) and in another participant CSF B-cells recovered back to baseline at the second measurable time point (Day 86). In Cohort 7, following single IV administration of 4000 mg, valid results are available for only one participant with a change from baseline in CSF B-cell levels of -81% observed on Day 15.There seems to be a dose dependent effect with higher levels of B-cell depletion seen in Cohort 6 (2000 mg). The cells stay depleted to different levels at least until approximately 2 months post dosing and seem to be recovering back to baseline overtime (latest on Day 86).

[0507] Antidruq antibodies (ADAs)

[0508] ADA samples were scheduled for Days 1, 8, 22, 29, 57, 85, and at follow-up (Day 169). For Cohorts 5, 6 and 7, additional ADA sample(s) were collected at the time of CSF sample(s). These samples were taken only if at the same visit no regular ADA sample collection was planned. The titres are shown in Table 5, below.

[0509] In all 7 cohorts, all pre-dose and Day 8 samples were ADA negative with the exception of one participant who showed a titre of 100 at pre-dose on Day 1 , which is a borderline signal.

[0510] In Cohort 3, one participant showed a decrease in titre at follow-up (titre of 800) compared to Day 85 (titre of 1600).

[0511] In Cohort 5, one participant showed a decrease in titre at follow-up (titre of 400) compared to Day 85 (titre of 800). Two participants were ADA negative at all available time-points.

[0512] In Cohort 6, 3 participants were ADA negative at follow-up , and on Day 57 for 3 further participants.

[0513] In Cohort 7, all participants were ADA negative at all available time-points.

[0514] Table 5 - ADA titres from CSF samples for Cohorts 1-6 (Part 1).

[0515]

[0516]

[0517] As the minimal required dilution of the ADA assay is 100, a titre of 100-200 is very low with a borderline signal. The data suggests trends of a lower prevalence and a later onset of ADAs at higher doses.

[0518] Conclusions from Part 1

[0519] These results summarise the overall favourable safety and tolerability assessment. The data from Cohorts 5, 6, and 7 confirmed that a single IV administration of 700, 2000, and 4000 mg RG6035 was well tolerated by all participants.

[0520] A geometric mean serum Cmax of 226 pg / mL (N=12) and 508 pg / mL (N=12) and a geometric mean AUCo-i68h of 14’400 pg*h / mL (1X1=12) and 44’500 pg*h / mL (N=12) were reached with the available PK data following a single IV administration of 700 and 2000 mg, respectively.

[0521] An initial drop in B-cells in blood on Day 2 was observed in cohorts 1-6. The mean drop (-16%) in B-cells in blood on Day 2 in Cohort 6 is of a smaller magnitude as compared to all the other cohorts (-23% to -41%). A trend for a prolonged effect beyond Day 2 was observed in some donors in high dose cohorts (Cohorts 4-6). Subsequently, B-cell levels gradually returned to baseline. Following single IV administration of 4000 mg RG6035, peripheral blood B-cell frequencies exceeded baseline levels at all time points. No significant changes were observed for monocytes, T cells, and NK cells.

[0522] A median of 57% CSF B-cell depletion was observed (range of change in B-cells: +13% to -62%) after a dose administration of 700 mg at early time points (Day 8-22). At 2000 mg, B-cell depletion ranges from -64% to -97% at early time points (Day 8-14). There seems to be a dose dependent effect with higher levels of B-cell depletion (up to a median of 88%) seen in the 2000 mg cohort. Following single IV administration of 4000 mg, valid results are available for only one participant with a change from baseline in CSF B-cell levels of -81% observed on Day 15.Example 5 - Clinical study with RG6035 for treatment of MS - Part 2

[0523] This is a Phase I, multiple-centre, non-randomized, open-label, adaptive, single ascending dose study of RG6035, subcutaneously (SC) administered to patients with MS. For details on the study, please refer to the study protocol presented in Example 4.

[0524] Study design for Part 2

[0525] Part 2 enrolled 6 participants: 3 x RRMS, 1 x PPMS, 2 x SPMS. All participants in Part 2 were all administered a single dose of 70 mg RG6035 subcutaneously.

[0526] Clinical observations

[0527] Expanded Disability Status Scale (EDSS)

[0528] One participant showed a change at day 85 (1 point decrease), others did show no change in EDSS at day 85 as compared to Screening.

[0529] Neurological examination

[0530] Two Participants showed changes in the neurological examination (NE), but these were not considered clinically significant changes.

[0531] Relapses

[0532] No clinical relapse(s) were reported in this cohort during the reporting interval for the dose escalation.

[0533] C-SSRS

[0534] All participants in this cohort had normal C-SSRS assessments at Screening and at pre-dose, no participant showed any sign of suicidal ideation or suicidal behaviour.

[0535] MRI

[0536] One participant has reached visit Day 169 and showed new / enlarging T2 lesions at the Follow Up MRI scan as compared to Screening.

[0537] Safety and tolerability evaluations

[0538] Adverse events in cohort 1

[0539] No adverse events have been reported to date in this cohort. There were no deaths or Non-Serious Adverse Events of Special Interest (NSAESI) reported to date.

[0540] Vital signs and ECG

[0541] Vital signs (body temperature, systolic blood pressure [SBP], diastolic blood pressure [DBP], pulse and respiratory rate) were generally performed according to the protocol.There was no apparent pattern of clinically significant abnormalities in VS (most of the absolute values within normal reference ranges and no significant change from baseline) after SC administration of 70 mg RG6035. There were no clinically relevant trends in the ECG parameters during the observed period.

[0542] Laboratory safety

[0543] Clinical laboratory evaluations were generally performed according to the protocol. Most laboratory safety parameter values were reported within normal ranges, with some isolated outlier values reported and not deemed clinically relevant because of the slight and transient change in respect to the normal ranges or because the abnormality was already present at screening or predose.

[0544] Overall, no clinically significant trend for changes in chemistry, haematology, immunology, or urinalysis laboratory assessments following single SC dose administration of 70 mg RG6035 was observed.

[0545] LISSA and VAS scales

[0546] No local injection site reaction was reported on the LISSA scale following a single SC administration of 70 mg RG6035.

[0547] According to the protocol, any score between 0 and 4 mm on the VAS scale should be considered as no pain. No score higher than 4 mm was reported in any participant.

