Methods of treatment

Abstract

Herein is reported the use of RG6035 as a medicament in the treatment of multiple sclerosis, wherein RG6035 is administered at a dose of 140 mg to 210 mg.

Description

[0001] The present application is in the field of disease treatment. In more detail, inter alia, dosing regimens for the treatment of multiple sclerosis using a brain-penetrating anti-CD20 antibody are reported herein. BACKGROUND

[0002] Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating and degenerative disease of the central nervous system (CNS) affecting approximately 2.5 million patients worldwide. MS creates a neurodegenerative environment leading to the accumulation of severe motor, sensory and cognitive impairments [1, 2] and is the most common inflammatory neurological disease in young adults.[3]

[0003] Over the past 15 years, the pathogenesis of MS has been closely linked to B cells. B cells are key players of the humoral immunity and bridge the gap between the innate and adaptive immune systems, playing a major role in the fight against pathogens and foreign antigens through antibody production.[4] However, B cell dysregulation has been identified as a stimulus for several autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus and myasthenia gravis.[5] Although no specific antibody can be identified as a hallmark of MS, studies have begun to characterize the distribution and function of B cells in patients with MS.[5] In addition to antibody production, B cells can contribute to the pathological process by stimulating T cell responses as antigen-presenting cells or secreting proinflammatory cytokines. In addition, memory B cells are considered key players in MS but are also susceptible to Epstein-Barr virus infection, which is considered a key factor in MS pathology

[47] .

[0004] The presence of CD20-positive (CD20+) B cells has been found in several subcompartments of the CNS of patients with MS, including the cerebrospinal fluid (CSF), parenchyma, meninges and perivascular space.[4, 6, 7, 8] In the CSF, more than 90% of patients with MS present with oligoclonal IgG bands, as well as cortical demyelination associated with meningeal inflammation, suggesting that B cells reside in CNS compartments from the early stages of the disease.[4, 9] In the context of persistent inflammation, B cells can infiltrate from the blood to the immune-privileged CNS, especially in the context of blood-brain barrier (BBB) disruption.

[10]

[0005] Drug delivery to the brain remains a major challenge in CNS drug development. The BBB is a physical, metabolic, and transport barrier that tightly controls the movement of substances from blood to neural tissue and vice versa, thereby ensuring CNS homeostasis but limiting the delivery of therapeutic monoclonal antibodies (mAbs) such as the anti-CD20 therapies obinutuzumab, ocrelizumab, and ofatumumab.[11-13] Thus, currently available anti-CD20 therapies target peripheral B cells and are effective in preventing acute relapse-remitting MS (RRMS) attacks (relapses). Anti-CD20 therapies have limited access to CNS B cells, which is believed to limit their effect on disease progression. To deplete compartmentalized B cells in the brain, a clinical candidate would need to substantially cross the BBB and kill B cells in a manner independent of effector function.

[0006] Brain-shuttling versions of therapeutic antibodies are reported, e.g., in WO 2017 / 055542. SUMMARY

[0007] The present invention is based, at least in part, on the use of a specific Brain Shuttle - CD20 RG6035 construct. RG6035 used in the methods of the present invention is a bispecific modular fusion protein of a human transferrin receptor 1 (TfRl)-directed brain- shuttling (TfRl-binding antigen-binding fragment (Fab)) and the paratope of the anti- CD20 antibody obinutuzumab. TfRl is abundant at the BBB and is a target for achieving receptor-mediated transport of macromolecules across the BBB, while obinutuzumab has a unique mechanism of action: inducing Fc effector-independent, non-apoptotic direct B cell death upon binding to CD20.[14-21] RG6035 has the function of (completely) depleting brain-localized B cells.

[0008] The present invention is based, at least in part, on the finding that a PK / PD model for RG6035 based on non-clinical data can be used to estimate pharmacologically active / therapeutically effective systemic RG6035 exposure. In more detail, it has been found that data observed in blood can be used as a surrogate for simulating human effective exposure.

[0009] In more detail, the present invention encompasses at least the following embodiments

[0010] 1. RG6035 for use as a medicament in the treatment of multiple sclerosis, wherein RG6035 is administered at a dose of 140 mg to 210 mg.

[0011] 2. RG6035, which is used to treat multiple sclerosis, is administered at doses of 140 mg to 210 mg.

[0012] 3. The use according to any one of Examples 1 to 2, wherein the multiple sclerosis is primary progressive multiple sclerosis or secondary progressive multiple sclerosis.

[0013] 4. The use according to any one of Examples 1 to 3, wherein the dose effectively depletes more than 95% of B cells in the CSF of a subject who has been administered RG6035.

[0014] 5. The use according to Example 4, wherein the depletion is achieved within 8 weeks after the start of application.

[0015] 6. The use according to any one of Examples 1 to 5, wherein the concentration of RG6035 in the CSF is 1% or more of the serum concentration of RG6035.

[0016] 7. The use according to any one of Examples 1 to 6, wherein the concentration of RG6035 in the CSF is 0.055 µg / mL or higher.

[0017] 8. The use according to any one of claims 4 to 7, wherein the B cell is a CD19 positive B cell.

[0018] 9. The use according to any one of Examples 1 to 8, wherein RG6035 is administered in a second dose after 95% or more of the B cells have been depleted from the CSF to maintain the depletion of B cells in the CSF at the stated level.

[0019] 10. The use according to any one of Examples 4 to 9, wherein the B cell depletion is determined by comparing the number of B cells in the CSF prior to the first application of RG6035.

[0020] 11. The use according to any one of Examples 1 to 10, wherein the application is performed once every four weeks.

[0021] 12. The use according to any one of Examples 1 to 10, wherein the application is initiated once a week and, after 95% or more of the B cells have been depleted from the CSF, is changed to once every four weeks to maintain the depletion of B cells in the CSF at the stated level.

[0022] 13. The use according to any one of Examples 1 to 12, wherein the RG6035 is administered intravenously or subcutaneously.

[0023] 14. The use according to any one of Examples 1 to 13, wherein the RG6035 is applied subcutaneously.

[0024] 15. The use according to any one of Examples 1 to 14, wherein RG6035 comprises: a first polypeptide having the amino acid sequence of SEQ ID NO: 01, a second polypeptide having the amino acid sequence of SEQ ID NO: 02, a third polypeptide having the amino acid sequence of SEQ ID NO: 03, and a fourth polypeptide having the amino acid sequence of SEQ ID NO: 05.

[0025] In addition to the various embodiments depicted and claimed, the disclosed subject matter also relates to other embodiments having other combinations of the features disclosed or claimed herein. Therefore, the specific features presented herein may be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of the features disclosed herein. For illustrative and descriptive purposes, the foregoing description of specific embodiments of the disclosed subject matter has been presented. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments. Detailed Implementation

[0026] Definitions

[0027] Unless otherwise defined herein, scientific and technical terms related to this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. The methods and techniques of this disclosure are generally performed according to conventional methods known in the art. Typically, terms and techniques related to biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are well-known and commonly used in the art.

[0028] General information about the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in: Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0029] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as “according to Kabat numbering”. Specifically, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647–660) is used for the constant domain CL of the κ and λ isoform light chains, and Kabat’s EU index numbering system (see pp. 661–723) is used for the constant heavy chain domains (CH1, hinge, CH2, and CH3, which are further classified herein by way of “according to Kabat’s EU index numbering”).

[0030] It is important to note that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly specifies otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and their equivalents known to those skilled in the art, and so on. Similarly, the terms “a,” “one or more,” and “at least one” are used interchangeably herein. It should also be noted that the terms “comprising,” “including,” and “having” are used interchangeably.

[0031] Unless otherwise defined herein, the term "comprising" shall include the term "consisting of".

[0032] When the term “about” is used in conjunction with specific values ​​(such as temperature, concentration, time, etc.) in this article, it should refer to a change of + / - 1% in the specific value referred to by the term “about”.

[0033] The blood-brain barrier (BBB) ​​refers to the physiological barrier between the peripheral blood circulation and the brain and spinal cord. This barrier is formed by tight junctions within the capillary endothelial cell membranes of the brain, creating a tight barrier that restricts the transport of molecules into the brain, even very small molecules such as urea (60 dorutons). The BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina constitute a continuous capillary barrier within the CNS, and are collectively referred to as the blood-brain barrier or BBB in this paper. The BBB also includes the blood-CSF barrier (choroid plexus), which is composed of ependymal cells, rather than capillary endothelial cells.

[0034] The terms “anti-human CD20 antibody” and “antibody that specifically binds to human CD20” refer to antibodies that can bind to human CD20 with sufficient affinity, thereby enabling the antibody to be used as a diagnostic and / or therapeutic agent targeting CD20.

[0035] The CD20 antigen is a non-glycosylated phosphoprotein of about 35 kDa that is present on the surface of more than 90% of B cells in peripheral blood or lymphoid organs. CD20 is expressed during early pre-B cell development and is retained until plasma cell differentiation. CD20 is present on both normal and malignant B cells. Other names for CD20 in the literature include “B lymphocyte-restricted antigen” and “Bp35” and “MS4A1”.

[46] For example, the CD20 antigen is described in Clark et al. Proc. Natl. Acad. Sci USA 82 (1985) 1766. See also SEQ ID NO: 06.

[0036] In this article, "autoimmune disease" refers to a non-malignant disease or condition that is caused by and affects the individual's own tissues. Examples of autoimmune diseases or conditions include, but are not limited to, multiple sclerosis.

[0037] An "antagonist" is a molecule that, upon binding to a marker on the surface of B cells, destroys, kills, or depletes B cells in mammals and / or interferes with one or more B cell functions, such as by reducing or preventing humoral responses induced by B cells. Antagonists can deplete B cells in mammals treated with them (i.e., reduce the number or level of circulating B cells). This depletion can be achieved through various mechanisms, such as 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).

[0038] Antagonists of "inducing apoptosis" are those that induce programmed cell death, such as in B cells, as determined by standard apoptosis assays, such as binding of annexin V, DNA fragmentation, cell contraction, endoplasmic reticulum expansion, cell fragmentation, and / or formation of membrane vesicles (called apoptotic bodies).

[0039] Antagonists of the antigens of interest (such as B cell surface markers) are compounds that can bind to the antigens with sufficient affinity and / or cohesion, so that the antagonists can be used as therapeutic agents targeting cells that express the antigens.

[0040] The “central nervous system” or “CNS” is a complex of neural tissues that control bodily functions and includes the brain and spinal cord.

[0041] Blood-brain barrier receptors (BBBRs) are extracellular membrane-connecting receptor proteins expressed on brain endothelial cells that can transport molecules across the BBB or be used to transport exogenous drug-administered molecules. Examples of BBBRs include transferrin receptor 1 (TfR1).

[0042] "Transferrin receptor 1" ("TfR1") is a transmembrane glycoprotein (molecular weight approximately 180,000 Da) composed of two disulfide-bonded subunits (each with an apparent molecular weight of approximately 90,000 Da) involved in iron uptake in vertebrates. In all aspects of the invention and in certain embodiments, the TfR1 referred to herein is human TfR1 including, for example, the amino acid sequence described by Schneider et al. (Nature 311 (1984) 675-678).

[0043] "Multispecific antibody" refers to an antibody that has binding specificity against at least two different antigens. An exemplary multispecific antibody may bind to both BBBR and brain antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., an F(ab')2 bispecific antibody) or combinations thereof (e.g., a full-length antibody with an additional scFv or Fab fragment). Engineered antibodies having two, three, or more (e.g., four) functional antigen-binding sites have also been reported (see, for example, US 2002 / 0004587).

[0044] The term "antibody" is used herein to encompass a variety of antibody structures, including but not limited to monoclonal antibodies and multispecific antibodies (e.g., bispecific antibodies), as long as they exhibit the desired antigen-binding activity.

[0045] The term "antibody-dependent cytotoxicity (ADCC)" refers to the lysis of target cells by an antibody mediated by Fc receptor binding, specifically in the presence of effector cells. ADCC can be measured by treating formulations containing CD19-expressing erythroblasts (e.g., K562 cells expressing recombinant human CD19) with relevant antibodies in the presence of effector cells (such as freshly isolated peripheral blood mononuclear cells (PBMCs)) or purified effector cells derived from the erythrocyte sedimentation rate (ESR) amber layer (e.g., monocytes or natural killer (NK) cells). Cr 51 Target cells were labeled and subsequently incubated with the relevant antibody. The labeled cells were then incubated with effector cells, and the release of Cr in the supernatant was analyzed. 51 The control group involved incubating target endothelial cells with effector cells but not with antibodies. The ability of the relevant antibodies to induce the initial steps of ADCC was investigated by measuring their binding to cells expressing Fcγ receptors, such as recombinant cells expressing FcγRI and / or FcγRIIA or NK cells (which essentially express FcγRIIIA).

[0046] An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0047] An antibody's "class" 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 some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are called α, δ, γ, ε, and μ, respectively.

[0048] The term "complement-dependent cytotoxicity (CDC)" refers to cell lysis induced by an associated antibody in the presence of complement. CDC can be measured by treating human endothelial cells expressing CD19 with an associated antibody in the presence of complement. Cells are labeled with calcein. CDC is detected if the associated antibody induces 20% or more of target cell lysis at a concentration of 30 µg / ml. Binding to complement factor C1q can be measured in an ELISA. In such an assay, an ELISA plate is coated with a concentration range of the associated antibody, and purified human C1q or human serum is added to it. C1q binding is detected by an antibody against C1q, followed by a peroxidase-labeled conjugate. Detection of binding (maximum binding B) max The optical density (OD405) of the peroxidase substrate ABTS® (2,2'-azono-di-[3-ethylbenzothiazoline-6-sulfonate(6)]) was measured at 405 nm.

[0049] "Effective functions" refer to those biological activities attributable to the Fc region of an antibody, which vary depending on 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; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0050] Fc receptor binding-dependent effector functions are mediated by the interaction of the antibody's Fc region with the Fc receptor (FcR), a specialized cell surface receptor on hematopoietic cells and their progeny. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes and to lyse antibody-coated erythrocytes and various other cellular targets (e.g., tumor cells) via antibody-dependent cell-mediated cytotoxicity (ADCC) (see, for example, Van de Winkel, JG and Anderson, CL, J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: the Fc receptor for IgG antibodies is called FcγR. Fc receptor binding is described, for example, in Ravetch, JV and Kinet, JP, Annu. Rev. Immunol. 9 (1991) 457-492; Capel, PJ, et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J. Lab. Clin. Med. 126 (1995) 330-341; and Gessner, JE, et al., Ann. Hematol. 76 (1998) 231-248.

[0051] Cross-linking of the Fc region receptor of IgG antibodies (FcγRs) triggers multiple effector functions, including phagocytosis, antibody-dependent cytotoxicity, release of inflammatory mediators, and regulation of immune complex clearance and antibody production. Three classes of FcγRs have been identified in humans, including:

[0052] -FcγRI (CD64) binds to monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils, and eosinophils. Modifications to at least one residue in the Fc region of IgG at amino acid residues E233-G236, P238, D265, N297, A327, and P329 (according to Kabat's EU index number) reduce binding to FcγRI. Substitution of the IgG2 residue at positions 233-236 to IgG1 and IgG4 reduces binding to FcγRI by 10³-fold and eliminates the response of human monocytes to antibody-sensitized erythrocytes (Armour, KL et al., Eur. J. Immunol. 29 (1999) 2613–2624).

[0053] FcγRII (CD32) binds to complexed IgG with medium to low affinity and is widely expressed. This receptor can be divided into two subtypes, FcγRIIA and FcγRIIB. FcγRIIA is present on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to activate the killing process. FcγRIIB appears to play a role in the inhibitory process and is present on B cells, macrophages, mast cells, and eosinophils. On B cells, it appears to inhibit further immunoglobulin production and isotype conversion, such as to IgE. On macrophages, FcγRIIB is used to inhibit phagocytosis mediated by FcγRIIA. On eosinophils and mast cells, the B subtype may contribute to the inhibition of these cell activation through the binding of IgE to its individual receptor. For example, antibodies containing the IgG Fc region have been found to bind less to FcγRIIA, which has a mutation at at least one of the following amino acid residues: E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, and R292 K414 (according to the Kabat EU index number).

[0054] -FcγRIII (CD16) binds to IgG with medium to low affinity and exists in two forms. FcγRIIIA is present on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. FcγRIIIB is highly expressed on neutrophils. For example, antibodies containing the IgG Fc region have been found to bind less to FcγRIIIA, which contains mutations at at least one of the amino acid residues E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376 (according to Kabat EU index number).

[0055] The location of the binding site on human IgG1 to the Fc receptor, the aforementioned mutation sites, and the method for measuring binding to FcγRI and FcγRIIA are described in Shields, RL et al., J. Biol. Chem. 276(2001) 6591-6604.

[0056] The "therapeutic effective amount" of a drug (e.g., a pharmaceutical preparation) refers to the amount that effectively achieves the desired therapeutic or preventive outcome at the necessary dosage and time period.

[0057] As used herein, the term "Fc receptor" refers to an activated receptor characterized by the presence of a receptor-associated cytoplasmic ITAM sequence (see, for example, Ravetch, JV and Bolland, S., Annu. Rev. Immunol. 19(2001) 275-290). Such receptors are FcγRI, FcγRIIA, and FcγRIIIA. The term "non-binding FcγR" indicates that, at an antibody concentration of 10 µg / ml, the binding of the associated antibody to NK cells is 10% or less of the binding to the anti-OX40L antibody LC.001 reported in WO 2006 / 029879.

[0058] Although IgG4 showed reduced FcR binding, antibodies against other IgG subclasses showed strong binding. However, Pro238, Asp265, Asp270, Asn297 (deficiency of Fc carbohydrate), Pro329 and 234, 235, 236 and 237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 are residues provided if a decrease in FcR binding also occurs (Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604; Lund, J. et al., FASEB J. 9 (1995) 115-119; Morgan, A. et al., Immunology 86 (1995) 319-324; and EP 0 307 434).

[0059] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes both native sequence Fc regions and variant Fc regions. The human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present.

[0060] The Fc region of an antibody is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. Although the effect of an antibody on the complement system depends on certain conditions, binding to C1q is caused by the binding site defined in the Fc region. Such binding sites are known in the art and described, for example, in Lukas, TJ et al., J. Immunol. 127(1981) 2555-2560; Brunhouse, R. and Cebra, JJ, Mol. Immunol. 16(1979) 907-917; Burton, DR et al., Nature 288(1980) 338-344; Thommesen, JE et al., Mol. Immunol. 37(2000) 995-1004; Idusogie, EE et al., J. Immunol. 164(2000) 4178-4184; Hezareh, M. et al., J. Virol. 75(2001) 12161-12168; Morgan, A. et al., Immunology 86(1995). 319-324; and EP 0 307 434. Such binding sites include, for example, L234, L235, D270, N297, E318, K320, K322, P331, and P329 (according to the Kabat EU index number). Antibodies against subclasses IgG1, IgG2, and IgG3 typically exhibit complement activation, C1q binding, and C3 activation, while IgG4 does not activate the complement system, does not bind C1q, and does not activate C3.

[0061] The term "antibody Fc region" is familiar to technical personnel and is defined based on the cleavage of antibodies by papain. The Fc region of RG6035 belongs to the human IgG1 subclass and includes effector functions for eliminating mutations L234A, L235A, and P329G (according to Kabat's EU index number).

[0062] The term "full-length antibody" is used herein to refer to an antibody having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein. A "full-length antibody" is an antibody containing antigen-binding variable regions VH and VL, and light chain constant domains (CL) and heavy chain constant domains CH1, CH2, and CH3. The constant domains may be natural sequence constant domains (e.g., human natural sequence constant domains) or amino acid sequence variants thereof. More specifically, a full-length antibody comprises two antibody light chains (each containing a variable domain and a constant domain) and two antibody heavy chains (each containing a variable domain, a hinge region, and three constant domains including the CH2 and CH3 domains). C-terminal amino acid residues K or GK may be present independently of each other or absent from the two antibody heavy chains of a full-length antibody.

[0063] 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 acids have been introduced, including progeny cells of these cells. Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny cells derived from them, regardless of the number of passages. Progeny cells may not be identical to the nucleic acid contents of the parent cells and may contain mutations. This document includes mutant progeny with the same function or biological activity as those screened or selected from the original transformed cells.

[0064] "Humanized" antibodies are chimeric antibodies that contain amino acid residues from non-human hypervariable regions and amino acid residues from the human backbone. "Humanized forms" of antibodies, such as non-human antibodies, refer to antibodies that have undergone humanization.

[0065] As used herein, the term “hypervariant region” or “HVR” refers to each of the following: regions of antibody variable domains that are hypervariable in sequence (“complementarity-determining region” or “CDR”) and / or form structurally defined loops (“hypervariant loops”) and / or contain antigen contact residues (“antigen contact sites”). Typically, an antibody contains six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3).

[0066] HVR includes

[0067] (a) Hyperchromatic rings present 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, AM, J. Mol. Biol. 196(1987) 901-917);

[0068] (b) CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.).

[0069] (c) Antigen contacts present 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

[0070] Combinations of (d)(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).

[0071] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domain are referenced in this paper to Kabat et al., with the same reference numbering above.

[0072] "Individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain preferred embodiments of all aspects and examples of the invention, the individual or subject is a human.

[0073] "Isolated" antibodies are antibodies isolated from components of their natural environment. In some embodiments of all aspects and examples of the invention, antibodies are purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0074] "Isolated" nucleic acids refer to nucleic acid molecules that have been isolated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain nucleic acid molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0075] "Isolated nucleic acid encoding anti-human CD20 / human transferrin receptor antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains, including such nucleic acid molecules in a single vector or in a separate vector, and such nucleic acid molecules are present at one or more locations in the host cell.

[0076] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that, apart from possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of a monoclonal antibody formulation, such variants are typically presented in small quantities), the individual antibodies comprising this group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies intended for use according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, 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 preparing monoclonal antibodies are described herein.

[0077] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also called a variable heavy chain domain or heavy chain variable domain), followed by three constant domains (CH1, CH2, and CH3), whereby the hinge region is positioned between the first and second constant domains. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light chain (CL) domain. The light chains of antibodies can be classified into one of two types based on the amino acid sequence of their constant domains, called kappa (κ) and lamuda (λ).

[0078] The term "pharmaceutical formulation" refers to a formulation in which the bioactive ingredient contained therein is in a form in which the activity is effective and which does not contain any additional components that would have unacceptable toxicity to a subject to whom the formulation will be administered.

[0079] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation that are non-toxic to the subject, excluding the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffer solutions, excipients, stabilizers, or preservatives.

[0080] As used herein, “treatment” (and its grammatical variations, such as “treat” or “treating”) refers to a clinical intervention that attempts to alter the natural course of the disease in an individual being treated, and may be performed for prevention or during the course of clinicopathological progression. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some embodiments of all aspects and examples of the invention, RG6035 is used to delay the development of disease or slow its progression, particularly the progression of multiple sclerosis.

[0081] The term "variable region" or "variable domain" refers to a domain of the heavy or light chain of an antibody that participates in the binding of the antibody to the antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved backbone regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt, TJ et al., Kuby Immunology, 6th ed., WH Freeman and Co., NY (2007), p. 91).

[0082] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0083] "Chemotherapy agents" are compounds used to treat cancer. Examples of chemotherapy agents include: alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates, such as busulfan, indomethacin, and piperazine; aziridines, such as benzodopa, carboquinone, metoprolol, and uredopala; ethylimine and methylmelamine, including hexamethylmelamine, triethylmelamine, triethylenephosphamide, triethylenethiophosphamide, and trihydroxymethylmelamine; nitrogen mustards, such as chlorambucil, naphthiamethoxam, cyclophosphamide, estradiol, ifosfamide, nitrogen mustard, methylambucil hydrochloride, melphalan, sine emblica, cholesterol phenylacetic acid nitrogen mustard, prednisolone, trefophosphatide, and uracil nitrogen mustard; and nitrosoureas, such as carmustine, chloramphenicol, and formoxetine. Tadalafil, lomustine, nimustine, ramustine; antibiotics such as aclarubicin, actinomycin, atrazosin, diazoserine, bleomycin, actinomycin C, chachiin, carrubicin, erythromycin, carcinogen, chromomycin, actinomycin D, doxorubicin, detoxin, 6-diazo-5-oxo-L-leucine, doxorubicin, epirubicin, isorubicin, idarubicin, maceromycin, mitomycin, mycophenolic acid, nogamycin, oligomycin, pepromycin, pofibromycin, puromycin, triamcinolone acetonide, rhodopsin, streptomycin, streptozotocin, tuberculin, ubenmex, fentostatin, zolrubicin; antimetabolites such as methotrexate and 5 - Fluorouracil (5-FU); folic acid analogs, such as folate, methotrexate, pteroxate, trimesartan; purine analogs, such as fludarabine, 6-mercaptopurine, thioimidine, thioguanine; pyrimidine analogs, such as cyclocytidine, azacitidine, 6-azouridine, carmoflurane, cytarabine, dideoxyuridine, deoxyfluorouridine, enoxabin, fluorouridine, 5-FU; androgens, such as calutidone, drotaloferrin propionate, cyclothrostanol, meandrolone, testrolide; anti-adrenergics, such as aminoglutethimide, mitotan, trilostan; folic acid supplements, such as folinic acid; acetolactone; aldehydephosphoramide glycosides; aminolevulinic acid; acridine; besbushes; bistocin; edatrazine Sand; Desphosphonamide; Demecocin; Desaccharin; Elonisen; Elimethicone; Etogluconol; Gallium nitrate; Hydroxyurea; Lentinan; Clonidamine; Mitoguanidine hydrazone; Mitoantrone; Moperadol; Nifuran; Pentostatin; Methamidomus acid; Pirarubicin; Podophyllotoxin; 2-Ethylhydrazide; Procarbazine; PSK®; Razosen; Sisophila; Germanium spiroamine; Alternaria ketoacid; Triaminoquinone; 2,2',2''-Trichlorotriethylamine; Uranium; Vinpocetine; Dacarbazine; Mannitol mustard; Dibromomannitol; Dibromoeusol; Piperbromo; Garcitocin; Arabinoside (“Ara-C”); Cyclophosphamide; Thiotepa; Taxanes, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ)Docetaxel (TAXOTERE®, Rhône-Poulenc Rorer, Antony, France); nitrogen mustard chlorhexidine; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); eporamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; norvincristine; nodarutamide; teniposide; doxorubicin; aminopterin; capecitabine; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esparmycin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes anti-hormonal agents that regulate or inhibit the effects of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, tripoxifene, raloxifene hydrochloride, LY117018, onanasone, and toremifene (Fareston); and anti-androgens, such as flutamide, nilutel, bicalutamide, leuprorelin, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0084] Recombinant Methods and Compositions

[0085] Antibodies can be produced using recombinant methods and compositions, such as those described in US 4,816,567. Isolated nucleic acids encoding RG6035 can be provided. One or more vectors (e.g., expression vectors) containing such nucleic acids can be provided. Host cells containing such nucleic acids can be provided. Host cells can be eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NSO, Sp2 / O cells). A method for preparing RG6035 can be provided, wherein the method includes culturing host cells containing nucleic acids encoding the antibody as provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cells (or host cell culture medium).

[0086] For recombinant production of RG6035, one or more nucleic acids encoding antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0087] Suitable host cells for RG6035 expression are mammalian cell lines adapted for growth in suspension. Useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220). For reviews of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0088] Multiple Sclerosis

[0089] Multiple sclerosis (MS) is a chronic, demyelinating, immune-mediated inflammatory disease of the central nervous system (CNS). [8a]

[0090] Historically, CNS tissue damage in MS was believed to be mediated by infiltrative pro-inflammatory CD4+ T cells [9a,10a]. However, B cells are now known to be key contributors to the immunopathology of MS, as evidenced by the clinical efficacy of CD20-targeted, peripheral B cell depletion therapy observed in relapsing-remitting MS [11a-17a]. Recent publications have shown that CNS compartmentalized B cells are particularly detrimental to progressive MS (PMS) [18a]. The presence of B cell-rich lymphoid structures in PMS is associated with an adverse course, including cortical demyelination, brain atrophy, microglial activation, and synaptic and neuronal loss [18a,19a].

[0091] Therefore, mounting evidence strongly suggests that B cells are involved in the pathophysiology of MS. While several B-cell depleted monoclonal antibody (mAb) therapies are currently approved for MS, they cannot actively cross the blood-brain barrier and resolve the disease in the CNS. Therefore, one of the key obstacles to developing novel B-cell depleted monoclonal antibody therapies remains their delivery to the brain.

[0092] Despite advances in the treatment of MS, delaying disease progression through efficient delivery of therapeutic drugs to the CNS remains an unmet medical need. Studies of post-mortem brain tissue from patients with secondary progressive MS (SPMS) have revealed the presence of ectopic lymphoid structures containing B cells, and these structures lack regulatory T cells. [7,8,29] This suggests that the latter promote autoantigen-specific adaptive immune responses, thereby exacerbating the chronic disease.

[30] Given this evidence, efficient depletion of B cells behind the BBB and blood-CSF barriers is crucial to halting persistent inflammation.

[0093] Recent scientific evidence suggests that compartmentalized B cells in the lymphoid follicle-like structures of the brain and spinal cord in MS patients are associated with disease progression. [29,31] In addition to approved MS therapies with different mechanisms of action, including T-cell targeted therapies, recently approved B-cell targeted therapies have also shown efficacy.

[32] The importance of this mode of action has been reinforced with the successful development of B-cell depletion monoclonal antibodies targeting the CD20 surface antigen, such as oligrizumab and olfamolumab.

[33] Although there are many drugs targeting the pathological inflammatory mechanisms associated with relapse and relapse-related exacerbations, only oligrizumab has been approved for primary progressive MS (PPMS) to date. Oligizumab is a recombinant humanized mAb that selectively targets B cells expressing CD20.

[15] A growing body of evidence supports the hypothesis that compartmentalized inflammation in the CNS contributes significantly to disability progression. [8,29,31] Therefore, therapeutic candidates targeting this region are needed to allow for effective therapies.

[0094] The brain barrier barrier (BBB) ​​prevents neurotoxic plasma components, blood cells, and pathogens from entering the brain. Simultaneously, the BBB strongly hinders the entry of systemically administered mAbs into the brain, limiting the delivery volume of therapeutically active mAbs and achieving therapeutic concentrations. This poses a significant challenge to the development of mAb-based brain barrier therapies.

[0095] Methods According to the Invention

[0096] Using PK / PD studies, a cynomolgus monkey model was designed to determine the therapeutic effect of RG6035, demonstrating that RG6035 can effectively enter the immune-exempt region of the CNS behind the BBB and deplete the B cell population.

[0097] RG6035 is an engineered trivalent bispecific monoclonal antibody containing a variant of obituzumab and an additional Fab specific to human TfR1. This additional binding to TfR1 allows for improved transcytosis of RG6035 across the BBB via TfR1-mediated transcytosis.

[0098] Unbound by this theory, it is assumed that TfR1-mediated uptake constitutes an important clearance pathway in the periphery and is considered to complement other clearance mechanisms, such as nonspecific clearance of antibody components.

[37] This would result in a higher overall clearance rate for brain shuttle constructs compared to conventional monoclonal antibodies.

[0099] The doses applied in the study were equivalent to 0.13% to 3.4% of the serum concentration in the brain. These are higher than those achievable with typical antibodies. Therefore, RG6035 exhibited higher brain penetration than normal IgG. That is, the brain AUC of steady-state brain exposure was 25 times higher than that of typical IgG (such as obbituzumab).

[0100] Multiple CSF sampling time points are required to generate complete PK curves for the compound in the CNS. Therefore, cannulation of the cerebellomedullary cistern was used and compared with lumbar puncture for intracerebrospinal fluid sampling. Indeed, cerebellomedullary cistern cannulation offers the advantage of repeated sampling without the bias caused by anesthesia.

[0101] It has been found that the concentrations in both compartments were similar over time, with the concentration ratio between the cerebellomedullary cistern and lumbar puncture ranging from 0.2 to 1.1, indicating no difference between the two sampling methods. The chronic CSF sampling model, which involves surgically implanting a catheter into the brain, allows for repeated CSF sampling in conscious, freely moving animals.

[0102] The ability of octotuzumab to induce direct B cell death was identified as a driving mechanism for the efficient depletion of immune-active B cells in an Fc region effector-independent manner. Compared with octotuzumab, the reduced Fc region efficacy in RG6035 resulted in less cytokine release and fewer adverse events in human whole blood assays.

[38] The Fc-silencing type II CD20 / TfR1 antibody (BS-octotuzumab-PGLALA) RG6035 has a more favorable safety profile, especially when bound to TfR1 brain shuttle. It can be shown that despite the combination of Fc region effector-silencing octotuzumab with the TfR1 brain shuttle module, both functions, namely brain shuttle function and CD20 binding function, including B cell killing properties (receptor binding and direct B cell depletion), are preserved, and brain exposure is increased compared to standard non-TfR1-binding IgG antibodies.

[0103] The increase in exposure was disproportionate to the increase in dose; as the dose increased from 0.3 mg / kg to 10 mg / kg, the dose-normalized C0.05 max The AUC increased by approximately 4 to 8 times. Unbound by this theory, it is assumed that these observations are consistent with TMDD and are believed to be related to TfR1 and / or CD20 binding and subsequent trafficking. It has been found that even at the highest single dose of 10 mg / kg, drug clearance is approximately 3 to 7 times higher than expected for similar molecules (such as standard IgG antibodies). Unbound by this theory, it is assumed that this increased clearance is consistent with TMDD via both TfR and CD20, however, they contribute differently to the nonlinearity in the dose-exposure relationship.

[0104] Although preclinical mouse MS models have been widely used in this field, their efficacy for RG6035 is extremely limited. There is leakage in the BBB in these models, and overall B cell exhaustion has shown marginal and inconsistent results in mouse MS models. [39,40] Furthermore, the difference between the type I and type II anti-CD20 antibody mechanisms that induce direct B cell death does not apply to wild-type rodents. Therefore, it can be expected that B cell exhaustion in the mouse CNS has limited value in inferring therapeutic effects in humans.

[40] In addition, and perhaps most importantly, clinical brain shuttles do not bind to rodent, canine, or porcine TfR1 homologs or nonprimate CD20. RG6035 has been shown to have similar binding affinity to human and cynomolgus monkey TfR1 via surface plasmon resonance (equilibrium dissociation constants at 37°C: KDhuTfR1 421±5 nM, KDcyTfR1 694±11 nM). Therefore, species selection was based on binding affinity limited to humans and cynomolgus monkeys. The constant regions of endogenous primate IgG also exhibit high homology compared to human therapeutic antibodies, thus reducing the degree of ADA formation. Taking these factors into account, the mouse MS model cannot be effectively used for dose prediction of human RG6035.

[0105] The CNS of cynomolgus monkeys is similar to that of healthy humans and contains very few (if any) B cells. [41,42] Therefore, healthy cynomolgus monkeys cannot be used as a model of B cell depletion in the human CNS.

[0106] It has now been found that the combination of peripheral pharmacodynamics (PD) of B cells in the blood and CNS exposure to RG6035 in cynomolgus monkeys can be used to simulate and predict the dose required to achieve therapeutic activity levels of RG6035 in the human brain. B cell depletion in lymphoid tissues suggests that B cell depletion is not limited to the blood.

[0107] Given the qualitative overlap between CD20 and CD19, CD19 has been used as a marker for total B cells, capturing the B cell maturation status from proto-B cells to plasmablasts.

[43] The sharp reduction in CD19+-B cells in replacement lymphoid organs (e.g., lymph nodes) after treatment with RG6035 illustrates their persistent and effective depletion. Free from this theory, it is hypothesized that several excretion mechanisms are believed to be involved in the clearance of RG6035. Specific clearance of RG6035, which binds to CD20 targets on B cells, can be mediated by the destruction of said B cells. RG6035 can be maintained in circulation by recirculation via neonatal Fc receptors.

[0108] Here, a transformed PK / PD model based on PK / PD data of blood B cell depletion in cynomolgus monkeys was used to identify the pharmacologically active dose range of RG6035.

[0109] Since there is no data on central (brain) B cell depletion in any relevant animal models, this invention is at least in part based on well-thought-out predictions that have been made:

[0110] (i)PK can actually be derived from cynomolgus monkeys;

[0111] (ii) Brain uptake of RG6035 was correctly estimated in cynomolgus monkeys and translated into humans;

[0112] (iii)RG6035 has similar potency in the brain as it does in the periphery because it is independent of the presence of effector cells or complement.

[0113] (iv) The ratio of in vitro measured potency in human and cynomolgus monkey blood (approximately 4 times higher in humans than in cynomolgus monkeys) can transfer the potency of B cell depletion to humans.

[0114] Using this transformed PK / PD model, the therapeutic efficacy of systemic exposure to pharmacologically active RG6035 can be estimated, defined in this paper as brain B cell depletion ≥ 60%, preferably ≥ 95% (as assessed in CSF).

[0115] Based on this assessment, it has been found that short dosing intervals (e.g., once a week, once every two weeks, or once every four weeks) are beneficial for maintaining B cell depletion in CSF.

[0116] Although the sample sizes used for some PK analyses in this study were small (n=2 per group), the ability of RG6035 to cross the BBB and deplete B cells in blood and secondary lymphoid tissues could still be assessed due to ethical restrictions associated with the cynomolgus monkey model, which enabled the identification of effective human doses.

[0117] It is estimated that a weekly dose of 70 mg for four weeks can achieve approximately 60% B-cell exhaustion, while a weekly dose of 200 mg for four weeks can achieve 80% to 90% B-cell exhaustion, and a weekly dose of 700 mg for four weeks can achieve over 95% B-cell exhaustion. This is illustrated in Figure 1.

[0118] Brain Shuttle – CD20 Construct

[0119] CD20 is a protein expressed on the surface of B cells (at low levels from proto-B cells to plasmablasts) and is a clinically validated therapeutic target for MS [20a]. Anti-CD20 antibodies are of two types: type I antibodies, such as rituximab, oligrin, or olfamumab, and type II antibodies, such as tosimomab or obbituzumab [22a, 23a]. Obbituzumab differs from rituximab and type I antibodies in that it mediates stronger direct cell death induction independent of Fc region effector function and enhances ADCC / ADCP due to Fc engineering, but reduces CDC

[48] .

[0120] To effectively target CNS compartmentalized B cells in progressive MS, CD20 mAbs would need to possess extravascular B cell depletion properties and the ability to cross the BBB. Free from this theoretical constraint, it is inferred that the type II CD20 antibody, obbituzumab, is particularly well-suited to disrupting brain-resident B cells and possesses effective extravascular B cell depletion properties due to its favorable B cell depletion potential, independent of limited effector function (ADCC / CDC) in the CNS. Obbituzumab not only mediates an Fc region effector function-dependent mechanism but, more importantly, induces Fc region effector function-independent, non-apoptotic B cell death upon CD20 receptor binding [22a, 23a]. RG6035 is a fusion of octotuzumab and a brain shuttle module in which the Fc region effector function of octotuzumab is silenced by the introduction of a P329G / L234A / L235A mutation (PGLALA mutation). This improves safety while preserving B cell depletion properties. [24a] Importantly, octotuzumab containing the PGALALA mutation is still able to deplete B cells in whole blood and mediate antitumor efficacy in xenograft models, in contrast to other antiCD20 antibodies that silence Fc region effector function and lose B cell depletion activity in the absence of Fc region effector function.

[0121] Therefore, compared to constructs with intact Fc region effector function, RG6035 has a favorable safety profile while maintaining the ability to cross the BBB and depleted B cells.

[0122] More specifically, RG6035 is a trivalent bispecific antibody that contains...

[0123] a) A full-length antibody comprising two pairs, each pair comprising a full-length antibody light chain and a full-length antibody heavy chain, wherein the binding site formed by each pair of full-length heavy chains and full-length light chains specifically binds to human CD20, and

[0124] b) An additional Fab fused to the C-terminus of one heavy chain of the full-length antibody of a), wherein the binding site of this additional Fab specifically binds to human transferrin receptor 1.

[0125] Each of the full-length antibody light chains contains the amino acid residue arginine (not wild-type glutamic acid residue; E123R mutation) at position 123 in the constant light chain domain, and the amino acid residue lysine (not wild-type glutamine residue; Q124K mutation) at position 124 (according to Kabat numbering).

[0126] Each of the full-length antibody heavy chains contains a glutamate residue (not a wild-type lysine residue; K147E mutation) at position 147 in the first constant heavy chain domain, and a glutamate residue (not a wild-type lysine amino acid residue; K213E mutation) at position 213 (according to Kabat numbering).

[0127] The additional Fab that specifically binds to human transferrin receptor 1 contains cross-domains, such that the constant light chain domain and the constant heavy chain domain 1 are interchanged.

[0128] Therefore, RG6035 is composed of four polypeptides having the amino acid sequences of SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03 and SEQ ID NO: 05.

[0129] In one embodiment of all aspects and embodiments of the invention, RG6035 is a trivalent bispecific antibody comprising two polypeptides comprising the amino acid sequence of SEQ ID NO: 01, a polypeptide comprising the amino acid sequence of SEQ ID NO: 02, optionally having additional N-terminal glutamine (Q) or pyrrolidone (pE) residues and / or optionally having additional C-terminal lysine (K) amino acid residues, a polypeptide comprising the amino acid sequence of SEQ ID NO: 03, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 04, optionally having additional N-terminal glutamine (Q) or pyrrolidone (pE) residues.