[0548] Results

[0549] Cohort 1 of 6 participants receiving a single SC dose of 70 mg RG6035. Sentinel participant dosed on 13-Aug-2024 and last participant dosed on 16-Dec-2024.

[0550] Subcutaneous administration of 70 mg RG6035 was safe and well tolerated in the 6 participants. No serious adverse events (SAE), Non-Serious Adverse Events of Special Interest or deaths were reported. There were no withdrawals due to an adverse event (AE), and no AE has been reported to date in this cohort. There were no deaths or Non-Serious Adverse Events of Special Interest (NSAESI) reported to date. No apparent pattern of clinically significant abnormalities in the electrocardiogram (ECG), vital sign (VS) or laboratory safety parameters have been reported.

[0551] Pharmacokinetics

[0552] Serum PK

[0553] Table 6 summarizes the computed RG6035 serum PK parameters (non-compartment analysis [NCA], Phoenix WinNonlin 8.3.4). The serum concentration-time profiles of 70 mg SC RG6035 are illustrated in Figure 6 (individual data).

[0554] Table 6 - Mean Serum PK Parameters of RG6035 Following a Single SC Administration of 70 mg

[0555]

[0556] | N=6 | (CV% geomean) | (100.7) | (70.3-167.4) | (161.6) |

[0557] * Median (Min-Max) for Tmax ; GeoMean = Geometric mean; CV = coefficient of variation; Cmax = maximum observed serum concentration; Tmax = time to maximum observed serum concentration; N = number of participants.

[0558] **N=4

[0559] PK concentrations and PK parameters (Cmax= 1.33 pg / mL) after a single SC dose administration of 70 mg RG6035 were in the expected range (predicted serum C max— 2.0 pg / mL).

[0560] Figure 7A and Figure 7B present, respectively, the individual and mean serum concentration-time profiles of 70 mg SC RG6035 (initial plotted RG6035 concentration of around 1 ug / mL) superimposed with the individual serum concentration-time profiles of 70 mg IV RG6035 (initial RG6035 concentration of around 10-50 ug / mL).

[0561] Median of observed serum concentrations are in line with the predictions from a preliminary population pharmacokinetic model, assuming approximately 50% bioavailability (BA), and an absorption half-life of approximately 3.9 days, as illustrated in Figure 8.

[0562] Typical PK profiles (i.e. not taking into account variability) were simulated assuming bioavailability of 35%, 50%, 73% or 93%, with associated absorption half-lives of approximately 5.3, 3.9, 3.0, or 1.7 days. Median of observed PK data available from 4 participants in this cohort best corresponds with the predicted profile assuming 50% BA.

[0563] Pharmacodynamics

[0564] Whole blood panel of T-cells, B-cells and natural killer [NK] cells (TBNK panel) was analysed by flow cytometry.

[0565] Figure 9 shows individual profiles of B-cells per patient presented as fold change in frequencies of cells from baseline following a single SC (Part 2-Cohort 1) and IV (Part 1-Cohort 3) administration of 70 mg. Baseline has been calculated as the mean of screening and pre-dose values.

[0566] In SAD SC Cohort 1 dosed with 70mg, an initial drop in blood B-cells on Day 2 was observed in all 6 participants (overall range -15% to -55%; mean -38%). Notably, two participants who showed higher B-cell depletion on Day 2 also showed prolonged depletion at least until Day 8. An increase in blood B-cells was observed at Day 22 for all participants which was already observed at Day 8 for one participant.

[0567] B-cells data observed following a single SC dose administration of 70 mg shows a similar pattern as data observed after a single IV dose administration of 70 mg (right panel) with mean depletion -38% in SC vs -23% in IV dose cohort.Antidrug antibodies (ADAs)

[0568] ADA samples were scheduled for Days 1 , 8, 22, 57, 85, and at follow-up (Day 169).

[0569] All pre-dose, Day 8 and Day 22 samples were ADA negative for all participants, and also on Day 57 for Participants 3 and 5.

[0570] Participants 54 and 57 showed on Day 57 a titre of 1600 and 800, respectively, and Participant 55 showed a titre of 100, which is a borderline signal (Table 7).

[0571] Table 7 - Antidrug Antibodies over Time per Participant

[0572]

[0573] As the minimal required dilution of the ADA assay is 100, a titre of 100-200 is very low with a borderline signal.

[0574] Conclusions from part 2

[0575] This report summarized the overall favourable safety and tolerability assessment. A single SC administration of 70 mg RG6035 was well tolerated by all participants.

[0576] A geometric mean serum Cmax of 1.33 pg / mL (N=6) was reached with the available PK data. Observed serum concentrations are in line with the predictions from a preliminary population pharmacokinetic model, assuming approximately 50% bioavailability.

[0577] An initial drop in B-cells in blood on Day 2 was observed in all six participants. The mean drop in B-cells in blood on Day 2 was -38% with range (-15% to -55%). Two patients that showed higher drop in B-cells on Day 2 also showed prolonged depletion (at least until Day 8). B-cell data following single SC dose of 70 mg shows very similar pattern as single IV dose of 70 mg.

[0578] Example 6 - Clinical study with RG6035 for treatment of MS - Part 3

[0579] This is a Phase I, multiple-centre, non-randomized, open-label, adaptive, multiple ascending dose study of RG6035, administered subcutaneously (SC) to patients with MS. For details on the study, please refer to the study protocol presented in Example 4.Study design for Part 3

[0580] Participants in Part 3 of the study received multiple doses of RG6035 for up to 4 dose administrations. A once-weekly dosing regimen was administered subcutaneously for 4 weeks (Q1W), but other dosing regimens (e.g., every 2 weeks) could be tested based on results from previous cohorts.

[0581] All participants in this study received a single IV dose of RG6035 in the morning of Day 1 .

[0582] Each cohort was split into two groups as follows: a sentinel participant was dosed on Day 1 , and the remaining participants were dosed no earlier than 7 days thereafter. If a dose level was repeated or if a lower dose (dose lower than one already studied and found safe) was investigated, the added group could consist of at least 3 participants (without a sentinel participant).