[0130] In a preferred embodiment of all aspects and embodiments of the invention, RG6035 is a trivalent bispecific antibody comprising two polypeptides comprising the amino acid sequence of SEQ ID NO: 01, a polypeptide comprising the amino acid sequence of SEQ ID NO: 02, optionally having additional N-terminal glutamine (Q) or pyrrolidone (pE) residues and / or optionally having additional C-terminal lysine (K) amino acid residues, a polypeptide comprising the amino acid sequence of SEQ ID NO: 03, and a polypeptide comprising the amino acid sequence of SEQ ID NO: 05, optionally having additional N-terminal glutamine (Q) or pyrrolidone (pE) residues.

[0131] SEQ ID NO: 01 has the following amino acid sequence:

[0132] DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVE IKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0133] SEQ ID NO: 02 has the following amino acid sequence:

[0134] VQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。

[0135] SEQ ID NO: 03 has the amino acid sequence:

[0136] AIQLTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNYASSNVDNTFGGGTKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC。

[0137] SEQ ID NO: 04 has the amino acid sequence:

[0138] SMQESGPGLVKPSQTLSLTCTVSGFSLSSYAMSWIRQHPGKGLEWIGYIWSGGSTDYASWAKSRVTISKTSTTVSLKLSSVTAADTAVYYCARRYGTSYPDYGDASGFDPWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0139] SEQ ID NO: 05 has the amino acid sequence:

[0140] VQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGSGGGGSGGGGSGGGGSQSMQESGPGLVKPSQTLSLTCTVSGFSLSSYAMSWIRQHPGKGLEWIGYIWSGGSTDYASWAKSRVTISKTSTTVSLKLSSVTAADTAVYYCARRYGTSYPDYGDASGFDPWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0141] In all aspects and in one embodiment of the present invention, RG6035 is a monoclonal trivalent bispecific antibody.

[0142] In CD20 binding assays, both the functional Fc region effector variant of RG6035 and RG6035 itself showed comparable CD20 binding in a concentration-dependent manner. The brain shuttle construct exhibited slightly higher affinity than the corresponding non-brain shuttle control antibodies (i.e., obbituzumab and obbituzumab-PGLALA). Unbound by this theory, it is speculated that this may be due to additional binding of the brain shuttle portion to TfR1 expressed on the surface of B cells.

[0143] RG6035 and opituzumab-PGLALA showed similar maximum B cell depletion in human CSF incorporating human peripheral blood mononuclear cells (PBMCs), and there were no significant differences in binding and B cell depletion between the two constructs in ex vivo human tonsil-derived cells, which confirms the ability of RG6035 to deplete B cells derived from lymphoid tissue.

[0144] More specifically, to assess the B cell death induction properties in cerebrospinal fluid (CSF), human peripheral blood mononuclear cells (PBMCs) were incubated with RG6035 and obituzumab-PGLALA from CSF of healthy human donors. After 22 hours of incubation, the mean maximum exhaustion (± standard deviation [±SD]) of RG6035 and obituzumab-PGLALA were 75.11% ± 5.94 and 69.87% ± 13.3, respectively. These results indicate a strong direct B cell exhaustion potency in CSF cell culture medium, confirming the sensitivity of B cells in CSF as compartmentalized B cells of the central nervous system (CNS) to a protease-independent (non-apoptotic) B cell exhaustion mechanism mediated by RG6035 and obituzumab-PGLALA.

[15]

[0145] The PBMC-CSF assay showed that the mechanism of action of RG6035 and ocbituzumab-PGLALA is a direct B-cell killing mechanism independent of Fc zone effector function, leading to effective exhaustion of B cells in CSF. Furthermore, B-cell exhaustion does not require the availability of the complement system.

[0146] In human tonsil-derived cells, RG6035 binding and B cell depletion were assessed using an in vitro autologous B cell depletion assay to evaluate the efficacy of direct cell death induction. Unbound by this theory, it is hypothesized that B cells from tonsil tissue better reflect the response of B cells in secondary lymphoid tissue compared to peripheral blood B cells.

[0147] The assessment showed that human tonsils contain a large proportion of B cells, and that B cell subsets exhibit great heterogeneity, particularly naive B cells, tissue-resident memory B cells, and immune-active germinal center (GC) B cells.

[0148] Obituzumab-PGLALA has been found to induce direct CD19+-B cell death and exhaustion at a half-maximal effective concentration (EC50) of 0.523 ± 0.134 nM. The direct cytotoxic capacity is retained in RG6035 (EC50 = 0.396 ± 0.177 nM). A concentration-dependent cytotoxic dose-response was observed 22 hours after the addition of both constructs to tonsillar-derived cells, demonstrating concentration-dependent CD19+-B cell exhaustion from three tonsillar donors. Maximum exhaustion was achieved at concentrations ranging from 25 nM to 100 nM for both compounds (see Table 1). There was no significant difference in maximum B cell exhaustion between the two constructs at any tested concentration. In this experiment, RG6035 was able to reduce the number of CD19+ human tonsillar B cells by a maximum average of 60%.

[0149] Table 1: Binding and B cell depletion of octotuzumab-PGLALA and RG6035 in ex vivo autologous human tonsil-derived B cell depletion assay.

[0150]

[0151] Using three tonsillar donors to determine EC50 values ​​for CD19+-B cell exhaustion and B cell subset exhaustion, different cell exhaustion morphologies were found among the three donors. Comparative analysis showed a correlation between maximum B cell subset exhaustion and surface CD20 expression, with the highest CD20 expression on transitional B cells and the lowest on naive B cells. The highest maximum exhaustion was observed on transitional B cells and the lowest on naive B cells. B cells with moderate CD20 expression levels also showed moderate levels of maximum B cell exhaustion. Therefore, the response to RG6035 is correlated with the degree of CD20 surface expression.

[0152] Therefore, RG6035 can induce direct cell death and deplete a subset of extravascular human B cells located in secondary lymphoid tissues.

[0153] Unbound by this theory, it is hypothesized that the explanation for incomplete B cell exhaustion may be the differential expression of CD19 and CD20 in late-stage B cells, particularly plasmablasts expressing low levels of CD20 and plasma cells that do not express CD20, both of which express CD19. Furthermore, the potential regulation of CD19 expression levels after CD20 antibody incubation could explain the under-detection of maximal exhaustion [Jones, DJ et al., Arthritis Rheum. 64 (2012) 3111-3118].

[0154] Therefore, the presence of the TfR1 binding moiety does not negatively interfere with B cell exhaustion efficacy. No significant differences in EC50 and maximum exhaustion were observed between the two constructs, demonstrating that RG6035 retains its direct B cell death induction ability despite BS-mediated TfR1 co-binding.

[0155] Compared to huTfR1-negative untransfected cells (MDCKII-parental), RG6035 and BS-obituzumab showed 30-fold higher intracellular uptake in Madin-Darby canine kidney II (MDCKII) cells transfected with huTfR1 (MDCKII-huTfR) after a 30-minute loading (pulse) phase. Both brain shuttle constructs exhibited comparable transcytosis activity following huTfR1-mediated uptake, with similar amounts of IgG released into the extracellular compartment. The non-target-binding huTfR1-positive control molecule BS-DP47-PGLALA showed similar huTfR1-mediated uptake and transcytosis. Data are mean ± SD from three replicates. Cells were extracted after a 30-minute loading (pulse) phase, and intracellular IgG levels were analyzed using ELISA. The results are expressed as IgG uptake (ng IgG / mg) normalized to total cellular protein in each well. The results are shown in Figures 2 to 3. Figure 4 As shown.

[0156] Transcytosis efficiency is shown in Figure 5. The transcytosis ng IgG was compared with the ng IgG in the corresponding intracellular compartment at the start of tracking (t=0) (expressed as %).

[0157] Therefore, Fc effector silencing and conjugation to the brain shuttle module do not impair CD20 binding or TfR1-mediated transcytosis. The extent to which these molecules cause direct B cell death upon binding is comparable and concentration-dependent.

[0158] Human CD20 transgenic mice were treated intravenously (IV) with an alternative antibody consisting of a human CD20 conjugate (obitutuzumab variable domain) and a mouse TfR-binding brain shuttle module (mBS-obitutuzumab) at doses of 0.6 mg / kg, 1.3 mg / kg, and 13.3 mg / kg, or with obitutuzumab (non-shuttle) at doses of 0.5 mg / kg, 1 mg / kg, and 10 mg / kg (equimolar concentrations). Both constructs achieved similar blood B-cell depletion on day 6, but there was no evidence of dose-dependent response. Obitutuzumab showed a higher degree of B-cell depletion compared to the mBS-obitutuzumab antibody. When comparing effects in lymphoid tissues, mBS-obitutuzumab exhibited stronger B-cell killing activity than mBS-obitutuzumab-PGLALA. As assessed using a two-tailed paired t-test, the effect in the spleen (p<0.05 at all doses) was more pronounced than the effect in the lymph nodes (p<0.05 only in the 0.6 mg / kg dose group). However, in the lymph nodes, a difference was observed only in animals receiving the lowest dose between constructs with silenced Fc region effector function and constructs with intact Fc region effector function, suggesting that considerable B cell exhaustion can be achieved through ADCC-independent molecules. The results are shown in Table 2.

[0159] Table 2: Kinetics of B cell depletion in blood, spleen, and lymph nodes: Kinetics of B cell depletion in the blood of huCD20 mice after administration of mBS-obituzumab or mBS-obituzumab-PGLALA (A) and parental (non-shuttle) antibody (B); data points represent mean ± SD for N=6 mice; kinetics of B cell depletion of mBS-huCD20 mAb in the spleen (C) and lymph nodes (D) of huCD20 and naïve mice on day 6; differences in mean B cell depletion in lymph nodes and spleen between mBS-huCD20-WT and mBS-huCD20-PGLALA treated mice and naïve mice were assessed; p-values ​​(one-way ANOVA) for spleen and lymph nodes in all dose groups (0.6, 1.3, and 13.3 mg / kg) were <0.05.

[0160]

[0161]

[0162]

[0163]

[0164] One potential problem with molecules that have fully functional Fc region effectors is the induction of infusion-related responses (IRR), which involves the crosslinking of TfR1 on peripheral cells with Fc γ receptors on immune cells and their subsequent activation. It has been previously shown that BS-antibody fusion constructs with full effector function can be transported in a “stealth mode” in the periphery due to steric hindrance while retaining full activity when binding to their CNS targets in preclinical in vitro and mouse models [27a].

[0165] To confirm the absence of risk of IRR associated with Fc region effector function and the presence of the BS module, in vitro whole blood assays (WBA) were performed. Additionally, the compound was administered to huCD20 and huCD20xHIGR3 mice, and cytokine release was determined in vitro, while temperature changes were determined in vivo.

[0166] More specifically, in human whole-body biopsies (WBA), no cytokine-related releases were observed in either obbituzumab-PGLALA or RG6035 across the examined concentration range (0.01 nM to 1000 nM). In contrast, moderate releases of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-8, and IL-1β were observed in obbituzumab. Moderate to strong releases of IL-6 and strong releases of IL-8 were observed at the highest concentrations of BS-obbituzumab. All tested constructs demonstrated concentration-dependent B cell exhaustion. The comparable B cell exhaustion potency of BS-obbituzumab and obbituzumab in the WBA indicates that the BS fraction has no effect on B cell killing function.

[0167] mBS-Obituzumab, mBS-Obituzumab-PGLALA, and obituzumab were evaluated in a single IV dose study in both huCD20 and huCD20xHIGR3 mice (n=5 animals / group for each compound and mouse model). Mild to moderate atrophy was recorded 15 minutes after administration of 10 mg / kg mBS-Obituzumab in all huCD20 and huCD20xHIGR3 mice. However, all animals recovered and exhibited normal behavior within two hours of administration. Temperature data indicated significant differences between treatments; all animals treated with mBS-Obituzumab experienced an acute hypothermia of approximately 2°C to 5°C, indicating acute IRR, followed by rapid recovery, while animals treated with obituzumab or mBS-Obituzumab-PGLALA did not experience a hypothermia. The cytokine levels in these groups showed a typical pattern of classical IRR [29a]; notably, serum levels of keratinocyte-derived cytokines (KC / CXCL1), monocyte chemoattractant protein-1 (MCP-1), granulocyte colony-stimulating factor (G-CSF), and macrophage inflammatory protein-1β (MIP-1β) were elevated.

[0168] Compared to mBS-obbituzumab-PGLALA, mBS-obbituzumab induced strong serum release of KC, G-CSF, MIP-1β, and MCP-1, and moderate release of IL-6. Other cytokines were largely unaffected, with TNF-α, IL-2, and IFN-γ levels below the detection limit (2.3 pg / mL, 1.0 pg / mL, and 1.1 pg / mL, respectively). In mice treated with mBS-obbituzumab-PGLALA, cytokine levels of KC, G-CSF, MIP-1β, and MCP-1 and MIP-2 were largely unaffected or moderately decreased.

[0169] Both mBS-Obiutuzumab and mBS-Obiutuzumab-PGLALA have a maximum tolerated dose (MTD) ≥10 mg / kg after a single IV administration.

[0170] Female huCD20xHIGR3 mice were administered mBS-obitutuzumab and the mBS-obitutuzumab-PGLALA variant via a single IV dose (0.6 mg / kg, 1.3 mg / kg, or 13.3 mg / kg). All mice survived until their scheduled sacrifice on day 3 (N=5), day 8 (N=5), or day 22 (N=5) post-administration. No clinical symptoms or effects on body weight were observed. IL-6 levels in all animals were below the lower limit of quantitation (1.94 pg / mL) on all study days (days 3, 8, and 22). Animals treated with mBS-obitutuzumab (13.3 mg / kg) showed a severe reduction in reticulocyte count and immature reticulocyte fraction on day 3, consistent with suppression / depletion of bone marrow erythrocyte (RBC) precursors. In a single animal treated with mBS-obitutuzumab (13.3 mg / kg), neutrophil count increased on day 3, consistent with the acute phase response. White blood cell (WBC) and lymphocyte counts decreased slightly on day 8, consistent with the expected pharmacology of B-cell killing. In contrast, in animals treated with mBS-obitutuzumab-PGLALA, only a slight decrease in mean corpuscular volume was observed on day 8.

[0171] In the blood, mBS-obitatuzumab-PGLALA increased B cell counts on day 3 at all three dose levels studied, followed by depletion from day 8 to day 22. The most significant B cell depletion (approximately 25%) was observed on day 22 at a dose of 1.3 mg / kg. Treatment with mBS-obitatuzumab resulted in approximately 20% B cell depletion on day 3, reaching a maximum of approximately 50% depletion on day 22 at a dose of 13.3 mg / kg. On day 8, treatment with 0.6 mg / kg and 1.3 mg / kg resulted in similar degrees of B cell depletion (10% to 20%), while a more pronounced depletion effect was observed with mBS-obitatuzumab at 13.3 mg / kg. The results are shown in Figures 6 through 8.

[0172] No effects on spleen and inguinal lymph node weight were recorded in any of the study constructs. In one animal treated with mBS-obituzumab, a gross observation included an increase in spleen size on day 3 at 13.3 mg / kg, which was histologically associated with increased extramedullary hematopoiesis. No gross observations were observed in the inguinal lymph nodes or sternal bone marrow after treatment with either mBS-obituzumab antibody.

[0173] In the inguinal lymph nodes, the most significant reduction in B cell count (approximately 50%) was observed on day 3. Free from this theory, it is hypothesized that this may not necessarily reflect B cell depletion, but rather leakage into the peripheral blood. Results are shown in Figures 9 to 9. Figure 11 middle.

[0174] In the spleen, no effect of mBS-obituzumab-PGLALA was observed, while only a minimal and dose-dependent effect was observed with mBS-obituzumab. Histological findings in the spleen included increased extramedullary hematopoiesis on days 3 and 8 with both mBS-obituzumab constructs, which returned to normal on day 22. There was no difference in the degree of hemosiderin deposition (evidence of iron accumulation) in the spleen between treated and control animals. No histological findings were observed in the inguinal lymph nodes and bone marrow. Results are shown in Figures 12 to 13. Figure 14 middle.

[0175] Following a single IV administration of 13.3 mg / kg of mBS-obbituzumab antibody, serum pharmacokinetic (PK) parameters were calculated based on composite concentration-time curves. PK parameters were similar between the two constructs. Both mBS-obbituzumab constructs were detected in brain tissue at 24, 48, and 168 hours post-administration, following injection of a blood tracer and perfusion, except in one animal treated with mBS-obbituzumab at 168 hours, where brain concentrations were below the limit of quantification. The brain-to-serum ratios for mBS-obbituzumab-PGLALA ranged from 0.0183 to 0.131, and for mBS-obbituzumab from 0.00951 to 0.153, where blood contamination in the brain was negligible in the latter.

[0176] Table 3: PK parameters of the mouse BS construct.

[0177]

[0178] Immunohistochemistry of human IgG in the blood tracer and two mBS-obbitutuzumab constructs showed that mice treated with the two mBS-obbitutuzumab constructs exhibited different parenchymal staining intensities at each time point, with comparable staining distribution and intensity, consistent with brain PK curves. The strongest diffuse staining of brain parenchyma and meningeal vessels appeared at 24 hours post-administration, while only vascular endothelial cell staining appeared at 168 hours, likely related to the presence of the blood tracer.

[0179] The cynomolgus monkey is the only species that exhibits cross-reactivity with the CD20 and TfR1 binding sites of RG6035. PK, pharmacodynamic (PD), and tolerability studies were conducted in cynomolgus monkeys to assess the PK / PD and safety of RG6035 following a single IV dose.

[0180] Two separate studies were conducted, each comparing four animals. Animals were treated with a single intravenous slow bolus of 10 mg / kg RG6035 or a mediator control for 15 days, followed by euthanasia and autopsy, or treated with 10 mg / kg BS-obituzumab for 8 weeks. In vivo parameters assessed included clinical observation, body weight, body temperature, clinicopathology (hematology, clinical chemistry, and coagulation), immunophenotyping, cytokine assessment, and macroscopic and histological examination.

[0181] Following a single IV administration of the construct at 10 mg / kg, PK parameters in cynomolgus monkey serum were similar. The concentration of RG6035 in CSF was also assessed, showing good brain penetration, with an area under the concentration-time curve of 242 nmol*h / L from 0 to 168 hours based on a composite curve.

[0182] Table 4: PK parameters of constructs in cynomolgus monkeys.

[0183]

[0184] RG6035 was well tolerated in all animals, and no treatment-related changes were observed in clinical observations, body weight, weight gain, or body temperature. There were no safety-related effects on blood cytokine levels, clinical chemistry (including transferrin, iron, ferritin, unsaturated iron binding capacity, and total iron binding capacity), blood soluble transferrin receptors, or macroscopic or histopathological features.

[0185] There is no evidence that a single dose of 10 mg / kg of RG6035 interferes with erythropoiesis.

[0186] In animals treated with RG6035, minimal to mild and transient clinicopathological changes were limited to a decrease in hemoglobin content in reticulocytes and a generally non-dose-related increase in C-reactive protein, both of which were observed as early as days 2–3 and / or 4. In all animals, including controls, transient minimal to moderate decreases in erythrocyte quality parameters (RBC count, hemoglobin, and hematocrit) and increases in reticulocyte counts were observed as early as days 2–8, with the trough typically occurring on days 3 / 4. These changes were attributed to multiple blood samplings. To assess the functional status of bone marrow erythropoietin, blood samples were collected before administration and on days 1, 2, 3, 4, 6, 8, and 44, and serum was measured to determine hepcidin and erythropoietin (EPO) concentrations. Mild to moderate increases in serum EPO concentrations were observed in all dose groups, including controls, and a marked trend toward hepcidin inhibition was observed. These findings indicate that regenerative erythropoiesis in response to multiple blood draws is normal, consistent with minimal to moderate decreases in observed erythrocyte quality parameters (RBC count, hemoglobin, and hematocrit levels) and an increase in reticulocyte count. The decrease in hemoglobin content in reticulocytes may indicate early iron restriction due to the binding of RG6035 to TfR1.

[0187] Based on these findings, there is no evidence that a single dose of 10 mg / kg of RG6035 interferes with normal regenerative erythrocyte production at relevant levels.

[0188] BS-obitatuzumab was generally well tolerated in cynomolgus monkeys, with the exception of one animal that exhibited transient clinical signs of diminished and / or reduced activity on days 2 to 3 post-administration. Overall, although some post-treatment changes in cytokine levels were observed in some animals, they did not appear to be proportional to the administered dose. Administration of 10 mg / kg BS-obitatuzumab caused transient changes in hematological, coagulation, and clinical chemistry parameters, consistent with repeated blood draws and acute-phase reactions.

[0189] In the mesenteric lymph nodes and spleen of some evaluated animals, a predicted PD effect of reduced lymphocyte counts was observed microscopically in both constructs, along with a decrease in peripheral blood CD19+-B cell and CD86 activated B cell counts starting 4 hours post-dose and continuing until day 15. CD3+-T cell and CD16+-NK cell counts decreased starting 4 hours post-dose and recovered from day 4 onwards. IL-6 levels increased at 1 and 4 hours post-dose, with only a slight increase in MCP-1 levels at 4 hours post-dose. Without being bound by this theory, it is hypothesized that this is related to the mode of action of RG6035.

[0190] In both cynomolgus monkey studies, blood B cell counts decreased rapidly after administration. The maximum mean reduction in B cell counts was approximately 89% for BS-obituzumab and approximately 83% for RG6035 at 4 hours post-administration. The maximum decrease in T cell counts was observed at 4 hours post-administration for both constructs (29%); this decrease was transient and returned to baseline levels after 24 to 96 hours. The maximum decrease in NK cells was also observed at 4 hours post-administration for both constructs (43%); this decrease was also transient and returned to baseline levels after 96 to 168 hours. Little change in monocyte levels was observed after administration. All animals developed ADA, independent of the Fc region effector functional status of the constructs. ADA in cynomolgus monkeys is considered not to predict human immunogenicity [29a].

[0191] The results are shown in Figure 15 and Table 5.

[0192] Table 5: B cell depletion after antibody administration in cynomolgus monkeys; the number of B cells after antibody administration is represented by the percentage of B cells measured at baseline (100%); data are expressed as mean measurements ± SD from N=4 animals.

[0193]

[0194] In cynomolgus monkeys treated with RG6035 on day 2, a slight to significant increase in absolute neutrophil and monocyte counts was observed, reflected in an increase in total white blood cell count. On day 8, a minimal decrease in red blood cell (RBC) quality parameters was observed, accompanied by a moderate increase in absolute reticulocyte count, indicating increased erythropoiesis secondary to repeat blood collection. Clinical chemical changes included a slight decrease in phosphorus concentration on day 2, and a slight to mild decrease in triglyceride and cholesterol concentrations on days 2 and / or 8. Additionally, slight increases in fibrinogen, C-reactive protein, haptoglobin, and ferritin concentrations, and a slight decrease in iron concentration were observed on days 2 and / or 8. These changes, along with the changes in absolute neutrophil and monocyte concentrations, indicate an acute-phase response. By day 49, the observed hematological, coagulation, and clinical chemical changes (except for changes in triglycerides at 1 mg / kg) were no longer present, indicating recovery. These findings indicate that regenerated erythropoiesis in response to multiple blood draws is normal, consistent with minimal to moderate decreases in observed erythrocyte quality parameters (RBC count, hemoglobin, and hematocrit levels) and an increase in reticulocyte count. Based on this, there is no evidence that a single dose of 10 mg / kg RG6035 interferes with normal regenerated erythropoiesis.

[0195] In summary, RG6035 has been shown to be well-tolerated with no indication of IRR risk while retaining B-cell killing properties. TfR1-dependent RG6035 uptake has been demonstrated in the brains of huCD20 transgenic mice. In conclusion, compared to currently available therapies, RG6035 possesses the advantageous property of being able to more effectively target and deplete B cells located in the CNS in MS. It has been shown that Fc region effector function silencing for peripherally expressed targets mitigates IRR risk and effects on reticulocytes, resulting in a favorable safety profile.

[0196] RG6035 (RO7121932) is currently undergoing clinical trials in human MS patients (NCT05704361).

[0197] Some specific embodiments of the present invention

[0198] This invention is based, at least in part, on the depletion of B cells in the brain using a specific brain shuttle-CD20 construct, RG6035 (BS-CD20, RO7121932). RG6035 used in the method of this invention is a bispecific modular fusion protein of human transferrin receptor 1 (TfR1) directed brain shuttle (TfR1 binding antigen-binding fragment (Fab)) and the complementary site of the anti-CD20 antibody obbituzumab. TfR1 is abundant at the BBB and is a target for receptor-mediated transport of macromolecules across the BBB, while obbituzumab has a unique mechanism of action: inducing Fc effector-independent, non-apoptotic direct B cell death upon binding to surface-exposed CD20. [14-21] Upon repeated administration, RG6035 has the function of (completely) depleting B cells in the brain.

[0199] This invention is based at least in part on the discovery that the PK / PD model of RG6035 based on MS pathophysiology and data observed in blood as a substitute can be used to simulate human conditions.

[0200] Therapeutic effective dose

[0201] The method of the present invention includes administering to a subject a composition comprising a therapeutically effective amount of RG6035. As used herein, the term "therapeuticly effective amount" means an amount of compound or pharmaceutical composition sufficient to produce the desired therapeutic effect.

[0202] Compared to vector-treated animals, RG6035-treated animals showed a sharp decrease in the percentage of CD19+-B cells and CD20+-B cells in total cells and in each tissue region across all studied lymphoid tissues, but no further significant decrease was observed between the different treatment groups. Compared to the vector control, all RG6035-treated animals showed a significant decrease in activated germinal center B cells in the spleen and tonsils.

[0203] Cell segmentation and subsequent thresholding of each marker allowed for precise quantification of each B cell subset. CD19+CD27-CD38-naïve B cells (n=2), CD19+CD38+- or CD19+CD21-- activated germinal center B cells (n=4), CD19+CD38-CD27+- memory B cells (n=2), CD19+CD21+-naïve / memory B cells (n=2), and CD138+CD21-- plasma cells (n=2) were found in the cervical lymph nodes, spleen, submandibular lymph nodes, and tonsils. Two-way ANOVA was performed for multiple comparisons within each row (simple effects within rows compared to the mediator control). The percentage of positive cell types was normalized to tissue area. *, p < 0.05 compared to the mediator control. Results are shown in Figures 16 through 19.

[0204] Those skilled in the art will understand that the therapeutically effective amount of RG6035 administered to a subject can depend on a variety of factors, including pharmacodynamic characteristics, route of administration, frequency of treatment, and the health status, age, and weight of the subject to be treated, and that, with the information disclosed herein, those skilled in the art will be able to determine the appropriate amount for each subject.

[0205] In all aspects and embodiments of the invention, in some embodiments, the therapeutically effective amount is a dose selected to improve efficacy and / or maintain efficacy and improve at least one of safety and tolerability. In some embodiments, the therapeutically effective amount is selected to reduce at least one side effect while simultaneously improving efficacy and / or maintaining efficacy.

[0206] In some embodiments, the subject is administered RG6035 at doses of 1 mg / kg to 4 mg / kg, 1 mg / kg to 3.5 mg / kg, 1 mg / kg to 3.0 mg / kg, 1 mg / kg to 2.9 mg / kg, 1 mg / kg to 2.8 mg / kg, 1 mg / kg to 2.7 mg / kg, 1 mg / kg to 2.6 mg / kg, 1 mg / kg to 2.5 mg / kg, 1 mg / kg to 2.4 mg / kg, 1 mg / kg to 2.3 mg / kg, 1 mg / kg to 2.2 mg / kg, 1 mg / kg to 2.1 mg / kg, or 1 mg / kg to 2 mg / kg relative to the subject's body weight.

[0207] In some embodiments, the subject was administered RG6035 at doses of 1.25 mg / kg to 4 mg / kg, 1.25 mg / kg to 3.5 mg / kg, 1.25 mg / kg to 3 mg / kg, 1.25 mg / kg to 2.9 mg / kg, 1.25 mg / kg to 2.8 mg / kg, 1.25 mg / kg to 2.7 mg / kg, 1.25 mg / kg to 2.6 mg / kg, 1.25 mg / kg to 2.5 mg / kg, 1.25 mg / kg to 2.4 mg / kg, 1.25 mg / kg to 2.3 mg / kg, or 1.25 mg / kg to 2.2 mg / kg relative to the subject's body weight.

[0208] In some embodiments, the subject was administered RG6035 at doses of 1.5 mg / kg to 4 mg / kg, 1.5 mg / kg to 3.5 mg / kg, 1.5 mg / kg to 3 mg / kg, 1.5 mg / kg to 2.9 mg / kg, 1.5 mg / kg to 2.8 mg / kg, 1.5 mg / kg to 2.7 mg / kg, 1.5 mg / kg to 2.6 mg / kg, 1.5 mg / kg to 2.5 mg / kg, 1.5 mg / kg to 2.4 mg / kg, 1.5 mg / kg to 2.3 mg / kg, or 1.5 mg / kg to 2.2 mg / kg relative to the subject's body weight.

[0209] In some embodiments, the subject was administered RG6035 at doses of 1.8 mg / kg to 4 mg / kg, 1.8 mg / kg to 3.5 mg / kg, 1.8 mg / kg to 3 mg / kg, 1.8 mg / kg to 2.9 mg / kg, 1.8 mg / kg to 2.8 mg / kg, 1.8 mg / kg to 2.7 mg / kg, 1.8 mg / kg to 2.6 mg / kg, 1.8 mg / kg to 2.5 mg / kg, 1.8 mg / kg to 2.4 mg / kg, 1.8 mg / kg to 2.3 mg / kg, or 1.8 mg / kg to 2.2 mg / kg relative to the subject's body weight.

[0210] In some embodiments, the subject was administered RG6035 at doses of 1.9 mg / kg to 4 mg / kg, 1.9 mg / kg to 3.5 mg / kg, 1.9 mg / kg to 3 mg / kg, 1.9 mg / kg to 2.9 mg / kg, 1.9 mg / kg to 2.8 mg / kg, 1.9 mg / kg to 2.7 mg / kg, 1.9 mg / kg to 2.6 mg / kg, 1.9 mg / kg to 2.5 mg / kg, 1.9 mg / kg to 2.4 mg / kg, 1.9 mg / kg to 2.3 mg / kg, or 1.9 mg / kg to 2.2 mg / kg relative to the subject's body weight.

[0211] In some embodiments, the subject was administered RG6035 at doses of 2 mg / kg to 4 mg / kg, 2 mg / kg to 3.5 mg / kg, 2 mg / kg to 3 mg / kg, 2 mg / kg to 2.9 mg / kg, 2 mg / kg to 2.8 mg / kg, 2 mg / kg to 2.7 mg / kg, 2 mg / kg to 2.6 mg / kg, 2 mg / kg to 2.5 mg / kg, 2 mg / kg to 2.4 mg / kg, 2 mg / kg to 2.3 mg / kg, or 2 mg / kg to 2.2 mg / kg relative to the subject's body weight.

[0212] In some embodiments, the subject was administered a dose relative to the subject's body weight of 1 mg / kg to 4 mg / kg, 1.1 mg / kg to 4 mg / kg, 1.2 mg / kg to 4 mg / kg, 1.3 mg / kg to 4 mg / kg, 1.4 mg / kg to 4 mg / kg, 1.5 mg / kg to 4 mg / kg, 1.6 mg / kg to 4 mg / kg, 1.7 mg / kg to 4 mg / kg, 1.8 mg / kg to 4 mg / kg, 1.9 mg / kg to 4 mg / kg, 2 mg / kg to 4 mg / kg, 2.1 mg / kg to 4 mg / kg, 2.2 mg / kg to 4 mg / kg, 2.3 mg / kg to 4 mg / kg, 2.4 mg / kg to 4 mg / kg, 2.5 mg / kg to 4 mg / kg, 2.6 mg / kg to 4 mg / kg, 2.7 mg / kg to 4 mg / kg, 2.8 ...8 mg / kg to 4 mg / kg, 2.8 mg / RG6035 in doses of 2.9 mg / kg to 4 mg / kg, 3 mg / kg to 4 mg / kg, or 3.5 mg / kg to 4 mg / kg.

[0213] In some embodiments, the subject was administered an dose relative to the subject's body weight of 1 mg / kg to 3 mg / kg, 1.1 mg / kg to 3 mg / kg, 1.2 mg / kg to 3 mg / kg, 1.3 mg / kg to 3 mg / kg, 1.4 mg / kg to 3 mg / kg, 1.5 mg / kg to 3 mg / kg, 1.6 mg / kg to 3 mg / kg, 1.7 mg / kg to 3 mg / kg, 1.8 mg / kg to 3 mg / kg, 1.9 mg / kg to 3 mg / kg, 2 mg / kg to 3 mg / kg, 2.1 mg / kg to 3 mg / kg, 2.2 mg / kg to 3 mg / kg, 2.3 mg / kg to 3 mg / kg, 2.4 mg / kg to 3 mg / kg, 2.5 mg / kg to 3 mg / kg, 2.6 mg / kg to 3 mg / kg, 2.7 mg / kg to 3 mg / kg, 2.8 ... RG6035 in doses of 2.9 mg / kg to 3 mg / kg or 2.9 mg / kg to 3 mg / kg.

[0214] In some embodiments, the subject was administered a dose relative to the subject's body weight of 1 mg / kg to 2.9 mg / kg, 1.1 mg / kg to 2.9 mg / kg, 1.2 mg / kg to 2.9 mg / kg, 1.3 mg / kg to 2.9 mg / kg, 1.4 mg / kg to 2.9 mg / kg, 1.5 mg / kg to 2.9 mg / kg, 1.6 mg / kg to 2.9 mg / kg, 1.7 mg / kg to 2.9 mg / kg, 1.8 mg / kg to 2.9 mg / kg, 1.9 mg / kg to 2.9 mg / kg, 2 mg / kg to 2.9 mg / kg, 2.1 mg / kg to 2.9 mg / kg, 2.2 mg / kg to 2.9 mg / kg, 2.3 mg / kg to 2.9 mg / kg, 2.4 mg / kg to 2.9 mg / kg, or 2.5 mg / kg to 2.9 mg / kg. RG6035 at doses of 2.6 mg / kg to 2.9 mg / kg, 2.7 mg / kg to 2.9 mg / kg, or 2.8 mg / kg to 2.9 mg / kg.

[0215] In some embodiments, the subject was administered a dose relative to the subject's body weight of 1 mg / kg to 2.8 mg / kg, 1.1 mg / kg to 2.8 mg / kg, 1.2 mg / kg to 2.8 mg / kg, 1.3 mg / kg to 2.8 mg / kg, 1.4 mg / kg to 2.8 mg / kg, 1.5 mg / kg to 2.8 mg / kg, 1.6 mg / kg to 2.8 mg / kg, 1.7 mg / kg to 2.8 mg / kg, 1.8 mg / kg to 2.8 mg / kg, 1.9 mg / kg to 2.8 mg / kg, 2 mg / kg to 2.8 mg / kg, 2.1 mg / kg to 2.8 mg / kg, 2.2 mg / kg to 2.8 mg / kg, 2.3 mg / kg to 2.8 mg / kg, 2.4 mg / kg to 2.8 mg / kg, or 2.5 mg / kg to 2.8 mg / kg. RG6035 at doses of 2.6 mg / kg to 2.8 mg / kg or 2.7 mg / kg to 2.8 mg / kg.

[0216] In some embodiments, the subject was administered a dose relative to the subject's body weight of 1 mg / kg to 2.7 mg / kg, 1.1 mg / kg to 2.7 mg / kg, 1.2 mg / kg to 2.7 mg / kg, 1.3 mg / kg to 2.7 mg / kg, 1.4 mg / kg to 2.7 mg / kg, 1.5 mg / kg to 2.7 mg / kg, 1.6 mg / kg to 2.7 mg / kg, 1.7 mg / kg to 2.7 mg / kg, 1.8 mg / kg to 2.7 mg / kg, 1.9 mg / kg to 2.7 mg / kg, 2 mg / kg to 2.7 mg / kg, 2.1 mg / kg to 2.7 mg / kg, 2.2 mg / kg to 2.7 mg / kg, 2.3 mg / kg to 2.7 mg / kg, 2.4 mg / kg to 2.7 mg / kg, or 2.5 mg / kg to 2.8 mg / kg. RG6035 at mg / kg or 2.6 mg / kg to 2.7 mg / kg.

[0217] In some embodiments, the subject was administered a dose relative to the subject's body weight of 1 mg / kg to 4 mg / kg, 1.1 mg / kg to 4 mg / kg, 1.2 mg / kg to 4 mg / kg, 1.3 mg / kg to 4 mg / kg, 1.4 mg / kg to 4 mg / kg, 1.5 mg / kg to 4 mg / kg, 1.5 mg / kg to 3.5 mg / kg, 1.5 mg / kg to 3 mg / kg, 1.5 mg / kg to 2.9 mg / kg, 1.5 mg / kg to 2.8 mg / kg, 1.5 mg / kg to 2.7 mg / kg, 1.6 mg / kg to 4 mg / kg, 1.6 mg / kg to 3.5 mg / kg, 1.6 mg / kg to 3 mg / kg, 1.6 mg / kg to 2.9 mg / kg, 1.6 mg / kg to 2.8 mg / kg, or 1.6 mg / kg to 2.7 mg / kg. mg / kg, 1.7 mg / kg to 4 mg / kg, 1.7 mg / kg to 3.5 mg / kg, 1.7 mg / kg to 3 mg / kg, 1.7 mg / kg to 2.9 mg / kg, 1.7 mg / kg to 2.8 mg / kg, 1.7 mg / kg to 2.7 mg / kg, 1.8 mg / kg to 4 mg / kg, 1.8 mg / kg to 3.5 mg / kg, 1.8 mg / kg to 3 mg / kg, 1.8 mg / kg to 2.9 mg / kg, 1.8 mg / kg to 2.8 mg / kg, 1.8 mg / kg to 2.7 mg / kg, 1.9 mg / kg to 4 mg / kg, 1.9 mg / kg to 3.5 mg / kg, 1.9 mg / kg to 3 mg / kg, 1.9 mg / kg to 2.9 mg / kg, 1.9 mg / kg to 2.8 mg / kg Or RG6035 at doses of 1.9 mg / kg to 2.7 mg / kg.

[0218] In some embodiments, the subject is administered RG6035 at a dose of 1 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 1.1 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 1.2 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 1.3 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 1.4 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 1.5 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 1.6 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 1.7 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 1.8 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 1.9 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 2 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 2.1 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 2.2 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 2.3 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 2.4 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 2.5 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 2.6 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 2.7 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 2.8 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 2.9 mg / kg relative to the subject's body weight. In some embodiments, the subject is administered RG6035 at a dose of 3 mg / kg relative to the subject's body weight.

[0219] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 300 mg, 70 mg to 280 mg, 70 mg to 260 mg, 70 mg to 250 mg, 70 mg to 240 mg, 70 mg to 230 mg, 70 mg to 220 mg, 70 mg to 210 mg, 70 mg to 200 mg, 70 mg to 190 mg, 70 mg to 180 mg, 70 mg to 170 mg, 70 mg to 160 mg, or 70 mg to 150 mg.

[0220] In some embodiments, subjects were administered RG6035 in doses of 75 mg to 300 mg, 75 mg to 280 mg, 75 mg to 260 mg, 75 mg to 250 mg, 75 mg to 240 mg, 75 mg to 230 mg, 75 mg to 220 mg, 75 mg to 210 mg, 75 mg to 200 mg, 75 mg to 190 mg, 75 mg to 180 mg, 75 mg to 170 mg, 75 mg to 160 mg, or 75 mg to 150 mg.

[0221] In some embodiments, subjects were administered RG6035 in doses of 100 mg to 300 mg, 100 mg to 280 mg, 100 mg to 260 mg, 100 mg to 250 mg, 100 mg to 240 mg, 100 mg to 230 mg, 100 mg to 220 mg, 100 mg to 210 mg, 100 mg to 200 mg, 100 mg to 190 mg, 100 mg to 180 mg, 100 mg to 170 mg, 100 mg to 160 mg, or 100 mg to 150 mg.

[0222] In some embodiments, subjects were administered RG6035 in doses of 110 mg to 300 mg, 110 mg to 280 mg, 110 mg to 260 mg, 110 mg to 250 mg, 110 mg to 240 mg, 110 mg to 230 mg, 110 mg to 220 mg, 110 mg to 210 mg, 110 mg to 200 mg, 110 mg to 190 mg, 110 mg to 180 mg, 110 mg to 170 mg, 110 mg to 160 mg, or 110 mg to 150 mg.

[0223] In some embodiments, subjects were administered RG6035 in doses of 120 mg to 300 mg, 120 mg to 280 mg, 120 mg to 260 mg, 120 mg to 250 mg, 120 mg to 240 mg, 120 mg to 230 mg, 120 mg to 220 mg, 120 mg to 210 mg, 120 mg to 200 mg, 120 mg to 190 mg, 120 mg to 180 mg, 120 mg to 170 mg, 120 mg to 160 mg, or 120 mg to 150 mg.

[0224] In some embodiments, subjects were administered RG6035 in doses of 130 mg to 300 mg, 130 mg to 280 mg, 130 mg to 260 mg, 130 mg to 250 mg, 130 mg to 240 mg, 130 mg to 230 mg, 130 mg to 220 mg, 130 mg to 210 mg, 130 mg to 200 mg, 130 mg to 190 mg, 130 mg to 180 mg, 130 mg to 170 mg, 130 mg to 160 mg, or 130 mg to 150 mg.

[0225] In some embodiments, subjects were administered RG6035 in doses of 140 mg to 300 mg, 140 mg to 280 mg, 140 mg to 260 mg, 140 mg to 250 mg, 140 mg to 240 mg, 140 mg to 230 mg, 140 mg to 220 mg, 140 mg to 210 mg, 140 mg to 200 mg, 140 mg to 190 mg, 140 mg to 180 mg, 140 mg to 170 mg, 140 mg to 160 mg, or 140 mg to 150 mg.