[0583] Treatment Groups for Part 3

[0584] • Cohort 1 - 70 mg RG6035

[0585] • Cohort 2 - 200 mg RG6035

[0586] • Cohort 3 - 700 mg RG6035

[0587] Dose decisions between cohorts were made based on comprehensive review by the Applicant and Investigators of the safety data (including AEs, ECGs, vital signs, laboratory safety test results) collected up to at least 14 days (± 2 days) after receiving the last RG6035 administration (i.e. , Day 36 [± 2 days]), and PK and PD data (whole blood flow cytometry) up to at least 7 days (± 2 day) after receiving last RG6035 administration (i.e., Day 29 [± 2 days]).

[0588] Multiple subcutaneous administration of 70 mg, 200 mg, or 700 mg RG6035 was safe and well tolerated in all participants. No Non-Serious Adverse Events of Special Interest (AESI) or deaths were reported. One participant in the 70 mg cohort was discontinued early from study drug after receiving 2 doses due to an ongoing Grade 2 SAE of influenza that required hospitalisation. One participant in the 200 mg cohort had a Grade 3 SAE of anxiety. Both events were assessed as unrelated to study drug. Two participants experienced an AE leading to dose modification / interruption, one each in the 70 mg (Influenza) and 700 mg (Coronavirus Disease 2019 [COVID-19]) cohorts.

[0589] 3 adverse events have been reported to date in 3 (out of 7) participants in the 70 mg cohort. One Grade 2 SAE of seasonal flu was reported, not considered related to the study drug by the PI and resolved. The other 2 AEs were non-serious, and one was Grade 1 and the other was Grade 2 in severity.

[0590] No apparent pattern of clinically significant abnormalities in the electrocardiogram (ECG), vital sign (VS) or laboratory safety parameters.

[0591] Participants characteristics

[0592] 23 participants completed this study.Seven participants were enrolled in the 1st MAD cohort with 4 weekly doses of 70 mg RG6035. The initial data provided for the 70 mg cohort disclosed in Figures 11-14 relates to participants 1-6 of 70 mg cohort.

[0593] 4 participants were diagnosed with RRMS and 2 with SPMS. Later data presented in Figures 16-18 includes one additional participant in this cohort. For discussion about this cohort. N=7 unless otherwise specified.

[0594] One participant of the 70 mg cohort received 2 doses only, first dose on 16-Dec-2024 and second dose on 23-Dec-2024. The third dose, planned on 30-Dec-2024, was not administered due to an AE of seasonal flu (including hospitalization) occurring on 29-Dec-2024. Given that the participant was still recovering, and that dosing would have been outside the pre-defined window, the Applicant decided to discontinue the participant from study treatment but to continue with the visit schedule (including CSF sampling).

[0595] Eight participants were enrolled in each of the 200 mg (cohort 2) and 700 mg (cohort 3) cohorts for Part 3.

[0596] 3 out of 7 participants in the 70 mg dose cohort completed the study. The remaining 4 participants in the 70 mg dose cohort and all participants in the 200 mg and 700 mg dose cohorts are still ongoing in the study.

[0597] Clinical observations

[0598] EDSS

[0599] One participant has reached visit Day 85, showing no change in EDSS as compared to Screening.

[0600] Neurological examination

[0601] No changes in the neurological examination (NE) have been reported, except for one participant (no clinically significant change).

[0602] Relapses

[0603] No clinical relapse(s) were reported in this cohort during the reporting interval for the dose escalation.

[0604] C-SSRS

[0605] All participants in this cohort had normal C-SSRS assessments at Screening and at pre-dose, no participant showed any sign of suicidal ideation or suicidal behaviour.

[0606] MRI

[0607] One Participant showed new / enlarging T2 lesions on the MRI scan performed at the D57 visit.

[0608] Safety and tolerability evaluations

[0609] Six of 23 participants (26.1%) experienced a total of 10 AEs related to study treatment: 70 mg cohort (skin reaction 1 participant), 200 mg cohort (erythema 2 participants, injection site pruritis 1 participant and intermenstrual bleeding 1 participant) and 700 mg cohort (rash 1 participant, Injection site reaction 1participant and CD19 lymphocytes decreased 1 participant) all of which were of Grade 1 intensity, except erythema of Grade 2 intensity. No Grade 4 or 5 AEs were reported.

[0610] Adverse events in cohorts 1-3

[0611] A total of 3 adverse events have been reported to date in cohort 1 in 3 (out of 6) participants.

[0612] One participant of the 70 mg cohort was withdrawn early from study drug after receiving 2 doses due to a SAE. The participant reported a grade 2 AE of seasonal flu on 29-Dec-2024 and was hospitalised due to the fact that the participant was from the countryside (limited access to the hospital). The participant received Tamiflu 75 mg, oral, twice a day and paracetamol 500 mg, oral, 3 times a day, from 29-Dec-2024 to 02-Jan-2025 and ringer solution 500 mL, IV, once a day, from 29-Dec-2025 to 31 -Dec-2024. The participant was discharged from the hospital on 31 -Dec-2024. The event resolved on 02-Jan-2025 and was not considered related to the study drug by the PI.

[0613] The other 2 AEs in cohort 1 were non-serious, and one was Grade 1 and the other was Grade 2 in severity. There were no deaths or Non-Serious Adverse Events of Special Interest (NSAESI) reported to date.

[0614] One participant in the 200 mg cohort experienced a Grade 3 SAE of anxiety with onset on Study Day 62. The event was assessed to be unrelated to the study drug by the Investigator and recovered / resolved with treatment.

[0615] Vital signs and ECG

[0616] Vital signs (body temperature, systolic blood pressure [SBP], diastolic blood pressure [DBP], pulse and respiratory rate) were generally performed according to the protocol. For some occasions, vital signs were measured in a sitting position instead of in a supine position and data is presented combined (supine and sitting).

[0617] There was no apparent pattern of clinically significant abnormalities in VS (most of the absolute values within normal reference ranges and no significant change from baseline) after multiple SC administration of 70 mg, 200 mg, or 700 mg RG6035. There were no clinically relevant trends in the ECG parameters (HR, PR, QRS, QT, QTcF, RR, T and U waves) during the observed period.

[0618] Laboratory safety

[0619] Clinical laboratory evaluations were generally performed according to the protocol. Most laboratory safety parameter values were reported within normal ranges, with some isolated outlier values reported and not deemed clinically relevant because of the slight and transient change in respect to the normal ranges or because the abnormality was already present at screening or predose.