[0226] In some embodiments, the dose administered to the subject is 70 mg to 300 mg, 75 mg to 300 mg, 100 mg to 300 mg, 110 mg to 300 mg, 120 mg to 300 mg, 130 mg to 300 mg, 140 mg to 300 mg, 150 mg to 300 mg, 160 mg to 300 mg, 170 mg to 300 mg, 180 mg to 300 mg, 190 mg to 300 mg, 200 mg to 300 mg, 210 mg to 300 mg, 220 mg to 300 mg, 230 mg to 300 mg, 240 mg to 300 mg, 250 mg to 300 mg, 260 mg to 300 mg, 270 mg to 300 mg, 280 mg to 300 mg, or 290 mg. Up to 300 mg of RG6035.

[0227] In some embodiments, subjects were administered RG6035 at doses of 70 mg to 290 mg, 75 mg to 290 mg, 100 mg to 290 mg, 110 mg to 290 mg, 120 mg to 290 mg, 130 mg to 290 mg, 140 mg to 290 mg, 150 mg to 290 mg, 160 mg to 290 mg, 170 mg to 290 mg, 180 mg to 290 mg, 190 mg to 290 mg, 200 mg to 290 mg, 210 mg to 290 mg, 220 mg to 290 mg, 230 mg to 290 mg, 240 mg to 290 mg, 250 mg to 290 mg, 260 mg to 290 mg, 270 mg to 290 mg, or 280 mg to 290 mg.

[0228] In some embodiments, subjects were administered RG6035 at doses of 70 mg to 280 mg, 75 mg to 300 mg, 100 mg to 280 mg, 110 mg to 280 mg, 120 mg to 280 mg, 130 mg to 280 mg, 140 mg to 280 mg, 150 mg to 280 mg, 160 mg to 280 mg, 170 mg to 280 mg, 180 mg to 280 mg, 190 mg to 280 mg, 200 mg to 280 mg, 210 mg to 280 mg, 220 mg to 280 mg, 230 mg to 280 mg, 240 mg to 280 mg, 250 mg to 280 mg, 260 mg to 280 mg, or 270 mg to 280 mg.

[0229] In some embodiments, subjects were administered RG6035 at doses of 70 mg to 270 mg, 75 mg to 270 mg, 100 mg to 270 mg, 110 mg to 270 mg, 120 mg to 270 mg, 130 mg to 270 mg, 140 mg to 270 mg, 150 mg to 270 mg, 160 mg to 270 mg, 170 mg to 270 mg, 180 mg to 270 mg, 190 mg to 270 mg, 200 mg to 270 mg, 210 mg to 270 mg, 220 mg to 270 mg, 230 mg to 270 mg, 240 mg to 270 mg, 250 mg to 270 mg, or 260 mg to 270 mg.

[0230] In some embodiments, subjects were administered RG6035 at doses of 70 mg to 260 mg, 75 mg to 260 mg, 100 mg to 260 mg, 110 mg to 260 mg, 120 mg to 260 mg, 130 mg to 260 mg, 140 mg to 260 mg, 150 mg to 260 mg, 160 mg to 260 mg, 170 mg to 260 mg, 180 mg to 260 mg, 190 mg to 260 mg, 200 mg to 260 mg, 210 mg to 260 mg, 220 mg to 260 mg, 230 mg to 260 mg, 240 mg to 260 mg, or 250 mg to 260 mg.

[0231] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 250 mg, 75 mg to 250 mg, 100 mg to 250 mg, 110 mg to 250 mg, 120 mg to 250 mg, 130 mg to 250 mg, 140 mg to 250 mg, 150 mg to 250 mg, 160 mg to 250 mg, 170 mg to 250 mg, 180 mg to 250 mg, 190 mg to 250 mg, 200 mg to 250 mg, 210 mg to 250 mg, 220 mg to 250 mg, 230 mg to 250 mg, or 240 mg to 250 mg.

[0232] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 240 mg, 75 mg to 240 mg, 100 mg to 240 mg, 110 mg to 240 mg, 120 mg to 240 mg, 130 mg to 240 mg, 140 mg to 240 mg, 150 mg to 240 mg, 160 mg to 240 mg, 170 mg to 240 mg, 180 mg to 240 mg, 190 mg to 240 mg, 200 mg to 240 mg, 210 mg to 240 mg, 220 mg to 240 mg, or 230 mg to 240 mg.

[0233] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 230 mg, 75 mg to 230 mg, 100 mg to 230 mg, 110 mg to 230 mg, 120 mg to 230 mg, 130 mg to 230 mg, 140 mg to 230 mg, 150 mg to 230 mg, 160 mg to 230 mg, 170 mg to 230 mg, 180 mg to 230 mg, 190 mg to 230 mg, 200 mg to 230 mg, 210 mg to 230 mg, or 220 mg to 230 mg.

[0234] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 220 mg, 75 mg to 220 mg, 100 mg to 220 mg, 110 mg to 220 mg, 120 mg to 220 mg, 130 mg to 220 mg, 140 mg to 220 mg, 150 mg to 220 mg, 160 mg to 220 mg, 170 mg to 220 mg, 180 mg to 220 mg, 190 mg to 220 mg, 200 mg to 220 mg, or 210 mg to 220 mg.

[0235] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 210 mg, 75 mg to 210 mg, 100 mg to 210 mg, 110 mg to 210 mg, 120 mg to 210 mg, 130 mg to 210 mg, 140 mg to 210 mg, 150 mg to 210 mg, 160 mg to 210 mg, 170 mg to 210 mg, 180 mg to 2100 mg, 190 mg to 210 mg, or 200 mg to 210 mg.

[0236] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 200 mg, 75 mg to 200 mg, 100 mg to 200 mg, 110 mg to 200 mg, 120 mg to 200 mg, 130 mg to 200 mg, 140 mg to 200 mg, 150 mg to 200 mg, 160 mg to 200 mg, 170 mg to 200 mg, 180 mg to 200 mg, or 190 mg to 200 mg.

[0237] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 190 mg, 75 mg to 190 mg, 100 mg to 190 mg, 110 mg to 190 mg, 120 mg to 190 mg, 130 mg to 190 mg, 140 mg to 190 mg, 150 mg to 190 mg, 160 mg to 190 mg, 170 mg to 190 mg, or 180 mg to 190 mg.

[0238] In some embodiments, subjects were administered RG6035 at doses of 70 mg to 180 mg, 75 mg to 180 mg, 100 mg to 180 mg, 110 mg to 180 mg, 120 mg to 180 mg, 130 mg to 180 mg, 140 mg to 180 mg, 150 mg to 180 mg, 160 mg to 180 mg, or 170 mg to 180 mg.

[0239] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 170 mg, 75 mg to 170 mg, 100 mg to 170 mg, 110 mg to 170 mg, 120 mg to 170 mg, 130 mg to 170 mg, 140 mg to 170 mg, 150 mg to 170 mg, or 160 mg to 170 mg.

[0240] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 160 mg, 75 mg to 160 mg, 100 mg to 160 mg, 110 mg to 160 mg, 120 mg to 160 mg, 130 mg to 160 mg, 140 mg to 160 mg, or 150 mg to 160 mg.

[0241] In some embodiments, subjects were administered RG6035 in doses of 70 mg to 150 mg, 75 mg to 150 mg, 100 mg to 150 mg, 110 mg to 150 mg, 120 mg to 150 mg, 130 mg to 150 mg, or 140 mg to 150 mg.

[0242] In some embodiments, the dose administered to the subject was 70 mg to 300 mg, 75 mg to 300 mg, 100 mg to 300 mg, 110 mg to 300 mg, 120 mg to 300 mg, 130 mg to 300 mg, 140 mg to 300 mg, 150 mg to 300 mg, 70 mg to 280 mg, 75 mg to 280 mg, 100 mg to 280 mg, 110 mg to 280 mg, 120 mg to 280 mg, 130 mg to 280 mg, 140 mg to 280 mg, 150 mg to 280 mg, 70 mg to 270 mg, 75 mg to 270 mg, 100 mg to 270 mg, 110 mg to 270 mg, 120 mg to 270 mg, 130 mg to 27 ...70 mg, 140 mg to 270 mg, 140 mg to 270 mg, 150 mg to 280 mg, 70 mg to 270 mg, 75 mg to 270 mg, 100 mg to 270 mg, 110 mg to 270 mg, 120 mg to 270 mg, 130 mg to 270 mg, 140 mg to 270 mg, 140 mg to 270 mg, 140 mg to mg to 270 mg, 150 mg to 270 mg, 70 mg to 260 mg, 75 mg to 260 mg, 100 mg to 260 mg, 110 mg to 260 mg, 120 mg to 260 mg, 130 mg to 260 mg, 140 mg to 260 mg, 150 mg to 260 mg, 70 mg to 250 mg, 75 mg to 250 mg, 100 mg to 250 mg, 110 mg to 250 mg, 120 mg to 250 mg, 130 mg to 250 mg, 140 mg to 250 mg, 150 mg to 250 mg, 70 mg to 240 mg, 75 mg to 240 mg, 100 mg to 240 mg, 110 mg to 240 mg, 120 mg to 240 mg, 13 ... mg to 240 mg, 140 mg to 240 mg, 150 mg to 240 mg, 70 mg to 230 mg, 75 mg to 230 mg, 100 mg to 230 mg, 110 mg to 230 mg, 120 mg to 230 mg, 130 mg to 230 mg, 140 mg to 230 mg, 150 mg to 230 mg, 70 mg to 220 mg, 75 mg to 220 mg, 100 mg to 220 mg, 110 mg to 220 mg, 120 mg to 220 mg, 130 mg to 220 mg, 140 mg to 220 mgRG6035 in doses of 150 mg to 220 mg, 70 mg to 210 mg, 75 mg to 210 mg, 100 mg to 210 mg, 110 mg to 210 mg, 120 mg to 210 mg, 130 mg to 210 mg, 140 mg to 210 mg, 150 mg to 210 mg, 70 mg to 200 mg, 75 mg to 200 mg, 100 mg to 200 mg, 110 mg to 200 mg, 120 mg to 200 mg, 130 mg to 200 mg, 140 mg to 200 mg, or 150 mg to 200 mg.

[0243] In some embodiments, a dose of 70 mg of RG6035 is administered to the subject. In some embodiments, a dose of 75 mg of RG6035 is administered to the subject. In some embodiments, a dose of 100 mg of RG6035 is administered to the subject. In some embodiments, a dose of 110 mg of RG6035 is administered to the subject. In some embodiments, a dose of 120 mg of RG6035 is administered to the subject. In some embodiments, a dose of 130 mg of RG6035 is administered to the subject. In some embodiments, a dose of 140 mg of RG6035 is administered to the subject. In some embodiments, a dose of 150 mg of RG6035 is administered to the subject. In some embodiments, a dose of 160 mg of RG6035 is administered to the subject. In some embodiments, a dose of 170 mg of RG6035 is administered to the subject. In some embodiments, a dose of 180 mg of RG6035 is administered to the subject. In some embodiments, a dose of 190 mg of RG6035 is administered to the subject. In some embodiments, a dose of 200 mg of RG6035 is administered to the subject. In some embodiments, a dose of 210 mg of RG6035 is administered to the subject. In some embodiments, a dose of 220 mg of RG6035 is administered to the subject. In some embodiments, a dose of 230 mg of RG6035 is administered to the subject. In some embodiments, a dose of 240 mg of RG6035 is administered to the subject. In some embodiments, a dose of 250 mg of RG6035 is administered to the subject. In some embodiments, a dose of 260 mg of RG6035 is administered to the subject. In some embodiments, a dose of 280 mg of RG6035 is administered to a relative of the subject. In some embodiments, a dose of 300 mg of RG6035 is administered to the subject.

[0244] Therapeutic applications

[0245] Achieving sufficient brain penetration while maintaining a safe therapeutic window is crucial for developing brain-targeting antibodies. To this end, RG6035, a huTfR1-guided brain shuttle and Fc region effector silencing bispecific modular fusion protein of obbituzumab, was designed. In head-to-head comparisons in human and mouse in vitro models, as well as in preclinical mouse and cynomolgus monkey models, RG6035 demonstrated superior safety compared to the Fc region effector-intact construct (BS-obbituzumab), while achieving higher brain exposure compared to "normal" non-TfR1 binding antibodies. The Fc region effector silencing PGLALA mutation prevents RG6035 from binding to the Fc γ receptor, thus inhibiting the activation of immune effector cells without affecting TfR1 and CD20 target binding. Compared to the Fc region effector-intact construct, RG6035 uptake and transcytosis were also maintained in TfR1-expressing cells. Furthermore, RG6035 exhibits potent direct B-cell death induction, demonstrated by rapidly and effectively reducing primary human B cells in human B-cell lymphoma cells, as well as in WBA, tonsil-derived cell cultures, and human CSF-cultured PBMCs. It must be noted that in assays evaluating direct B-cell killing in B-cell lymphomas lacking immune effector function, RG6035 demonstrated comparable efficacy to BS-obituzumab. In ex vivo CSF ​​assays, RG6035 depleted B cells as effectively as BS-obituzumab. Therefore, the data presented in this paper suggest that Fc region effector silencing mutations and the BS module do not impede CD20 target binding or direct B-cell killing activity, and that anti-CD20 targeting does not interfere with active brain uptake in RG6035.

[0246] TfR1 is widely expressed in multiple tissues, which may lead to systemic target-mediated drug disposal (TMDD) and safety implications due to TfR1 receptor binding at unwanted sites in the body. [30a, 31a] It has now been found that the potential safety concerns associated with peripheral targeting of TfR1 are mitigated in RG6035 [see also 25a].

[0247] IRR is an inherent risk of antibody-based therapies and represents a major obstacle to mAb clinical development. Therefore, IRR assessment and steps to reduce IRR are crucial for mAb clinical development. In RG6035, IRR and its effect on RBC precursors have been favorably affected by a specific design of RG6035 with Fc region effector function silenced, i.e., resolved, as shown in humanized CD20 mice. Decreased activity, reduced temperature, and cytokine release, all of which indicate that IRR, as well as reduced reticulocyte count, occurred only in mice treated with BS-obituzumab with intact Fc region effector function [see also 27a]. It must be noted that steric hindrance may attenuate Fc-γ receptor interactions, but only in cases where the brain restricts the therapeutic target, and may not hold true when both targets (e.g., CD20 and TfR1) are expressed peripherally. Interestingly, obbituzumab without the BS molecule did not induce IRR in transgenic mouse models, despite the fact that the molecule retains its effector function and typically exhibits IRR in humans.

[0248] In human whole-body brain tissue (WBA), BS-obituzumab with intact Fc region effector function indicates the release of pro-inflammatory cytokines, while this is not observed in RG6035 with silenced Fc region effector function. Importantly, RG6035 with silenced Fc region effector function still possesses B-cell cytotoxic properties due to type II B cell killing. Free from this theory, it is hypothesized that direct type II B cell killing plays a major role in the CNS, since a smaller number of effector cells exist in the brain. Furthermore, from a safety perspective, Fc region effector function-mediated effector activation and subsequent B-cell killing in the CNS via the same mechanism would induce the release of adverse cytokines.

[0249] Compared to non-shuttle anti-CD20 antibodies, RG6035 exhibits improved BBB penetration properties and can enter the CNS. In mouse pharmacokinetic studies, vascular uptake and brain penetration were demonstrated in vivo via enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Unbound by this theory, it is hypothesized that the improved brain penetration, combined with the effective Fc region effector function-independent extravascular B cell depletion through direct B cell killing, translates into clinical efficacy in MS patients by preventing or slowing clinical progression.

[0250] In cynomolgus monkey and transgenic mouse models, secondary lymphoid tissue containing B cell populations has been used as a substitute for models of refractory B cells, such as in MS. In single-dose PK / PD studies of huCD20 transgenic mice, no severe depletion of B cells in the blood, spleen, or lymph nodes was observed, but multiple-dose studies in primates showed B cell depletion in these organs. Free from this theory, it is hypothesized that huCD20 transgenic mice require multiple administrations of anti-CD20 antibodies to achieve severe B cell depletion compared to non-human primates. Possible reasons include the lower density of human CD20 on mouse B cells in huCD20 transgenic mice, or the higher affinity of the alternative anti-mouse TfR1 brain shuttle module in mice compared to the anti-human TfR1 brain shuttle module in cynomolgus monkeys, leading to increased TMDD via TfR1. In huCD20 mice, RG6035 cells with Fc region effector silencing have been shown to effectively deplete GC class-switching B cells, which have been described as an important component of MS pathophysiology. [33a]

[0251] The potency of RG6035 in depleting B cells was evaluated in cynomolgus monkeys. In a single-dose PK study, RG6035 was well tolerated and showed improved safety compared to BS-obbituzumab with intact Fc region effector function, based on clinical symptoms, cytokine levels, and clinicopathology. This supports in vitro and in vivo mouse data suggesting an increased risk of adverse events with BS constructs with intact Fc region effector function. Following administration of RG6035, B cell counts in the blood of cynomolgus monkeys decreased rapidly and strongly. Although all animals produced ADA independent of the Fc variant, immunogenicity in cynomolgus monkeys is known to be difficult to predict in humans. [34a]

[0252] Unbound by this theory, it is hypothesized that RG6035 will enter the ventricularized neuroinflammation of the brain and, through effective Fc area effector-independent extravascular B cell depletion, will produce better clinical efficacy in patients with MS, preventing or slowing clinical progression.

[0253] This article assumes that subjects requiring treatment with RG6035 will have a weight in the range of 70 kg to 75 kg to allow the indicated dose to be changed from mg to mg / kg, and vice versa.

[0254] Therefore, the present invention covers at least the following independent and dependent embodiments.

[0255] 1. RG6035, which is used to treat multiple sclerosis.

[0256] 1a.RG6035, which is used as a drug to treat multiple sclerosis.

[0257] 1b.RG6035 is used for the treatment of multiple sclerosis.

[0258] 2. Use of RG6035 in the manufacture of medicines for the treatment of multiple sclerosis.

[0259] 2a.RG6035 Use in the manufacture of medicaments for the treatment of multiple sclerosis.

[0260] 3.RG6035, which is used for depleted CD20-expressing isolated B cells in the brain.

[0261] 4. A method for treating an individual suffering from multiple sclerosis, the method comprising administering an effective amount of RG6035 to the individual.

[0262] 5. A method for depleting CD20-expressing circulating B cells, CD20-expressing brain-isolated B cells, or CD20-expressing B cells, or all of the aforementioned cells, in an individual, the method comprising administering an effective amount of RG6035 to the individual to deplete the CD20-expressing circulating B cells, CD20-expressing brain-isolated B cells, or CD20-expressing B cells, or all of the aforementioned B cells, in the CSF.

[0263] 6. The method according to Example 5, wherein the individual suffers from multiple sclerosis.

[0264] 7. A method for treating multiple sclerosis in a human, the method comprising administering to the human a therapeutically effective amount of RG6035, the RG6035 binding to human CD20 and depleting B cells, wherein the antibody is not conjugated to a cytotoxic agent.

[0265] 8. The method or use according to any one of Examples 1 to 7, wherein the multiple sclerosis is relapsing multiple sclerosis or progressive multiple sclerosis.

[0266] 9. The method or use according to any one of Examples 1 to 8, wherein the multiple sclerosis is primary progressive multiple sclerosis or secondary progressive multiple sclerosis.

[0267] 10. The method or use according to any one of Examples 1 to 9, wherein the multiple sclerosis is secondary progressive multiple sclerosis.

[0268] 11. The method or use according to any one of Examples 1 to 10, wherein RG6035 is administered at an effective dose to deplete more than 60%, more than 80%, or more than 95% of B cells.

[0269] 12. The method or use according to any one of Examples 1 to 10, wherein RG6035 is administered at an effective dose to achieve ≥60% or ≥80% or ≥95% B cell depletion.

[0270] 12a. The method or use according to any one of Examples 1 to 10, wherein RG6035 is administered at an effective dose for a prolonged period (at least 1 year) to achieve ≥ 95% B cell depletion.

[0271] 13. The method or use according to any one of Examples 11 to 12a, wherein the depletion is in the brain and / or CSF.

[0272] 14. The method or use according to any one of Examples 11 to 13, wherein the depletion occurs in the CSF.

[0273] 15. The method or use according to any one of Examples 11 to 14, wherein the depletion is achieved within 8 weeks after the start of application.

[0274] 16. The method or use according to any one of Examples 11 to 15, wherein the depletion is achieved within 6 weeks after the start of application.

[0275] 17. The method or use according to any one of Examples 11 to 16, wherein the depletion is achieved within 5 weeks after the start of application.

[0276] 18. The method or use according to any one of Examples 11 to 17, wherein the depletion is achieved within 4 weeks after the start of application.

[0277] 19. The method or use according to any one of Examples 1 to 18, wherein RG6035 is administered in a non-toxic dose to achieve a reduction in compartmentalized inflammation of the CNS.

[0278] 20. The method or use according to any one of Examples 1 to 19, wherein RG6035 is administered in a non-toxic dose to reduce the influx of B cells from the blood into the brain and to achieve direct local killing of B cells in the brain.

[0279] 21. The method or use according to any one of Examples 1 to 20, wherein RG6035 is administered in a non-toxic dose to achieve a slowing of disability progression.

[0280] 22. The method or use according to any one of Examples 1 to 21, wherein RG6035 is administered in a non-toxic dose to achieve a serum concentration of RG6035 in the cortex of 0.1% or higher, 0.5% or higher, 0.75% or higher, 0.85% or higher, 0.9% or higher, or 1% or higher.

[0281] 23. The method or use according to any one of Examples 1 to 22, wherein RG6035 is administered in a non-toxic dose to achieve a serum concentration of RG6035 in the CSF of 0.1% or higher, 0.5% or higher, 0.75% or higher, 0.85% or higher, 0.9% or higher, 1% or higher, 1.25% or higher, or 1.5% or higher.

[0282] 24. The method or use according to any one of Examples 1 to 22, wherein RG6035 is administered in a non-toxic dose to achieve serum concentrations of RG6035 of 0.1% or higher, 0.5% or higher, 0.75% or higher, 0.85% or higher, 0.9% or higher, 1% or higher, 1.25% or higher, or 1.5% or higher in different brain regions (excluding the meninges) and the CSF.

[0283] 25. The method or use according to any one of Examples 1 to 24, wherein RG6035 is administered in a non-toxic dose to achieve a concentration of RG6035 in the CSF of 0.006 µg / mL or higher, 0.025 µg / mL or higher, 0.035 µg / mL or higher, 0.045 µg / mL or higher, 0.055 µg / mL or higher, or 0.07 µg / mL or higher.

[0284] 26. The method or use according to any one of Examples 1 to 25, wherein RG6035 is administered in a non-toxic dose to achieve depletion of the resident B cell population in the brain.

[0285] 27. The method or use according to Example 26, wherein the depletion is achieved by inducing direct B cell death of immune-active B cells in a manner independent of Fc region effector function.

[0286] 28. The method or use according to any one of Examples 1 to 27, wherein the B cell is a CD19 positive B cell.

[0287] 29. The method or use according to any one of Examples 1 to 28, wherein RG6035 is administered in a second dose after more than 60% or more than 80% or more than 95% of B cells have been depleted from the CSF, in order to maintain the depletion of B cells in the CSF at the said level.

[0288] 29a. The method or use according to any one of Examples 1 to 28, wherein RG6035 is administered at a second dose for an extended period (at least 1 year) after more than 95% of B cells have been depleted from the CSF to maintain the depletion of B cells in the CSF at the stated level.

[0289] 30. The method or use according to any one of Examples 1 to 29a, wherein the B cell depletion is determined by comparison with the number, level or concentration of B cells prior to the first application of RG6035.

[0290] 31. The method or use according to any one of Examples 1 to 30, wherein RG6035 is administered as a single therapy.

[0291] 32. The method or use according to any one of Examples 1 to 30, wherein RG6035 is administered in combination with a second therapeutic agent for depleted B cells.

[0292] 33. The method or use according to Example 32, wherein the second therapeutic agent is applied once prior to the application of RG6035.

[0293] 34. The method or use according to Example 32, wherein the second therapeutic agent is administered simultaneously with RG6035.

[0294] 35. The method or use according to any one of Examples 1 to 34, wherein RG6035 is administered in an effective amount ranging from 75 mg to 300 mg, or 100 mg to 275 mg, or 125 mg to 250 mg, or 140 mg to 210 mg, or 150 mg to 200 mg per administration.

[0295] 36. The method or use according to any one of Examples 1 to 35, wherein RG6035 is administered in an effective amount of about 150 mg or about 200 mg per administration.

[0296] 37. The method or use according to any one of Examples 1 to 34, wherein RG6035 is administered in an effective amount relative to the said body weight in the range of 1 mg / kg to 4 mg / kg, or 1.25 mg / kg to 3.75 mg / kg, or 1.5 mg / kg to 3.25 mg / kg, or 1.75 mg / kg to 3 mg / kg, or 2 mg / kg to 2.85 mg / kg per administration.

[0297] 38. The method or use according to any one of Examples 1 to 34 and 37, wherein RG6035 is administered at an effective amount of about 2 mg / kg or about 2.85 mg / kg relative to the subject's body weight per administration.

[0298] 39. The method or use according to any one of Examples 1 to 38, wherein the effective amount is administered as a dose.

[0299] 40. The method or use according to any one of Examples 1 to 39, wherein the application is once a week, once every two weeks, or once every four weeks.

[0300] 41. The method or use according to any one of Examples 1 to 40, wherein the application is performed once every four weeks.

[0301] 42. The method or use according to any one of Examples 1 to 41, wherein the application is initiated once a week and changed to once every four weeks after more than 95% of the B cells have been depleted from the CSF, in order to maintain the depletion of B cells in the CSF at the said level.

[0302] 42a. The method or use according to any one of Examples 1 to 42, wherein the application is initiated once every two weeks and changed to once every four weeks after more than 95% of the B cells have been depleted from the CSF, in order to maintain the depletion of B cells in the CSF at the said level.

[0303] 43. The method or use according to any one of Examples 41 to 42a, wherein the application once a week or once every two weeks is for a maximum of eight applications.

[0304] 44. The method or use according to any one of Examples 41 to 43, wherein the application once a week or once every two holes is for a maximum of 6 applications.

[0305] 45. The method or use according to any one of Examples 41 to 44, wherein the application once a week or once every two weeks is for a maximum of 5 applications.

[0306] 46. ​​The method or use according to any one of Examples 41 to 45, wherein the application once a week or once every two weeks is for a maximum of four applications.

[0307] 47. The method or use according to any one of Examples 29 to 46, wherein the second dose is in the range of 20 mg to 300 mg, or 50 mg to 275 mg, or 75 mg to 250 mg, or 100 mg to 210 mg, or 150 mg to 200 mg or about 150 mg per administration.

[0308] 48. The method or use according to any one of Examples 29 to 46, wherein the second dose is in the range of 0.25 mg / kg to 4 mg / kg, or 0.5 mg / kg to 3.75 mg / kg, or 0.75 mg / kg to 3.25 mg / kg, or 1.00 mg / kg to 3 mg / kg, or 1.25 mg / kg to 2.75 mg / kg, or 1.50 mg / kg to 2.50 mg / kg, or about 2 mg / kg, relative to the subject's body weight per administration.

[0309] 49. The method or use according to any one of Examples 1 to 48, wherein the RG6035 is administered intravenously or subcutaneously.

[0310] 50. The method or use according to any one of Examples 1 to 48, wherein the RG6035 is applied subcutaneously.

[0311] Pharmacokinetic and pharmacodynamic experimental results

[0312] This article demonstrates that RG6035 improves BBB penetration and B cell killing. This has been confirmed using pharmacokinetic (PK) and pharmacodynamic (PD) studies in cynomolgus monkeys. To our knowledge, there are no known reliable disease models of B cell depletion in the brain, let alone using brain shuttle antibodies.

[0313] Serum Pharmacokinetics

[0314] In the first experiment, RG6035 was administered intravenously to cynomolgus monkeys to establish its pharmacokinetic / pharmacodynamic relationship in both cerebrospinal fluid and blood. RG6035 was found to be well-tolerated and showed efficacy in reaching the brain and depleting B cells in the blood and secondary lymphoid tissues.

[0315] Serum PK levels were quantified within 288 hours after a single IV administration of RG6035 to three groups (groups 3, 4, and 6, respectively) of RG6035 at doses of 0.3 mg / kg, 1 mg / kg, or 10 mg / kg. RG6035 exhibited nonlinear PK behavior attributable to target-mediated drug disposal (TMDD), with accelerated drug clearance at 0.3 mg / kg compared to 10 mg / kg. Serum NCA PK parameters for each animal were calculated and are shown in Table 6.

[0316] Table 6: Individual mean PK parameters of serum RG6035 after IV administration of 0.3 mg / kg, 1 mg / kg and 10 mg / kg RG6035 in cynomolgus monkeys; AUC 0-inf Area under the concentration-time curve from time 0 to infinity; C0, initial concentration or retrospective concentration after rapid IV injection; CL, clearance rate; C max Maximum observed concentration; IV, intravenous; PK, pharmacokinetics; V ss The volume distribution under steady-state conditions.

[0317]

[0318] Two-compartment models, including target-mediated treatment and immunogenicity-related clearance, have been found to effectively capture PK (see Figure 20). These are known for other molecules. [22,23]

[0319] At high doses, the estimated linear clearance was 0.818 mL / (h*kg), which is 3 to 4 times the expected clearance of human IgG antibodies in cynomolgus monkeys.

[23] For target-mediated treatment, assuming rapid binding to equilibrium and a constant target pool to fix KD to an in vitro measured CD20 value of 3.3 nM, the estimated binding capacity was approximately 400 pmol / kg.

[24]

[0320] Pharmacokinetics in CSF

[0321] CSF samples were collected via cerebellomedullary cistern and lumbar puncture at different time points and across dose groups. For each animal and for each collection, the ratio of RG6035 concentration in CSF measured via cerebellomedullary cistern to that measured via lumbar puncture was calculated, as shown in Table 7.

[0322] Table 7: Individual CSF concentrations of RG6035 after single IV administration of 0.3 mg / kg, 1 mg / kg and 10 mg / kg in cynomolgus monkeys; BLQ: below the limit of quantitation; CSF: cerebrospinal fluid; CSF-CM: CSF in the cerebellomedullary cistern; CS-LP: CSF in lumbar puncture.

[0323]

[0324] The ratio of RG6035 concentration in CSF samples taken from the cerebellomedullary cistern to those taken by lumbar puncture ranged from 0.2 to 1.1. PK parameters from CSF samples taken from the cerebellomedullary cistern were calculated based on composite curves for each dose group. Similar to serum, exposure to RG6035 in CSF increased slightly with dose at the 0.3 mg / kg, 1 mg / kg, and 10 mg / kg dose groups, with the area under the curve (AUC) increasing from zero to infinity. 0_inf The concentrations were 0.241, 2.19, and 35.7 h*µg / mL, respectively. The highest observed concentration (C) was... max The C value also increased proportionally with the dose, with the 0.3 mg / kg, 1 mg / kg, and 10 mg / kg dose groups showing the highest C values. max The concentrations were 0.0076, 0.0283, and 1.06 µg / mL, respectively. The CSF concentrations ranged from 0.13% to 3.4% of the serum concentrations.

[0325] It has been found that models consisting of single-compartment CSF compartments with inflow and first-order outflow rates proportional to serum concentration can capture data well (Figure 21).

[0326] The equilibrium CSF to serum concentration ratios in the lumbar vertebrae and cisterns were estimated to be 0.00537 and 0.00628, respectively. Assuming equal first-order efflux rates in both ventricles (and brain tissue), the estimated half-life was approximately 16 hours.

[0327] Pharmacokinetics in brain regions

[0328] Following intravenous administration of RG6035 at 10 mg / kg, RG6035 concentrations in different brain regions and tissues were measured at autopsy on day 3 (Group 2) or day 6 (Group 6). Using an exploratory approach, RG6035 concentrations in different brain regions were corrected for blood contamination based on results of blood tracers in serum and brain tissue at autopsy. Results of blood tracer analysis in serum and brain tissue showed that the concentration of the blood tracer in serum was two orders of magnitude higher than that in any brain tissue. This indicates that blood was effectively removed from most brain regions during brain perfusion at autopsy. It has been found that models consisting of single-compartment units, each with inflow and first-order outflow proportional to serum concentration, capture data well.

[0329] RG6035 uptake was confirmed in brain vascular endothelial cells and parenchyma, consistent with the expected TfR1-mediated transbasal transcytosis. The highest RG6035 concentration was detected in the meninges, while concentrations were comparable in all other brain tissues. Brain AUC exposures in different brain regions (excluding the meninges) and CSF ranged from 1% (cortical) and 0.8% to 1.5% of serum exposure (AUC), representing approximately 25-fold higher brain AUC at steady state compared to typical IgG (such as obbituzumab) (2.5-fold to 60-fold higher in different brain regions and CSF).

[0330] It has been found that the binding of RG6035 to TfR1 is expected to be similar in cynomolgus monkeys and humans. Therefore, significant species differences in net brain uptake are not anticipated. This invention is based, at least in part, on the assumption that the increased brain uptake observed in cynomolgus monkeys translates into a clinical condition.

[0331] Pharmacodynamics of B cells in blood

[0332] RG6035 induced a rapid, dose-dependent decrease in peripheral B cells as early as 4 hours after administration. Four phases of the PD response of hematopoietic B cells to RG6035 exposure were observed: an initial rapid decline (up to 85% exhaustion, without significant dose dependence), followed by a rebound (3 to 7 days after administration), then a dose-dependent sustained exhaustion phase (approximately 2 weeks after administration), with B cell counts eventually returning to baseline. Similar declines were observed in all B cell subsets (initial, non-converted memory, and CD27+ converted memory B cells), and appeared to be more pronounced and persistent in the high-dose group and the memory population (unconverted and CD27+ converted B cells).

[0333] This invention is based, at least in part, on the finding that a PK / PD model that combines concentration-driven B cell depletion with redistribution between two exchange pools of B cells can well capture B cell dynamics (see Figure 22). In this model, the observed B cells are part of a blood pool that receives B cells from a hidden, tissue-resident B cell pool.

[0334] Based on this model, a stable concentration of 0.0758 µg / mL is estimated to be sufficient to deplete 50% of B cells. The half-life of the tissue-resident B cell pool is estimated to be approximately 18 days, consistent with previously published observations. [25-28] Based on this model, it has been found that the B cell depletion effect of RG6035 in the blood is delayed relative to depletion in the main pool. This delayed half-life is approximately 3 days for memory B cells and approximately 8 days for naive B cells, resulting in different PD curves for these cell types.

[0335] The brief, dose-independent initial depletion with a half-life of 1.1 days adds complexity to PD. Unbound by this theory, it is assumed to be caused by a redistribution phenomenon, but this has no significant pharmacological implications. The parameter estimates for this model have been found to be largely robust.

[0336] Pharmacodynamics of peripheral tissues

[0337] The relative percentage of B cells in the spleen, submandibular lymph nodes, and tonsils decreased 48 hours after RG6035 induction administration, and in the mesenteric lymph nodes decreased 120 hours after administration (Figure 23 and ·). Figure 24 However, it had no significant effect on the relative percentages of total T cells and natural killer (NK) cells.

[0338] Efficient and durable B cell depletion in surrogate organs

[0339] The staining patterns of the four tissues assessed (spleen, tonsils, cervical lymph nodes, and mandibular lymph nodes) were similar. One animal in group 2 was missing its tonsils. One animal in both groups 1 and 6 was missing its cervical lymph nodes. Due to the highly nonspecific background, no further analysis was performed on the mandibular lymph node samples from group 2. The staining patterns of CD19 and CD20 on serial sections were qualitatively very similar (Fig. 23). Both markers labeled the total B cell population: for further staining, the B cell population was identified using an anti-CD19 antibody, which was found to be equivalent in serial sections of tonsils, cervical lymph nodes, and mandibular lymph nodes across all treatment groups. Compared to the vector-treated animals, the RG6035-treated animals showed a decrease in the percentage of CD19+-B cells and the percentage per tissue area in all lymphoid tissues studied, without further significant decreases between different time points (Fig. 23A to Fig. 23D; Table 8; Fig. 25).

[0340] Table 8: CD19+-B cells in all organs and total stained cells.

[0341]

[0342] Human body prediction

[0343] This invention is based, at least in part, on the finding that human PK / PD predictions show ≥95% B cell depletion after 4 weeks. Figure 26 shows a simulation of PK, brain uptake, blood B cells, and CSF B cells after four months of subcutaneous injection of 150 mg RG6035.

[0344] Blood B cells showed a gradual, cumulative decrease during administration and congestion after administration cessation. The kinetics of CSF integrate both i) the reduction of B cells in the brain due to decreased blood inflow, and ii) the transport of RG6035 to brain tissue mediated by TfR1 and enhanced local depletion through direct local cell killing. Unbound by this theory, it is hypothesized that the effects of direct cell killing should lead to a deeper and more rapid depletion of B cells in CSF than a purely passive depletion driven solely by blood depletion.

[0345] ****

[0346] Examples, sequences, and drawings are provided to aid in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the described procedures without departing from the spirit of the invention.

[0347] Quotation

[0348] All documents cited in this article are explicitly incorporated herein by reference.

[0349] 1. Compston, A. and Coles, A., The Lancet 372 (2008) 1502-1517.

[0350] 2. Reich, DS et al., Multiple Sclerosis. 378 (2018) 169-180.

[0351] 2a. Niewoehner, J. et al., Neuron 81 (2014) 49-60.

[0352] 3. Wallin, MT et al., Neurol. 92 (2019) e1029-e1040.

[0353] 4. Cencioni, MT et al., Nat. Rev. Neurol. 17 (2021) 399-414.

[0354] 5. Lee, DSW et al., Nat. Rev. Drug Discov. 20 (2021) 179-199.

[0355] 6. Obermeier, B. et al., Nat. Med. 14 (2008) 688-693.

[0356] 7. Serafini, B. et al., Brain Pathol. 14 (2004) 164-174.

[0357] 8. Magliozzi, R. et al., Brain 130 (2007) 1089-1104.

[0358] 8a. Wallin, MT et al., Neurol. 92 (2019) e1029-e1040.

[0359] 9.Lucchinetti, CF, New Engl. J. Med. 365 (2011) 2188-2197.

[0360] 9a. Furuzawa-Carballeda, J. et al., Autoimmun. Rev. 6 (2007) 169-175.

[0361] 10. Obermeier, B. et al., Nat. Med. 19 (2013) 1584-1596.

[0362] 10a. Sospedra, M. and Martin, R., Annu. Rev. Immunol. 23 (2005) 683-747.

[0363] 11. Abbott, NJ et al., Neurobiol. Dis. 37 (2010) 13-25.

[0364] 11a. Chisari, CG et al., J. Neurol., 8 (2021) 1-25.

[0365] 12. Daneman, R. and Prat, A., Cold Spring Harbor perspectives inbiology 7 (2015) a020412.

[0366] 12a. Gasperi, C. et al., Dis. Manag. 6 (2016) 37-47.

[0367] 13. Freskgård, PO and Urich, E., Neuropharmacol. 120 (2017) 38-55.

[0368] 13a. Gelfand, JM et al., Neurotherap. 14 (2017) 835-841.

[0369] 14. Johnsen, KB et al., Prog. Neurobiol. 181 (2019) 101665.

[0370] 14a. Greenfield, AL and Hauser, SL, Ann. Neurol. 83 (2018) 13-26.

[0371] 15. Klein, C. et al., MAbs 5 (2013) 22-33.

[0372] 15a. Jakimovski, D. et al., Expert Opin. Biol. Ther. 17 (2017) 1163-1172.

[0373] 16. Al-Sawaf, O. et al., Drug Des. Devel. Ther. 11 (2017) 295-304.

[0374] 16a. Montalban, X. et al., N. Eng. J. Med. 376 (2017) 209-220.

[0375] 17. Herter, S. et al., Mol. Canc. Ther. 12 (2013) 2031-2042.

[0376] 17a. Sabatino, JJJr. et al., Cold Spring Harb. Perspect. Med. 9 (2019)a032037.

[0377] 18. Kern, DJ et al., Leukem. Lymph. 54 (2013) 2500-2505.

[0378] 18a. Howell, OW et al., Brain 134 (2011) 2755-2771.

[0379] 19. Lim, SH et al., Blood 118 (2011) 2530-2540.

[0380] 19a.Reali, C. et al., Brain Pathol. 30 (2020) 779-793.

[0381] 20. Honeychurch, J. et al., Blood 119 (2012) 3523-3533.

[0382] 20a.Florou, D. et al., Brain Sci. 10 (2020) 758.

[0383] 21. Niederfellner, G. et al., Blood 118 (2011) 358-367.

[0384] 22. Dirks, NL and Meibohm, B., Clin. Pharmacokinet. 49 (2010) 633-659.

[0385] 22a. Klein, C. et al., mAbs 5 (2013) 22-33.

[0386] 23. Wang, J. et al., Biopharm. Drug Dispos. 37 (2016) 51-65.

[0387] 23a. Tobinai, K. et al., Adv. Ther. 34 (2017) 324-356.

[0388] 24. Gibiansky, L. et al., J. Pharmacokinet. Pharmacodyn. 35 (2008) 573-591.

[0389] 24a. Herter, S. et al., Haematologica 103 (2018) e78-e81.

[0390] 25. Macallan, DC et al., Blood 105 (2005) 3633-3640.

[0391] 25a.Couch, J, A et al., Sci. Transl. Med. 5 (2013) 183ra157.

[0392] 26. De Boer, RJ et al., J. Immunol. 170 (2003) 2479-2487.

[0393] 27. Pan, S. et al., Br. J. Clin. Pharmacol. 85 (´2019) 1790-1797.

[0394] 27a. Weber, F. et al., Cell Rep. 22 (2018) 149-162.