[0620] The participant of the 70 mg cohort who reported an AE of seasonal flu on Day 14, showed an increase in bilirubin (53.0 pmol / L, reference range: 5.13-20.5 pmol / L - baseline 37.6 pmol / L) and an increase in iron (37.6 pmol / L, reference range: 11.6-31.4 pmol / L) on Day 8. This participant also showed an increase in gamma glutamyl transferase (122 U / L, reference range: 0-55 U / L) and an increase in potassium (6.1 mmol / L, reference range: 3.5-5.1 mmol / L) on Day 22.Overall, no trend for changes in chemistry, haematology, immunology, or urinalysis laboratory assessments following multiple SC dose administration of 70 mg, 200 mg, and 700 mg RG6035 was observed. However, a trend for an increase in soluble transferrin receptor in the 70 mg and 200 mg dose cohorts, and returning towards baseline at follow-up. There was also a trend for a decrease in erythrocytes was observed after Day 1 for all cohorts. The reduction began early in the treatment period, persisted throughout the study, and levels went back towards baseline at follow-up.

[0621] LISSA and VAS scales

[0622] No local injection site reaction was reported on the LISSA scale following multiple SC administration of 70 mg RG6035.

[0623] According to the protocol, any score between 0 and 4 mm on the VAS scale should be considered as no pain and any score between 5-44 mm should be considered as mild pain. One participant reported a score of 11 mm on Day 1 , 1 hour post-dose and a score of 2 mm 7 hours later.

[0624] MRI

[0625] All 23 participants in Part 3 had an MRI scan at screening, of whom 17 reached the Day 57 visit and out of these 7 reached the final follow-up MRI visit (Day 197). One participant in the 700 mg dose cohort had new / enlarging T2 lesions at screening. Two participants in the 70 mg dose cohort reported an abnormal MRI scan at Day 57, which indicated new or enlarging T2 lesion(s). All follow-up MRI scans were reported as normal (i.e. , no new T1 Gadolinium enhanced and / or new / enlarging T2 lesion(s)).

[0626] Results

[0627] Pharmacokinetics

[0628] Serum PK

[0629] Table 8 summarizes the computed RG6035 serum PK parameters (non-compartment analysis [NCA], Phoenix WinNonlin 8.3.4). The serum concentration-time profiles of multiple 70 mg SC RG6035 administration are illustrated in Figure 11 (individual data). Mean serum RG6035 concentration-time profiles following multiple SC administration of 70 mg, 200 mg and 700 mg RG6035 Q1 W for 4 weeks (at Day 1 are displayed in Figure 17.

[0630] Table 8 Mean Serum PK Parameters of RG6035 Following Multiple SC Administration of 70 mg, 200 mg and 700 mg

[0631]

[0632]

[0633] * Median (Min-Max) for Tmax ; GeoMean = Geometric mean; CV = coefficient of variation; Cmax = maximum observed serum concentration; Tmax = time to maximum observed serum concentration; N = number of participants.

[0634] Note: the area under the serum concentration versus time curve until time 168 hour (AUCo-i68h) is not reportable due to the limited data.

[0635] **N=6 ; ***N=4

[0636] PK concentrations and PK parameters after multiple SC dose administration of 70 mg RG6035 were in the expected range (predicted serum Cmax,ss= 4.6 pg / mL and AUCtau,ss= 719 hr.pg / mL).

[0637] The serum time course data initially observed for the 70 mg cohort (N=6) are in line with the predictions of a preliminary population PK / PD model, under the scenarios of low (35%) to moderate (50%) bioavailability, as illustrated in the Figure 12.

[0638] Medians (squares) and error bars (representing the range between 25th and 75th quantiles) of observed data indicate that bioavailability and accumulation rates of RG6035 after 70 mg weekly SC dosing are consistent with model predictions assuming bioavailability between 35% and 50%. Model updates with emerging data are planned to better characterize the overall absorption kinetics.

[0639] As the Part 3 study progressed, it was found that after multiple dose SC administration of 70 mg RG6035 (N=7), CSF to serum concentration ratio was reportable for 2 participants only, with a value of 1.32% and 2.69%. After multiple dose SC administration of 200 mg and 700 mg RG6035, mean (±SD) CSF to serum concentration ratios were 2.54% (±1.20%) and 1.10% (±0.34%), respectively.

[0640] Note: the results at 700 mg should be considered with caution due to the limited data (N=3) available in this dose cohort.

[0641] Pharmacodynamics

[0642] Blood B-cells

[0643] Whole blood panel of T-cells, B-cells and natural killer [NK] cells (TBNK panel) was analysed by flow cytometry.

[0644] Data is preliminary and quality control is ongoing including checks for cell gating. Thus, preliminary data might deviate from final data.Figure 13A shows individual profiles of B-cells per patient in blood presented as fold change in frequencies of cells from baseline following SC administration of multiple doses of 70mg RG6035. Baseline has been calculated as the mean of screening and pre-dose values.

[0645] An initial drop in B-cells in blood on Day 2 was observed in 4 out of 5 participants (N=6). The mean drop in B-cells in blood on Day 2 was -36% with range (+19% to -70%). One participant showed highest drop in B-cells on Day 2 (-70%) and also prolonged depletion approximately -34% (at least until Day 22).

[0646] Mean profiles of blood B cells after multiple SC administration of 70 mg, 200 mg and 700 mg RG6035 are presented as fold change in frequencies of cells from baseline in Figure 18A. Following multiple SC administration of RG6035, a decline in peripheral blood B cells was observed in all dose cohorts on Day 2): mean= -31% [range +19% to -70%]; Cohort 2: mean= -28% [range +9% to -43%]; Cohort 3: mean= -33% [range -13% to -44%]), with no clear dose-dependent trend. However, a trend for sustained depletion during the treatment period was observed at 700 mg.

[0647] CSF B-cells

[0648] In CSF CD19+ B-cells were assessed by flow cytometry using A872 panel. A minimum of 15 B-cell events need to be measured on flow cytometer to reliably report resulting frequencies of B-cells in the CSF sample.

[0649] The CSF B-cell change from baseline is reported for 2 participants in Figure 13B: no change at Day 56 for one participant and -26% post treatment at Day 68 for another participant (Figure 13B and Figure 18B). Figure 18B additionally shows -22% CSF B-cell change from baseline post treatment at Day 29.