[0395] 28. Grimm, HP et al., J. Pharm. Sci. 108 (2019) 3729-3736.

[0396] 28a. Bessa, J. et al., Pharm. Res. 32 (2015) 2344-2359.

[0397] 29. Howell, OW et al., Brain 134 (2011) 2755-2771.

[0398] 29a. Leon, LR, Front. Biosci. 9 (2004) 1877-1888.

[0399] 30. Bell, L. et al., Front. Immunol. 10 (2020) 3090.

[0400] 30a. Candelaria, PV et al., Front. Immunol. 12 (2021) 607-692.

[0401] 31. Reali, C. et al., Brain Pathol. 30 (2020) 779-793.

[0402] 31a. Neiveyans, M. et al., mAbs 11 (2019) 593-605.

[0403] 32.Bittner, S. and Wiendl, H., Neurotherapeutic. 13 (2016) 4-19.

[0404] 33.Sabatino, JJ et al., Proc. Natl. Acad. Sci. 116 (2019) 25800-25807.

[0405] 33a.Palanichamy, A. et al., Sci. Transl. Med. 6 (2014) 248ra106-248ra106.

[0406] 34. Brell, J. et al., Commun. Biol. 3 (2020) 621.

[0407] 34a. van Meer, PJK et al., mAbs 5 (2013) 810-816.

[0408] 35. Trenor, CC, 3rd et al., Blood 96 (2000) 1113-1118.

[0409] 35a. Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-681.

[0410] 36. Jabara, HH et al., Nat. Gen. 48 (2016) 74-78.

[0411] 36a. Klein, C. et al., mAbs 8 (2016) 1010-1020.

[0412] 37. Pulgar, VM, Front. Neurosci. 12 (2019) 1019-1019.

[0413] 37a. Schaefer, W. et al., Proc. Natl. Acad. Sci. 108 (2011) 11187.

[0414] 38. Schumacher, V. et al., unpublished results.

[0415] 38a. Schlothauer, T. et al., Protein Eng. Des. Sel. 29 (2016) 457-466.

[0416] 39. Bell, L. et al., Histochem. Cell Biol. 151 (2019) 489-499.

[0417] 40. Sefia, E. et al., Mult. Sclerosis Rel. Disord. 14 (2017) 46-50.

[0418] 40a. Clavarino, G. et al., PLoS One 11 (2016) e0162209.

[0419] 41. Anthony, IC et al., Brain 126 (Pt 5, 2003) 1058-1067.

[0420] 41a. Gong, Q. et al., J. Immunol. 174 (2005) 817.

[0421] 42. Bischoff, T. et al., Cytomet. A 79 (2011) 436-445.

[0422] 43. Pieper, K. et al., J. Allergy Clin. Immunol. 131 (2013) 959-971.

[0423] 44. Cross, A. et al., Neurol. 92 (15 Supplement, 2019) S56.008.

[0424] 45. Syková, E., Neurochem. Int. 45 (2004) 453-466.

[0425] 46. ​​Comi, G. et al., Ann. Neurol. 89 (2021) 13–23.

[0426] 47. Bjornevik, K. et al., Science 375 (2022) 296-301.

[0427] 48. Moessner, E. et al., Blood 3 (2010) 4393-4402, 17. Attached Figure Description

[0428] Figure 1: Prediction of B cell depletion based on multiple subcutaneous doses (once a week for four weeks).

[0429] Figure 2: Binding of human B-cell lymphoma cell lines, such as RG6035, obbituzumab-PGLALA, and rituximab, to CD20 as measured by flow cytometry. Detection was performed using a fluorescein isothiocyanate-labeled F(ab)'2 goat anti-human Fcγ specific antibody, and median fluorescence intensity (MFI) was measured.

[0430] Figure 3: Percentage of annexin V and PI double-positive B-cell lymphoma cells as determined by flow cytometry after 24 hours of incubation with different concentrations (0.01 nM to 1000 nM) of RG6035, obbituzumab-PGLALA, or rituximab.

[0431] Figure 4: Internalization of RG6035, obituzumab-PGLALA, and BS-DP47-PGLALA mediated by human TfR in MDCKII-TfR and MDCKII parental cells.

[0432] Figure 5: Time course of transcytosis efficiency: Comparison between BS-Orbituzumab, RG6035 and BS-DP47-PGLALA

[0433] Figure 6: FACS analysis of B cell exhaustion in response to treatment with mBS-obituzumab mAb; the frequency of remaining B220+-B cells in the blood was calculated by setting the percentage of B220+-B cells at baseline to 100%; data points represent mean ± SD blood B cell exhaustion measured in 5 mice across 3 groups at a dose of 0.6 mg / kg.

[0434] Figure 7: FACS analysis of B cell exhaustion in response to treatment with mBS-obituzumab mAb; the frequency of remaining B220+-B cells in the blood was calculated by setting the percentage of B220+-B cells at baseline to 100%; data points represent mean ± SD blood B cell exhaustion measured in 5 mice across 3 groups at a dose of 1.3 mg / kg.

[0435] Figure 8: FACS analysis of B cell exhaustion in response to treatment with mBS-obituzumab mAb; the frequency of remaining B220+-B cells in the blood was calculated by setting the percentage of B220+-B cells at baseline to 100%; data points represent mean ± SD blood B cell exhaustion measured in 5 mice across 3 groups at a dose of 13.3 mg / kg.

[0436] Figure 9: FACS analysis of B cell depletion in response to treatment with mBS-obituzumab antibody; the percentage of remaining B220+-B cells in lymph nodes was calculated by setting the frequency of B220+-B cells in the vector-treated animals (N=5) to 100%; data represent mean ± SD measurements for each group of N=5 mice; Day 3.

[0437] Figure 10: FACS analysis of B cell depletion in response to treatment with mBS-obituzumab antibody; the percentage of remaining B220+-B cells in lymph nodes was calculated by setting the frequency of B220+-B cells in the vector-treated animals (N=5) to 100%; data represent mean ± SD measurements for each group of N=5 mice; day 8.

[0438] Figure 11: FACS analysis of B cell depletion in response to treatment with mBS-obituzumab antibody; the percentage of remaining B220+-B cells in lymph nodes was calculated by setting the frequency of B220+-B cells in the vector-treated animals (N=5) to 100%; data represent mean ± SD measurements for each group of N=5 mice; day 22.

[0439] Figure 12: FACS analysis of B cell depletion in response to treatment with mBS-obituzumab antibody; the percentage of remaining B220+-B cells in the spleen was calculated by setting the frequency of B220+-B cells in the mediator-treated animals (N=5) to 100%; data represent mean ± SD measurements for each group of N=5 mice; Day 3.

[0440] Figure 13: FACS analysis of B cell depletion in response to treatment with mBS-obituzumab antibody; the percentage of remaining B220+-B cells in the spleen was calculated by setting the frequency of B220+-B cells in the mediator-treated animals (N=5) to 100%; data represent mean ± SD measurements for each group of N=5 mice; day 8.

[0441] Figure 14: FACS analysis of B cell depletion in response to treatment with mBS-obituzumab antibody; the percentage of remaining B220+-B cells in the spleen was calculated by setting the frequency of B220+-B cells in the mediator-treated animals (N=5) to 100%; data represent mean ± SD measurements for each group of N=5 mice; day 22.

[0442] Figure 15: B cell depletion after antibody administration in cynomolgus monkeys; the number of B cells after antibody administration is represented by the percentage of B cells measured at baseline (100%); data represent the average measurement for N=4 animals.

[0443] Figure 16: Percentage of positive cell types in cervical lymph nodes normalized to tissue area. Bars marked with "*" represent p < 0.05 compared to the mediator control.

[0444] Figure 17: Percentage of positive cell types in the spleen normalized to tissue area. Bars marked with "*" represent p < 0.05 compared to the control group.

[0445] Figure 18: Percentage of positive cell types in the mandibular lymph nodes normalized to tissue area. Bars marked with "*" represent p < 0.05 compared to the mediator control.

[0446] Figure 19: Percentage of positive cell types in tonsils normalized to tissue area. Bars marked with "*" represent p < 0.05 compared to the mediator control.

[0447] Figure 20: PK model of cynomolgus monkeys using target-mediated drug treatment and additional time-dependent clearance to capture the effect of immunogenicity; observations are shown as circles above 1E-4, and observations below the quantitation limit are shown as 1E-1, with each model simulation shown as a line; the superposition of selected animals at 0.3 mg / kg (top row), 1 mg / kg (middle row), and 10 mg / kg (bottom row) is shown.

[0448] Figure 21: The brain distribution of RG6035 in cynomolgus monkeys was modeled as a single chamber, with uptake from circulating compounds in the blood and first-order efflux; observations are shown as circles above 1E-4, and observations below the quantitation limit are shown as circles at 1E-4, with each model simulation shown as a line; the superposition of lumbar CSF in the selected animals at 0.3 mg / kg (top row), 1 mg / kg (middle row), and 10 mg / kg (bottom row) is shown.

[0449] Figure 22: PK / PD model of the effect of RG6035 on blood B cells in cynomolgus monkeys; observations are shown as circles above 1E1, and observations below the quantitative limit are shown as circles at 1E1. The simulations of each model are shown as lines, and the baseline estimates are shown as cyan dashed lines; the superposition of memory B cells switched in the selected animals at 0.3 mg / kg (top row), 1 mg / kg (middle row), and 10 mg / kg (bottom row) is shown.

[0450] Figure 23: Depletion of effective and persistent subsets of B cells in lymphoid organs; A: Representative overview images (A–C) and insets (a–c) of the spleen and corresponding mandibular lymph nodes in animals treated with the medium (A, a) and RG6035 after staining with anti-CD19 antibody (green) for 48 hours (B, b) and 120 hours (C, c), showing a significant reduction in B cells; DAPI (blue) shows the cell nuclei.

[0451] Figure 24: Quantification of B cells in lymphoid organs by cell segmentation and subsequent CD19+ cell thresholding.

[0452] Figure 25: Representative images of equivalents of CD19 (green; A, a) and CD20 (reference; B, b) in the tonsils, along with nuclear staining (DAPI, blue); staining with anti-CD20 and anti-CD19 antibodies on serial sections shows that the staining patterns in the lymphoid tissues studied are very similar; therefore, for the B cell subset, characterization of CD19 is used to identify the B cell population.

[0453] Figure 26: Human PK / PD predictions after 4 months of subcutaneous (SC) injection of RG6035; Bottom right panel: Effects of B cells in CSF, TfR-mediated brain uptake, and direct B cell killing by RG6035 (linear line) and passive / non-active transport resulting from reduced B cell migration from blood to brain (dashed line).

[0454] Figure 27: Nuclear segmentation (B, b) based on DAPI channels (A, a), including the definition of cytoplasmic and membrane compartments around the nucleus (1 µm around the nucleus); thresholding of CD19 (green; C, c) is adjusted based on the minimum intensity of the mediator animal found in the cytoplasmic / membrane compartment, resulting in CD19+ cells depicted in yellow (D, d).

[0455] Figure 28: Structure of a complete model used to predict PK / PD of RG6035 in humans; PK (purple box) is controlled by a two-compartment model with target-mediated treatment, represented by a central compartment (serum) and a peripheral compartment; optionally, subcutaneous absorption can be modeled as a first-order process; precipitation absorption is described by a linear distribution process; it is assumed that the distribution within brain tissue is limited to interstitial spaces; B cells (blue box) exchange between the blood and a “hidden pool” (“tissue”); the latter maintains a balance between proliferation and loss in the absence of drug intervention; RG6035 increases B cell loss by killing cells in the “tissue” pool; B cells in the brain are either derived from the blood or from local proliferation and are lost by returning to the blood or cell death; local cytotoxicity is driven by the interstitial concentration of RG6035 in the brain; PD: pharmacodynamics; PK: pharmacokinetics.

[0456] Sequence Description

[0457] SEQ ID NO: 01RG6035 Light chain 1.

[0458] SEQ ID NO: 02RG6035 Heavy chain 1.

[0459] SEQ ID NO: 03RG6035 Light chain 2.

[0460] SEQ ID NO: 04 RG6035 FAB fragment

[0461] SEQ ID NO: 05 RG6035 heavy chain 2

[0462] SEQ ID NO: 06 Human CD20.

[0463] Experimental section

[0464] To demonstrate the favorable safety and efficacy characteristics of RG6035, the safety and efficacy profiles of BS were directly compared with those of a control antibody in preclinical models.

[0465] Antibody and bispecific antibody production

[0466] The BS construct was engineered by fusing an anti-huTfR1 cross-Fab (human; clone 1026) to the C-terminus of the heavy chain of obinutuzumab or an obinutuzumab-PGLALA variant. [36a, 38a] The "stub-into-hole" technique [35a] was used to facilitate heterodimer pairing of the heavy chain carrying the BS module with the non-fused heavy chain. The CrossMab technique [36a, 37a] was used to avoid mispairing of different light chains on the antibody complex. In the same manner, the huTfR1 positive control reference conjugate 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 murine 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 an obinutuzumab-PGLALA variant, again using the "stub-into-hole" technique. A glycine-serine peptide linker was used to construct the single-chain Fab fragment.

[0467] The genes of the different chains of the different antibodies were cloned into an expression cassette that contains regulatory components for gene expression comprising a CMV promoter, a bGH poly A signal, and an hGH transcription terminator. The expression cassette was cloned into an expression vector. According to the manufacturer's protocol, Expi293 TM or ExpiCHO-S TM cells (ThermoFisher Scientific; A14527 and A29127, respectively) were co-transfected with the appropriate vector combination and the antibodies were purified from the cell culture supernatant as previously described [2a].

[0468] CD20 binding

[0469] Cells were incubated with the test antibody and subjected to FACS analysis to assess CD20 binding.

[0470] Human B-cell lymphoma cells were maintained at a concentration of 0.3 to 0.9 * 1E6 cells / mL in a humidified incubator at 37°C and 5% CO2, using a solution containing 10% fetal bovine serum (FCS; Gibco 16140) and 1% (v / v) 2 mM N-acetyl-L-alanyl-L-glutamine (GlutaMAX). TM Cells were cultured in RPMI 1640 medium (ThermoFisher, 11875093) at ThermoFisher (35050061). Harvested cells (98.2% viability) were centrifuged (4 min, 400 × g) to pellet and resuspended at approximately 0.6 × 1E6 viable cells / mL in fluorescence activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) containing 2% (v / v) FCS, 5 nM EDTA, and 0.25% sodium azide). Cells were seeded at 1 × 1E5 cells / well in 96-well U-shaped plates. The test antibody was added to the cells at 4°C for 30 minutes to a final concentration ranging from 1000 nM to 0.0128 nM (1:5 dilution, triplicate). 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) at 4°C for 30 min. Cells were then washed twice with FACS buffer and fixed with 2% paraformaldehyde in FACS buffer before FACS acquisition (BD FACS CantoII flow cytometer). FACS gating was performed using FACS Diva software to determine median fluorescence intensity. EC50 values ​​were calculated using GraphPad Prism based on S-shaped dose-response (variable slope) analysis. No further statistical analysis was performed.

[0471] Cell death induction

[0472] Human B-cell lymphoma cells were incubated with test antibodies (0.0128 nM to 1000 nM) for 24 hours, and cell death was then measured by assessing annexin V (annV) expression and propidium iodide (PI) uptake.

[0473] Human B-cell lymphoma cells were seeded at 1*1E5 cells per well in 96-well U-shaped plates. Test antibodies (0.0128 nM to 1000 nM, triplicate) were incubated with the cells at 37°C for 24 hours. The cells were then resuspended and centrifuged (400×g for 4 minutes), followed by washing with annV binding buffer (BB; 10 mM HEPES / NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). The final cell pellet was resuspended in annV-FLUOS (Roche Diagnostics GmbH, Mannheim, Germany; 11828681001; annV BB 1:100) and incubated at room temperature in the dark for 15 minutes. Cells were then washed with annV BB and then added with annV BB containing PI (Merck, P4864; 1:4000). Signal acquisition was performed immediately after PI addition using a BD FACS CantoII flow cytometer. FACS gating was performed using BD FACS Diva software to determine the percentage of annV / PI double-positive cells. No further statistical analysis was performed.

[0474] TfR-mediated cellular internalization and transcytosis

[0475] 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 humidified incubator with 5% CO2. Cells were seeded at 7.5*1E4 cells per well in 24-well plates and transiently transfected 24 hours post-seeding. For transient transfection of MDCKII cells, Opti-MEM was used... TM The vector DNA mixture was prepared at a concentration of 0.02 μg / μL in low serum medium (ThermoFisher, 31985070), and 0.5 μg DNA / 25 μL volume was transferred per well (i.e., the total amount of DNA transfected per 24 wells). Lipofectamine TM The 2000 reagent (ThermoFisher, 11668027) was also diluted in Opti-MEM low-serum medium to achieve a transfer volume of 1.5 μL of Lipofectamine per well. TM A concentration of 25 μL was prepared separately using diluted DNA and Lipofectamine. TMThe reagents were mixed in a 1:1 ratio and incubated at room temperature for 10 minutes to allow the formation of DNA / Lipofectamine. TM The transfection complex was carefully transferred into each well after incubation. Parallel simulated transfection was included, in which the same transfection procedure was performed, but without vector DNA.

[0476] MDCKII cells were transiently transfected with huTfR 24 hours prior to the transcytosis assay. HuTfR-mediated internalization (uptake) and transcytosis (pulse-tracking) assays were performed 24 hours post-transfection; each treatment condition was evaluated in triplicate. The assay buffer (AB) used consisted of Hanks balanced salt solution (Mg++ / Ca++; ThermoFisher, 14065072) supplemented with 20 mM HEPES (ThermoFisher, #15630056), pH 7.4. All assay incubation steps were performed at 37°C and 5% CO2 without shaking. Before the start of the pulse phase (uptake / cell loading), cells were washed twice with AB, and 0.2 mL of AB containing the test antibody was added to each well. The antibody was incubated with cells at the specified time points (30 or 45 minutes) according to the experiment. At the end of incubation, the added AB was immediately aspirated to stop uptake, followed by two rapid washes with AB containing 0.1% (v / v) bovine serum albumin (BSA), and a final wash with BSA-free AB to remove any residual protein. The pulsed step was performed in duplicate plates; one plate was immediately lysed to represent the intracellular compartment IgG content at the start of the tracking phase (time 0), and the other plate was evaluated at multiple time points. After the final wash (as described above), the cells were incubated with preheated AB (37°C) to begin tracking. AB (extracellular compartment) samples were then collected at the end of each time point (5, 10, and 30 minutes). At the final time point, the cells were washed three times as described in the pulsed phase, then lysed and the intracellular IgG content was determined.

[0477] To measure intracellular IgG levels, cells were lysed in RIPA lysis and extraction buffer (ThermoFisher Scientific, 89900) containing a protease inhibitor (Sigma Aldrich, 11697498001) and incubated at 4°C for 30 minutes (with frequent stirring). The dissolved 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 follow-up phase were immediately diluted in 96-well LoBind deep-well plates with assay buffer. Pierce was used... TM BCA protein assay (ThermoFisher, 23225) determined the total cellular protein content in cell lysates according to the manufacturer's protocol. IgG content was quantitatively assessed using a universal IgG ELISA based on chemiluminescence detection.

[0478] B cell depletion in human CSF

[0479] Human PBMCs and human CSF were used as the substrates for the B cell exhaustion assay. PBMCs from five healthy donors across all age groups and CSF from twelve individual donors (female and male) were obtained from Discovery Life Sciences in Huntsville, USA. CSF samples were thawed once, pooled, aliquoted, and stored at -80°C until needed. PBMCs were thawed in RPMI (containing 10% (v / v) FCS, 1% penicillin / streptomycin (PS), and 1% non-essential amino acids (NEAA)) and equilibrated at 37°C and 5% CO2 for at least 2 hours. Approximately 2*1E6 cells / mL (2*1E5 cells per well) were seeded into 96-well plates. After incubation for 2 hours, the plates were centrifuged at 300×g for 5 minutes at room temperature. The cells were then resuspended in 100 μL of human CSF.

[0480] The treatment antibodies were diluted and added to PBMCs in CSF in the range of 0.002 nM to 100 nM for 22 hours. The cell pellet was then FACS stained with the following antibodies: anti-CD45 antibody (APC, F20; BDBiosciences, 560973), anti-CD19 antibody (PE, F20; BioLegend, 982402), and anti-CD3 antibody (PE / Cy7, F20; BioLegend, 300419). The staining was performed using LIVE / DEAD. TMFixable Aqua Dead Cell StainKit (ThermoFisher, L34957), Zombie Aqua TM Cell viability was measured using the Fixable Viability Kit (BioLegend, 423101) and the F1000 BioLegend. In volume-based acquisition, cell viability was measured using BD Biosciences FACSLyric software with FACS Suite. TM Samples were measured by flow cytometry. Raw FACS data were analyzed using FlowJo software. B cell exhaustion was calculated using the B cell / T cell ratio of the untreated control versus the B cell / T cell ratio of the samples. Statistical evaluation was obtained using GraphPad Prism. The statistical significance of the differences in maximum CD19+-B cell exhaustion between different antibodies was calculated using one-way ANOVA (multiple comparisons).