[0650] Individual profiles of B cells in CSF following multiple SC administration of 70 mg, 200 mg and 700 mg RG6035 are presented as fold change in frequencies from baseline overtime in Figure 18B.

[0651] Following multiple SC administration of 200 mg, changes from baseline in CSF B-cell levels were +38%, -20% and -77%. Following multiple SC administration of 700 mg, valid results are available for only one participant with a change from baseline in CSF B-cell levels of -56% observed on Day 29.

[0652] Antidruq antibodies (ADAS)

[0653] ADA samples were scheduled for Days 1 , 8, 15, 22, 29, 57, 85, 120 and at follow-up (Day 197). The available ADA data from the first four participants was ADA negative at all visits (Table 9).

[0654] Part 3, no participants had pre-existing ADAs. Emergent ADAs were observed in 6 out of 20 participants after multiple SC doses of RG6035 from 70 mg to 700 mg, with titres from 100 to 400. Participants with emergent ADAs, who had available data at the follow-up visit, showed the same titre or decreased titre, suggesting a transient effect. One participant at 70 mg was ADA negative at all visits, except at the followup visit (titre of 200).

[0655] No impact on safety related to the presence of ADAs has been identified.Table 9 Antidrug Antibodies over Time per Participant for 70 mg RG6035

[0656]

[0657] As the minimal required dilution of the ADA assay is 100, a titre of 100-200 is very low with a borderline signal.

[0658] CSF sampling time points

[0659] In Cohort 1 of the MAD, two mandatory CSF samples will be obtained (one at pre-dose and one post-dose) with an optional third CSF sample (at post-dose, if consent is given) to investigate RG6035 CSF concentrations and to assess the effect of RG6035 on CSF PD related to changes in B-cell count (as a proxy for the brain).

[0660] Measurable RG6035 concentrations in CSF are expected within the first 1-2 weeks after the last dose. Thus, from a PK point of view, CSF samples in the first 1-2 weeks after the last dose would be most meaningful.

[0661] In the CSF, based on the results to date and reported herein, it is predicted that maximum B-cell depletion would be observed 1-2 months after the last dose of 70 mg RG6035. This is presented in Figure 14.

[0662] It was suggested to collect in Cohort 1 CSF samples for

[0663] • 3 participants: Day 29 (+ / - 2 days) or Day 36 (+ / - 2 days)*

[0664] • 3 participants: Day 57 (+ / - 12 days)*

[0665] • Optional 2nd post-dose sample: combined Day 29 or 36 + Day 57

[0666] • sampling windows per protocol

[0667] Conclusions from part 3

[0668] This report summarized the overall favourable safety and tolerability assessment. Multiple SC administration of 70 mg, 200 mg and 700 mg RG6035 was well tolerated by all participants.

[0669] Following multiple SC administration, serum exposures increased with dose. PK concentrations and PK parameters after multiple SC dose administration of 70 mg RG6035 (N=6) were in the expected range. Observed serum concentrations are in line with the predictions from a preliminary population pharmacokinetic model, assuming a bioavailability between 35% and 50%.

[0670] Geometric mean Cmax after the first dose (Week 1) increased from 1.91 pg / mL (70 mg) to 18.8 pg / mL (700 mg), and geometric mean AUCtau at Week 4 increased from 492 pg*hr / mL (70 mg) to 4630 pg*hr / mL (700mg). Accumulation was observed, with AUCtau at Week 4 being substantially higher than AUCiast at Week 1. Inter-subject variability (CV%) for AUCtau (Week 4) was moderate at 46.6%.

[0671] After multiple SC administration (200 mg and 700 mg), mean CSF to serum concentration ratios were 2.54% and 1.10%, respectively.

[0672] An initial drop in B-cells in blood on Day 2 was observed in 4 out of 5 participants following multiple SC administration of 70 mg RG6035. The mean drop in B-cells in blood on Day 2 was -36% with range (+19% to -70%) for this cohort.

[0673] In fact, a decline in peripheral blood B cells was observed in all dose cohorts on Day 2 following multiple SC administration of RG6035, with no clear dose-dependent trend. However, a trend for sustained depletion during the treatment period was observed at 700 mg.

[0674] The CSF B-cell change from baseline is reported for 2 participants: no change at Day 56 for Participant 59 and -26% post treatment at Day 68 for Participant 60. -22% CSF B-cell change from baseline was reported for another participant following multiple SC administration of 70 mg RG6035. Following multiple SC administration of 200 mg, changes from baseline in CSF B-cell levels were +38%, -20% and -77%. Following multiple SC administration of 700 mg, valid results are available for only one participant with a change from baseline in CSF B-cell levels of -56% observed on Day 29.

[0675] None of the dose escalation stopping rules were reached after multiple SC administration of 70 mg, 200 mg or 700 mg of RG6035.

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[0709] Ravetch, J.V. and Kinet, J.P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P.J., et al., Immunomethods 4 (1994) 25-34.

[0710] Capel, P.J., et al., Immunomethods 4 (1994) 25-34.

[0711] de Haas, M„ et al., J. Lab. Clin. Med. 126 (1995) 330-341.

[0712] Gessner, J.E., et al., Ann. Hematol. 76 (1998) 231-248.

[0713] Shields, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604.

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[0715] Chothia, C. and Lesk, A.M., J. Mol. Biol. 196 (1987) 901-917.

[0716] Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

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[0719] Kindt, T.J. et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91.

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[0721] Cross, A., et al., Neurol. 92 (15 Supplement, 2019) S56.008.

[0722] Klein, C„ et al., mAbs 8 (2016) 1010-1020.

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[0726] Clavarino, G., et al., PLoS One 11 (2016) e0162209.

[0727] Jones, D.J., et al., Arthritis Rheum. 64 (2012) 3111-3118.

[0728] Shankar G, Arkin S, Cocea L, et al. Assessment and reporting of the clinical immunogenicity of therapeutic proteins and peptides harmonized terminology and tactical recommendations. AAPS J. 2014;16:658 73.Embodiments

[0729] The present invention provides at least the following numbered embodiments:

[0730] 1. RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg, 2000 mg or 4000 mg.

[0731] 2. RG6035 for use according to embodiment 1 , wherein the dose of RG6035 is administered intravenously.

[0732] 3. RG6035 for use according to embodiment 1 or embodiment 2, wherein the dose of RG6035 is effective to deplete B-cells in the cerebral spinal fluid (CSF) of the subject.

[0733] 4. RG6035 for use according to any one of embodiments 1 to 3, wherein the dose of RG6035 is effective to deplete B-cells in the cerebral spinal fluid (CSF) of the subject by at least 50%.

[0734] 5. RG6035 for use according to any one of embodiments 1 to 4, wherein the dose of RG6035 is effective to deplete B-cells in the cerebral spinal fluid (CSF) of the subject by at least 80%.

[0735] 6. RG6035 for use according to any preceding embodiment, wherein depletion of B-cells in the CSF is achieved within 14 days following administration of the dose of RG6035.

[0736] 7. RG6035 for use according to any preceding embodiment, wherein depletion of B-cells in the CSF is achieved within 8 days following administration of the dose of RG6035.

[0737] 8. RG6035 for use according to any preceding embodiment, wherein the depletion of B-cells is maintained for at least 14, at least 21 or at least 50 days.

[0738] 9. RG6035 for use according to any preceding embodiment, wherein the CSF to serum ratio of RG6035 following the administration of the dose of RG6035 is 0.5% or greater.

[0739] 10. RG6035 for use according to any preceding embodiment, wherein the CSF to serum ratio of RG6035 following the administration of the dose of RG6035 is 1% or greater.

[0740] 11. RG6035 for use according to any preceding embodiment, wherein the dose is administered on day 1.

[0741] 12. RG6035 for use according to any one of embodiments 3 to 11 , wherein the B-cells are CD19 positive B-cells.

[0742] 13. RG6035 for use according to any one of embodiments 3 to 12, wherein the B-cell depletion is determined as the decrease in the number or concentration of B-cells in the CSF prior to theadministration of the dose of RG6035, compared to the number or concentration of B-cells in the CSF after the administration of RG6035.

[0743] 14. RG6035 for use according to any preceding embodiment, wherein the treatment comprises administering the RG6035 at a first dose, which is an induction dose, and is followed by one or more subsequent maintenance doses of RG6035.

[0744] 15. RG6035 for use according to embodiment 14, wherein the induction dose is administered intravenously, and the one or more subsequent maintenance doses are administered subcutaneously.

[0745] 16. RG6035 for use according to embodiment 14 or embodiment 15, wherein the 700 mg or 2000 mg or 4000 mg dose is the induction dose, and the treatment further comprises administering one or more subsequent maintenance doses of RG6035.

[0746] 17. RG6035 for use according to any one of embodiments 14 to 16, wherein the one or more subsequent maintenance doses of RG6035 are each 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg.

[0747] 18. RG6035 for use according to embodiment 14 to 17, wherein the one or more subsequent maintenance doses of RG6035 are each 300 mg.

[0748] 19. RG6035 for use according to embodiment 14 to 17, wherein the one or more subsequent maintenance doses of RG6035 are each 100 mg.

[0749] 20. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 100 mg.

[0750] 21. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 150 mg.

[0751] 22. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 300 mg.

[0752] 23. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 600 mg.24. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 700 mg.

[0753] 25. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 100 mg.

[0754] 26. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 150 mg.

[0755] 27. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 300 mg.

[0756] 28. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 600 mg.

[0757] 29. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 700 mg.

[0758] 30. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 150 mg.

[0759] 31. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 300 mg.

[0760] 32. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 600 mg.

[0761] 33. RG6035 for use according to any of embodiments 14 to 17, wherein the treatment comprises administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 700 mg.

[0762] 34. RG6035 for use according to any of embodiments 14 to 33, wherein the one or more subsequent maintenance doses of RG6035 are Q1W.35. RG6035 for use according to any of embodiments 14 to 33, wherein the one or more subsequent maintenance doses of RG6035 are Q2W.

[0763] 36. RG6035 for use according to any of embodiments 14 to 33, wherein the one or more subsequent maintenance doses of RG6035 are Q3W.

[0764] 37. RG6035 for use according to any of embodiments 14 to 33, wherein the one or more subsequent maintenance doses of RG6035 are Q4W.

[0765] 38. RG6035 for use according to any of embodiments 14 to 34, wherein the induction dose is administered on day 1 , and the maintenance doses are administered on day 8 and every 7 days thereafter.

[0766] 39. RG6035 for use according to any of embodiments 14 to 33 and 35, wherein the induction dose is administered on day 1 , and the maintenance doses are administered on day 15 and every 14 days thereafter.

[0767] 40. RG6035 for use according to any of embodiments 14 to 33 and 36, wherein the induction dose is administered on day 1 , and the maintenance doses are administered on day 22 and every 21 days thereafter.

[0768] 41. RG6035 for use according to any of embodiments 14 to 33 and 37, wherein the induction dose is administered on day 1 , and the maintenance doses are administered on day 29 and every 28 days thereafter.

[0769] 42. RG6035 for use according to any preceding embodiment, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every week starting on day 15 and every 14 days thereafter.

[0770] 43. RG6035 for use according to any preceding embodiment, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 3 weeks starting on day 22 and every 21 days thereafter.

[0771] 44. RG6035 for use according to any preceding embodiment, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0772] 45. RG6035 for use according to any preceding embodiment, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every week starting on day 15 and every 14 days thereafter.46. RG6035 for use according to any preceding embodiment, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1, followed by maintenance doses of 300 mg once every 2 weeks starting on day 15 and every 14 days thereafter.

[0773] 47. RG6035 for use according to any preceding embodiment, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1, followed by maintenance doses of 300 mg once every 3 weeks starting on day 22 and every 21 days thereafter.

[0774] 48. RG6035 for use according to any preceding embodiment, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1, followed by maintenance doses of 300 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

[0775] 49. RG6035 for use according to any of embodiments 14 to 48, wherein the maintenance dose is administered for at least 3 cycles.

[0776] 50. RG6035 for use according to any of embodiments 14 to 48, wherein the maintenance dose is administered for at least 10 cycles.

[0777] 51. RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg.

[0778] 52. RG6035 for use according to embodiment 51, wherein the dose of RG6035 is Q1W, Q2W, Q3W, or Q4W.

[0779] 53. RG6035 for use according to embodiment 51, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q1 W.

[0780] 54. RG6035 for use according to embodiment 51, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q2W.

[0781] 55. RG6035 for use according to embodiment 51, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q3W.

[0782] 56. RG6035 for use according to embodiment 51, wherein the treatment comprises administering RG6035 at a dose of 100 mg Q4W.

[0783] 57. RG6035 for use according to embodiment 51, wherein the treatment comprises administering RG6035 at a dose of 150 mg Q1 W.

[0784] 58. RG6035 for use according to embodiment 51, wherein the treatment comprises administering RG6035 at a dose of 150 mg Q2W.59. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 150 mg Q3W.

[0785] 60. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 150 mg Q4W.

[0786] 61. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 300 mg Q1W.

[0787] 62. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 300 mg Q2W.

[0788] 63. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 300 mg Q3W.

[0789] 64. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 300 mg Q4W.

[0790] 65. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 600 mg Q1W.

[0791] 66. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 600 mg Q2W.

[0792] 67. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 600 mg Q3W.

[0793] 68. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 600 mg Q4W.

[0794] 69. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 700 mg Q1W.

[0795] 70. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 700 mg Q2W.

[0796] 71. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 700 mg Q3W.

[0797] 72. RG6035 for use according to embodiment 51 , wherein the treatment comprises administering RG6035 at a dose of 700 mg Q4W.73. RG6035 for use according to any one of embodiments 51 to 72, wherein the RG6035 is administered subcutaneously.

[0798] 74. RG6035 for use according to any one of embodiments 51 to 73, wherein the subject has previously been administered ocrelizumab.

[0799] 75. RG6035 for use according to embodiment 74, wherein the ocrelizumab has been administered intravenously and / or subcutaneously.

[0800] 76. RG6035 for use according to embodiments 74 or 75, wherein the subject is refractory to ocrelizumab.

[0801] 77. RG6035 for use according to any one of embodiments 74 to 76, wherein the subject is experiencing clinical progression after treatment with ocrelizumab.

[0802] 78. RG6035 for use according to any one of embodiments 74 to 77, wherein the subject is experiencing relapse.

[0803] 79. RG6035 for use according to any preceding embodiment, wherein RG6035 is a trivalent, bispecific antibody comprising:

[0804] a. one full-length antibody comprising two pairs each of a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding sites formed by each of the pairs of the full-length heavy chain and the full-length light chain specifically bind to human CD20, and

[0805] b. one additional Fab that is fused to the C-terminus of one heavy chain of the full-length antibody of a), wherein the binding site of the additional Fab specifically binds to human transferrin receptor 1 ,

[0806] wherein each of the full-length antibody light chains comprises in the constant light chain domain at position 123 the amino acid residue arginine (instead of the wild-type glutamic acid residue; E123R mutation) and at position 124 the amino acid residue lysine (instead of the wildtype glutamine residue; Q124K mutation) (numbering according to Kabat),

[0807] wherein each of the full-length antibody heavy chains comprises in the first constant heavy chain domain at position 147 a glutamic acid residue (instead of the wild-type lysine residue; K147E mutation) and at position 213 a glutamic acid residue (instead of the wild-type lysine amino acid residue; K213E mutation) (numbering according to Kabat),

[0808] wherein the additional Fab specifically binding to human transferrin receptor 1 comprises a domain crossover such that the constant light chain domain and the constant heavy chain domain 1 are replaced by each other.

[0809] 80. RG6035 for use according to any one of the proceeding embodiments, wherein RG6035 comprises a first polypeptide with the amino acid sequence of SEQ ID NO: 01 , a second polypeptide with theamino acid sequence of SEQ ID NO: 02, a third polypeptide with the amino acid sequence of SEQ ID NO: 03 and a fourth polypeptide with the amino acid sequence of SEQ ID NO: 05.

[0810] 81. RG6035 for use according to any one of the proceeding embodiments, wherein RG6035 comprises two copies of a first polypeptide with the amino acid sequence of SEQ ID NO: 01 , a second polypeptide with the amino acid sequence of SEQ ID NO: 02, a third polypeptide with the amino acid sequence of SEQ ID NO: 03 and a fourth polypeptide with the amino acid sequence of SEQ ID NO: 05.

[0811] 82. RG6035 for use according to any preceding embodiment, wherein the multiple sclerosis is relapsing multiple sclerosis or progressive multiple sclerosis.

[0812] 83. RG6035 for use according to any preceding embodiment, wherein the multiple sclerosis is relapsing multiple sclerosis.

[0813] 84. RG6035 for use according to any preceding embodiment, wherein the multiple sclerosis progressive multiple sclerosis.

[0814] 85. RG6035 for use according to any preceding embodiment, wherein the progressive multiple sclerosis is primary progressive MS (PPMS), or secondary progressive MS (SPMS).

[0815] 86. RG6035 for use according to any preceding embodiment, wherein no anti-drug antibodies to RG6035 are detected in the CSF following administration of the dose of RG6035.

[0816] 87. RG6035 for use according to any preceding embodiment, wherein the RG6035 does not induce the production of anti-drug antibodies in the subject.

[0817] 88. RG6035 for use according to any preceding embodiment, wherein RG6035 is for administration in combination with ocrelizumab.

[0818] 89. RG6035 for use according to any preceding embodiment, wherein RG6035 is for subsequent, sequential or separate administration with ocrelizumab.

[0819] 90. RG6035 for use according to any preceding embodiment, wherein the depleted B-cells are CNS- compartmentalized and / or meningeal B-cells.

[0820] 91. RG6035 for use according to any preceding embodiment, wherein RG6035 induces Fc-effector- independent B-cell death upon binding CD20 on B-cells.

[0821] 92. RG6035 for use in a method of depleting B-cells in the CNS of a multiple sclerosis patient.93. RG6035 for use according to embodiment 92, as further defined according to any one of embodiments 1 to 91.

[0822] 94. A composition comprising RG6035.

[0823] 95. A pharmaceutical composition comprising RG6035 and one or more pharmaceutically acceptable excipients.

[0824] 96. The composition of embodiment 94 or the pharmaceutical composition of embodiment 95, wherein the RG6035 is present in an amount of 700 mg, 2000 mg, or 4000 mg.

[0825] 97. The composition of embodiment 94 or the pharmaceutical composition of embodiment 95, wherein the RG6035 is present in an amount of 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg.

[0826] 98. The composition or pharmaceutical composition of embodiment 96 or 97, wherein the composition or pharmaceutical composition is for subcutaneous administration.

[0827] 99. The composition or pharmaceutical composition of embodiment 96, wherein the composition or pharmaceutical composition is for intravenous administration.

[0828] 100. The composition of embodiment 94 or any one of embodiments 96 to 99, or the pharmaceutical composition of any one of embodiments 95 to 99 for use in a method as further defined in any one of embodiments 1 to 93.

[0829] 101. A method of treating multiple sclerosis, comprising administering RG6035 to a subject in need thereof.

[0830] 102. A method of depleting B-cells in the CNS of a multiple sclerosis patient comprising administering a therapeutically effective amount of RG6035

[0831] 103. The method of embodiment 101 or 102, wherein the method is further defined as in any one of embodiments 1 to 93.

[0832] 104. The method of embodiment 101 or 102, wherein the RG6035 is administered in the form of a composition or pharmaceutical composition as defined in any one of embodiments 94 to 100.

Claims

1. Claims1. RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 700 mg, 2000 mg, or 4000 mg.

2. RG6035 for use according to claim 1 , wherein the treatment comprises administering the RG6035 at a first dose, which is an induction dose, and is followed by one or more subsequent maintenance doses of RG6035.

3. RG6035 for use according to claim 2, wherein the induction dose is administered intravenously, and the one or more subsequent maintenance doses are administered subcutaneously.

4. RG6035 for use according to any one of claims 2 or 3, wherein the one or more subsequent maintenance doses of RG6035 are each 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg.

5. RG6035 for use according to any one of claims 2 to 4, wherein the one or more subsequent maintenance doses of RG6035 are each 300 mg.

6. RG6035 for use according to any one of claims 2 to 4, wherein the one or more subsequent maintenance doses of RG6035 are each 100 mg.

7. RG6035 for use according to any of claims 1 to 4, wherein the treatment comprises:a. administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 100 mg;b. administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 150 mg;c. administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 300 mg;d. administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 600 mg;e. administering the RG6035 at an induction dose of 700 mg, followed by one or more subsequent maintenance doses of 700 mg;f. administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 100 mg;g. administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 150 mg;h. administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 300 mg;i. administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 600 mg;j. administering the RG6035 at an induction dose of 2000 mg, followed by one or more subsequent maintenance doses of 700 mg;k. administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 100 mg;l. administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 150 mg;m. administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 300 mg;n. administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 600 mg; oro. administering the RG6035 at an induction dose of 4000 mg, followed by one or more subsequent maintenance doses of 700 mg.

8. RG6035 for use according to any preceding claim, wherein the treatment comprises administering RG6035 at a first dose of 2000 mg, followed by one or more subsequent doses of 300 mg each.

9. RG6035 for use according to any preceding claim, wherein the treatment comprises administering RG6035 at a first dose of 2000 mg, followed by one or more subsequent doses of 100 mg each.

10. RG6035 for use according to any one of claims 2 to 9, wherein the one or more subsequent maintenance doses of RG6035 are Q1W, Q2W, Q3W, or Q4W.

11. RG6035 for use according to any one of claims 2 to 7, 9 and 10, wherein the treatment comprises:a. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every week starting on day 8 and every 7 days thereafter;b. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 2 weeks starting on day 15 and every 14 days thereafter;c. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 3 weeks starting on day 22 and every 21 days thereafter; ord. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

12. RG6035 for use according to any one of claims 2 to 7 and 10, wherein the treatment comprises:a. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every week starting on day 8 and every 7 days thereafter;b. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every 2 weeks starting on day 15 and every 14 days thereafter;c. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every 3 weeks starting on day 22 and every 21 days thereafter; ord. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 150 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

13. RG6035 for use according to any one of claims 2 to 8 and 10, wherein the treatment comprises:a. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every week starting on day 8 and every 7 days thereafter;b. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every 2 weeks starting on day 15 and every 14 days thereafter;c. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every 3 weeks starting on day 22 and every 21 days thereafter; ord. administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every 4 weeks starting on day 29 and every 28 days thereafter.

14. RG6035 for use according to any one of claims 2 to 8, 10 and 13, wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 300 mg once every 2 weeks starting on day 15 and every 14 days thereafter.

15. RG6035 for use according to any one of claims 2 to 7 and 9 to 11 , wherein the treatment comprises administering the RG6035 at an induction dose of 2000 mg on day 1 , followed by maintenance doses of 100 mg once every 2 weeks starting on day 15 and every 14 days thereafter.

16. RG6035 for use according to any one of the proceeding claims, wherein RG6035 comprises two copies of a first polypeptide comprising the amino acid sequence of SEQ ID NO: 01 , a second polypeptide comprising the amino acid sequence of SEQ ID NO: 02, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 03 and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 05.

17. RG6035 for use according to any preceding claim, wherein the multiple sclerosis is relapsing multiple sclerosis or progressive multiple sclerosis.

18. RG6035 for use according to any preceding claim, wherein RG6035 is for administration in combination with ocrelizumab.

19. RG6035 for use in the treatment of multiple sclerosis, wherein the treatment comprises administering RG6035 at a dose of 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg.

20. RG6035 for use according to claim 19, wherein the dose of RG6035 is Q1 W, Q2W, Q3W, or Q4W.

21. RG6035 for use according to claims 19 or 20, wherein the subject has previously been administered ocrelizumab.

22. RG6035 for use according to claim 21 , wherein the ocrelizumab has been administered intravenously and / or subcutaneously.

23. A pharmaceutical composition comprising RG6035 and one or more pharmaceutically acceptable excipients, wherein the RG6035 is present in an amount of 700 mg, 2000 mg, or 4000 mg and the pharmaceutical composition is for intravenous administration.

24. A pharmaceutical composition comprising RG6035 and one or more pharmaceutically acceptable excipients, wherein the RG6035 is present in an amount of 100 mg, 150 mg, 300 mg, 600 mg, or 700 mg and the pharmaceutical composition is for subcutaneous administration.

25. A method of treating multiple sclerosis, comprising administering RG6035 to a subject in need thereof.

26. A method of depleting B-cells in the CNS of a multiple sclerosis patient comprising administering a therapeutically effective amount of RG6035.

27. The method of claim 25 or 26, wherein the method is further defined as in any one of claims 1 to 22.

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