[0481] B cell depletion in human tonsil-derived cells

[0482] Freshly excised tonsil tissue was obtained from adult donors who underwent routine tonsillectomy at the Hirslanden Clinic Muenchenstein Birshof in Switzerland. Written consent was obtained from each patient-derived sample. Following collection, the tissue was stored in Hanks' balanced salt solution (Mg++ / Ca++, ThermoFisher, 14065-072) at 4°C. Subsequently, the tonsil tissue was mechanically and enzymatically digested to obtain a cell suspension, which was used directly for B cell depletion assays. Concentration dependence and B cell subset depletion were determined as described below.

[0483] Total B cell depletion

[0484] B cells were transferred to 96-well U-plates in RPMI medium (containing 10% FBS, 1% PS, and 1% NEAA at a concentration of 2.5 x 1E5 cells / well). Treatment antibodies were diluted and added in the range of 0.03 nM to 100 nM. Further experimental conditions followed B cell depletion in human CSF (as described above).

[0485] Depletion of B cell subsets

[0486] B cells were transferred to 96-well U-plates (1*1E6 cells / well). Treatment antibodies were diluted and added at 1 nM to 100 nM, and incubated at 37°C, 5% CO2 for 8 hours. Cell pelleting was then performed and FACS staining was performed. Live / dead cells were then used. TM and Zombie Aqua TM The kit, the antibodies listed in Table 9, and BDHorizon TM Cell viability was measured using Brilliant staining buffer (BD Biosciences, 563794). FACSDiva was used. TM Software (BD Biosciences) in FACSymphony TM or FACS LSRFortessa TM The samples were analyzed. Phenotypic identification of a subset of B cells was performed based on previously described surface marker expression and staining strategies [40a].

[0487] Table 9: Antibodies used for B cell depletion and human whole blood assays.

[0488]

[0489] Lymphocytes were identified based on size (forward scattering and granular lateral scattering) and single-cell gating. After live-to-dead differentiation, live CD45+ lymphocytes were further gated into CD3− and CD3+ cells. B cell subset exhaustion was then normalized using CD3+-T cells. CD19 positivity was used to identify the total B cells of interest. The CD19+-B cell subset was further identified based on surface marker expression. Therefore, the distinct expression of CD38 and CD10 on CD19+-B cells served as an initial identifying factor for the three major B cell populations.

[0490] Plasma cells express the highest levels of CD38 but not CD10 (CD38+++ / CD10−), while CD38-low and CD10−CD19+-B cells produce CD27+ memory cells and CD27− naive B cells. CD38-intermediate (CD38++) and CD10+CD19+-B cells can further differentiate into GC B cells, transitional B cells, and immature B cells. CD5 expression is upregulated during the transformation of immature bone marrow-derived B cells into peripheral blood transitional cells. Therefore, CD38++CD10+CD5+ cells are classified as transitional B cells. Furthermore, immature B cells have been distinguished from GC B cells based on their surface IgM expression. IgD−CD38++CD10+ cells are considered GC B cells, while IgD− but IgM+CD38++CD10+ cells are considered immature B cells.

[0491] Human whole blood assay (WBA)

[0492] Fresh, undiluted human blood samples from six healthy individuals were incubated for 24 hours with test and control antibodies at concentrations of 0.01, 0.1, 1.0, 10, 100, 500, and 1000 nM (Table 9). Cytokine release (IL-1β; IL-6; IL-8; TNF-α, and IFN-γ) and B cell depletion (at concentrations of 0.01, 1.0, 100, and 1000 nM only) were then measured.

[0493] Venous blood from six healthy donors was collected in vacuum blood collection tubes containing lithium heparin as an anticoagulant (Roche Medical Center, Basel, Switzerland) and stored at room temperature until the start of the assay (1 to 3 hours to avoid erythrocyte lysis). Antibodies were incubated with the blood samples in U-bottom 96-well plates (1:40); antibody concentrations of RG6035, BS-obbituzumab, obbituzumab, and obbituzumab-PGLALA ranged from 0.01 to 1000 nM (see above), and the negative comparator Erbitux was used. (R) (Final concentration 100 µg / ml) and positive comparator Lemtrada (R) (Final concentration 100 µg / ml). The tested 1000 nM compound concentrations covered the predicted human C... maxConcentration (maximum serum concentration). These conditions ensure optimal performance in terms of practicality and efficiency, obtaining at least 70 μL of plasma and sufficient cells for multi-cytokine analysis. Endogenous activation and reactivity of blood cells were assessed using controls including phosphate-buffered saline, a mediator, or lipopolysaccharide. After incubation at 37°C for 24 hours, cells and plasma were separated by centrifugation at 1,800 × g for 5 min. Plasma samples were stored at -80°C until cytokine levels were analyzed. Preliminary testing revealed no difference in cytokine levels between fresh and thawed samples. Cytokine concentrations were determined from diluted (1:5) plasma samples. The Ciraplex Multi-Cytokine Chemiluminescence Assay Kit was used. TM Chemiluminescence array kit; Aushon BioSystems, 101-3EF-1-AB) and SignaturePLUS TM The imaging system and PROarray analysis software (Aushon BioSystems) were used to determine the analyte concentration using enzyme-linked immunosorbent assay (ELISA).

[0494] For data points indicated as "less than" (<), the highest indicated value was used. For data points indicated as "greater than" (>), the lowest indicated value was used. Data are presented as average cytokine measurements from the supernatant of three replicate wells. Cell staining for flow cytometry was performed after a 24-hour incubation period in cell culture medium containing individual test items or controls. The cell suspension was incubated with the antibody of interest for 30 minutes at room temperature and washed twice with PBS (phosphate-buffered saline) to remove any unbound antibody. Cell samples were then analyzed on a BD Biosciences FACSCantoII flow cytometer. Flow cytometry data were analyzed using FlowJo.2, Microsoft Excel, and Graph Pad Prism 7.

[0495] Animal care and handling

[0496] All research plans and any revisions or procedures involving animal care and use in the experiments were reviewed and approved by the relevant institutional animal care and use committees. All mouse studies were conducted by F. HOFFMANN-LA ROCHE AG (Basel, Switzerland). The Basel pharmaceutical research and early development facility is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), and all procedures complied with relevant Swiss regulations and were approved by the cantonal animal research ethics committee. Crab-eating macaque studies were conducted under contract research with German and British organizations, sponsored by F. HOFFMANN-LA ROCHE AG; all procedures complied with the German Animal Welfare Act or EU Directive 2010 / 63 / EU.

[0497] Retention potency of Fc-silent BS-CD20

[0498] The transgenic mouse model is based on the C57BL / 6 background. The C57BL / 6 humanized CD20 mouse (huCD20) is generated by random integration of human CD20 bacterial artificial chromosome transgenes, as previously described [41a]. The C57BL / 6-Tg(hIg-γ1,κ,λ)ait mouse (HIGR3) was generated as previously described [28a]. Three transgenic constructs were co-injected into prokaryotes; these constructs contained microloci of unrearranged human Ig heavy chain γ1, human Ig light chain κ, and λ, respectively. The transgenic mice inherited these transgenes at a single locus and subsequently expressed human IgG1 antibodies in their plasma [28a]. The C57BL / 6 huCD20xC57BL / 6-Tg(hIg-γ1,κ,λ)ait mouse (huCD20xHIGR3) is the result of crossing the huCD20 and HIGR3 mouse strains; these mice are transgenic mice targeting human CD20 drugs and do not produce ADA antibodies against humans.

[0499] Flow cytometry immunopotency and potency studies

[0500] Humanized CD20 transgenic mice (huCD20) were intravenously administered 0.6, 1.3, and 13.3 mg / kg of mouse BS-obbituzumab (mBS-obbituzumab or mBS-obbituzumab-PGLALA) or 0.5, 1, and 10 mg / kg (equimolar concentration) of parental (non-shuttle) obbituzumab (obbituzumab or obbituzumab-PGLALA). Blood samples were collected on days -1, 2, and 6 to determine the frequency of B cells (B220+-B cells). On day 6, mice were sacrificed and the frequency of B220+-B cells in the spleen and inguinal draining lymph nodes (LN) was determined. Spleens and inguinal LNs were harvested from 6 primordial mice (i.e., untreated mice) to establish baseline B cell frequencies.

[0501] IRR and tolerability studies

[0502] Using Trucount TM Absolute total B cell count was determined using a bead (BD Biosciences, 340335). Fc receptors were blocked by an anti-mouse CD16 / CD32 antibody (BioLegend 101320). LSR Tortessa was used to determine the absolute total B cell count. TM Cells were sorted using a cytometer (BD Biosciences) and analyzed using FlowJo software (BD Biosciences).

[0503] PK and PD studies

[0504] A one-day tolerability study was conducted in huCD20xHIGR3 mice. Five groups of mice (two males and two females per group) were administered a single IV bolus (5 mL / kg) of 0 (mediator control), 3, or 10 mg / kg mBS-obbituzumab-PGLALA, or mBS-obbituzumab. Toxicity was assessed based on mortality and clinical observation on the day of administration. Cytokine release and exposure were assessed in terminal orbital blood samples collected approximately 2 hours after administration. Serum cytokines IL-2, IL-6, IL-10, MCP-1, MIP-1β, MIP-2, G-CSF, IFN-γ, TNF-α, and KC were measured using a Luminex assay (R&D system).

[0505] To determine the underlying causes of any differences in the toxicity profile of the MTD and whether the efficacy depended on Fc region activity and / or the BS moiety, mBS-obbituzumab, mBS-obbituzumab-PGLALA, and obbituzumab were evaluated in a single IV-dose study in huCD20 or huCD20xHIGR3 transgenic mice. Acute-phase responses were characterized by assessing cytokine levels (IL-2, MIP-2, IL-6, G-CSF, IL-10, IFN-γ, MCP-1, TNF-α, MIP-1β, and KC, measured in serum 2 hours after terminal orbital blood collection via Luminex assay) and body temperature (measured via telemetry; BMDS IPTT300 system (Plexx)) to identify the potential for IRR generation. Cytokine release and exposure assessment were evaluated in terminal orbital blood samples collected approximately 2 hours after administration.

[0506] ​

[0507] Single-dose pharmacokinetic study of huCD20xHIGR3 mice

[0508] The PK of mBS-obitutuzumab and mBS-obitutuzumab-PGLALA was evaluated in 12 female huCD20xHIGR3 mice following a single IV bolus administration (13.3 mg / kg). Serum sampling was performed up to 168 hours post-dose (based on eight time points in serum: 1, 3, 7, 24, 48, 72, 96, and 168 hours post-dose (composite, 3 samples per animal)) and terminal brain sampling was performed at 24, 48, or 168 hours post-dose. Blood samples (0.05 mL) were collected from the tail vein and heart via puncture at the terminal sampling time. Five minutes prior to sacrifice, DP47 (a human IgG blood tracer for brain contamination) was administered via IV injection (30 mg / kg). PK parameters were derived from composite concentration data and estimated using non-compartmental analysis with the Phoenix@ version 1.4 kinetic assessment procedure.

[0509] Serum and brain blood tracer levels were measured, and serum and brain samples were analyzed using two different ELISA methods. For the CD20-specific ELISA method (Bioanalytical Method 1), capture antibody (anti-IgG mAb M-6.28.530-IgG-Bi), positive control standard or diluted sample, and detection antibody (anti-IgG mAb M.1.19.31-IgG-Dig) were sequentially added to streptavidin-coated microtiter plates (SA-MTP). Immobilized immune complexes were detected using a polyclonal anti-digoxin-wasabi peroxidase (POD) conjugate (Merck, 11633716001). Finally, ABTS was added... TM The formed immobilized immune complexes were visualized using a solution (Merck, 11684302001) (a POD substrate). The intensity of the color change was analyzed at 405 nm (490 nm reference wavelength) using a BioTek MTP Reader ELx808. TM The concentration of the analyte was directly proportional to the concentration of the analyte in the test sample. Five minutes before euthanasia, animals were treated with an intravenous injection of a blood tracer at 30 mg / kg. Autopsy samples (serum and brain lysates) were analyzed using the following ELISA method. Prior to analysis, tissue samples were mechanically lysed in 800 µl of Tissue Extraction Reagent I (ThermoFisher, FNN0071) containing a protease inhibitor (Merck, P8340) using a MagNA Lyser instrument (Roche Diagnostics GmbH, Mannheim, Germany).

[0510] For the blood tracer-specific ELISA method (Bioanalytical Method 2), the capture antibody (mAb 2B01-6D01-Bi), diluted calibrator and diluted quality control and sample, detection reagent (anti-IgG mAb M1.7.24-IgG-Dig), and anti-digoxin-POD were sequentially added to the SA-MTP. This was achieved by adding ABTS. TM The solution visualizes the formed immobilized immune complexes. As described in Bioanalytical Method 1 above, the intensity of the color change is analyzed for luminance. Absorbance quantification is performed using a calibration curve with a nonlinear four-parameter Wiemer-Rodbard curve fitting function. The analytical sensitivity for 100% serum and brain lysate in Method 1 is 7.8 ng / mL, and the analytical sensitivity for 100% serum and brain lysate in Method 2 is 11 ng / mL.

[0511] For the mouse pharmacokinetic studies, due to the composite study design, no formal statistical analysis was performed. The data are summarized only as the arithmetic mean (or the median time to peak drug concentration).

[0512] PK / PD Study in huCD20xHIGR3 Mice

[0513] One control group (0 mg / kg) and six groups of 15 female huCD20xHIGR3 mice were treated with mBS-obitutuzumab or mBS-obitutuzumab-PGLALA via a single intravenous slow bolus (5 mL / kg). The control group received only the delivery medium; a pH 6.0 solution containing 20 mM histidine and 140 mM NaCl. Five animals from each group were sacrificed at 48 hours (day 3), 168 hours (day 8), and 504 hours (day 22) post-administration.

[0514] Subsequently, exposure assessment, mortality, clinical observation, body weight, hematology, determination of cytokine (IL-6) levels, inguinal lymph node and spleen organ weight, gross findings, and histopathology of inguinal lymph nodes, bone marrow, and spleen were evaluated. FACS analysis was performed in blood, spleen, and inguinal lymph nodes to assess the efficacy of the two BS-obbituzumab constructs in exhausting systemic and lymph node-resident B cells (CD19+ B220+). Marginal zone (CD21+) and follicular B cells were also assessed in the spleen. Approximately 50 µl of blood was collected from the tail vein at each sampling time point for FACS analysis at administration (day 1; all 105 mice), 48 hours (day 3; all 105 mice), 168 hours (day 8; 70 mice), and 504 hours (day 22; 35 mice). The following FACS antibody groups were assessed: anti-B220, anti-CD19, anti-huCD20, anti-TCRβ, anti-CD4, anti-CD8, anti-CD45, anti-CD21, anti-CD23, and anti-IgM / IgD. Blood was collected from behind the eye under anesthesia (ketamine / toluidine 150 / 9 mg / kg) shortly before pleurodesis on days 3, 8, or 22 for hematological, exposure, ADA assessment, and IL-6 determination. One blood sample was collected in an EDTA tube to measure hematological parameters (RBC, WBC, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, RBC distribution width, neutrophils, eosinophils, basophils, monocytes, lymphocytes, reticulocytes, platelets, and peripheral blood smear). IL-6 levels were determined using a 20 µl serum sample. Two 50 µl serum samples were also collected for exposure and ADA assessment. The maternal material of the samples was analyzed by ELISA. Inguinal lymph nodes, bone marrow, and spleen were collected, formalin-fixed, and embedded in paraffin. Slides were prepared and stained with hematoxylin and eosin, and examined under an optical microscope by a research toxicology pathologist and a peer-reviewed toxicology pathologist. Spleen samples were sectioned and stained with Prussian blue iron stain, and the percentage of iron-positive area was assessed using HALO v1.2 for image analysis. For Prussian blue iron staining and assessment of the percentage of iron-positive area using HALO v1.2 image analysis, the first step of the analysis included identifying spleen tissue from non-tissue using a random forest classifier. The identified spleen tissue was defined as the region of interest (ROI). The brightfield region quantification module was used to define the Prussian blue positive region within the ROI by adjusting the applied threshold parameter in real time. Areas were expressed in µm. 2 / Tissue and Positive Staining / Percentage of Tissue Report.

[0515] Single-dose study of cynomolgus monkeys

[0516] Four female cynomolgus monkeys were administered BS-obituzumab and RG6035 via a single IV bolus dose (10 mg / kg), followed by observation periods of 15 days and 8 weeks, respectively. Throughout the observation period, clinical observation, body weight and weight changes, body temperature, clinicopathology (hematology, coagulation, and clinical chemistry), immunophenotyping, and cytokine assessment were evaluated. Organ weight and macro / micro outcomes related to RG6035 were also assessed. Blood hepcidin levels were assessed only in the BS-obituzumab study. For RG6035, blood samples were collected pre-administration, 24 hours post-administration, and on the day of necropsy for hematology (0.5 mL, EDTA-soluble), clinical chemistry (2.0 mL), coagulation (1.0 mL trisodium citrate), and soluble transferrin (0.2 mL). Blood samples were collected twice for cytokine analysis: pre-administration and 1 hour and 4 hours post-administration on day 1.

[0517] Cytokine levels were analyzed using a Luminex multiplex kit (R&D system; IFNγ, TNFα, MCP-1, IL-6, IL-8). For immunophenotyping analysis on day 1 (before administration) and at 4, 24, 72, 168, and 336 hours post-administration, blood samples (600 μL) were drawn from the cephalic forearm vein and placed in EDTA anticoagulant. Immunophenotyping was performed using specific monoclonal antibodies consisting of T cells (T helper cells and cytotoxic T cells), B cells, monocytes, and NK cells.

[0518] Total lymphocyte count was determined on the same day as the relative cell count analysis. Absolute numbers of lymphocyte subsets were determined based on the relative and total counts. For the BS-Orbituzumab study, blood samples were collected for hematology (0.5 mL EDTA), coagulation (0.9 mL trisodium citrate), clinical chemistry (1.5 mL lithium heparin), and hepcidin (0.4 mL). Blood was collected from the femoral vein before treatment and at 2, 24, and 72 hours after administration, and on days 8 and 49 for cytokine analysis (0.3 mL, EDTA). Samples were evaluated using a BioRad Bio-Plex 200 reader with a customized IL-6, IL-8, IFNγ, TNFα, and MCP-1 multiplex kit. Blood was also collected from the femoral vein for flow cytometry (0.5 mL heparin sodium) before treatment and at 4, 24, and 73 hours after administration, as well as on days 5, 6, 8, 11, 13, 15, 20, 23, 28, 30, 35, 37, 42, 44, and 49.

[0519] Immunophenotyping and FACS in cynomolgus monkeys:

[0520] FACS was used to assess B cell exhaustion using flow cytometry and other lymphocyte immunophenotyping assays. The cell antigens CD45 and CD19 of the B lymphocyte population were quantified using specific antibodies against the labeled antigens and reported as absolute counts (cells / μL blood). ADVIA was used. (R) The 120 hematology system (Siemens Healthineers) determined WBC counts (total and absolute differences) from whole blood samples. Total lymphocyte counts with antigenic markers CD45+, CD14−, CD3+, and CD159α− were reported as cells / μL of blood and used to calculate the absolute count of the lymphocyte population of interest. The change in B cells relative to baseline for each animal was calculated as follows: Percentage change in B cells = Absolute B cell count at T(x) (hours) × 100 / Absolute B cell count at T(0) (hours). The SD of the mean for four animals was also calculated.

[0521] Statistical analysis:

[0522] For CD20 binding and direct B cell death induction, FACSDiva was used. TM FACS gating was performed using software (BDBiosciences), and median fluorescence intensity and the percentage of positive cells were determined. The half-maximum effective concentration (WMC) was calculated using GraphPad Prism based on S-type dose-response (variable slope) analysis. For the IRR study, temperature measurements were performed in triplicate for each animal and time point. Temperature changes were measured based on the pre-drug temperature.

[0523] Crab-eating macaque PK / PD study

[0524] This PK / PD study was conducted in cynomolgus monkeys to determine the pharmacokinetic properties of RG6035 and its distribution in the brain, and to quantify B cell depletion in the blood and tissues of interest.

[0525] The research plan and procedures concerning animal care and use in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the CR MTL. Throughout the study, animal care and use were conducted under the guidance of the U.S. National Research Council and the Canadian Council on Animal Care (CCAC).

[0526] animal:

[0527] The cynomolgus macaque is the only species that cross-reacts with the CD20 and TfR1 binding sites of RG6035. Cynomolgus macaques from Mauritius were provided by Research Models Houston (Texas, USA). Twelve females were used in the study, with a target age of 2–4 years and a target weight of 2–3.5 kg at the start of administration, with two animals as backups. All animals were weighed and socialized in enclosures equipped with automatic watering valves before administration began. They were fed twice daily. Animals were randomly assigned to one of six administration groups using a computer randomization procedure (Table 10). Each group consisted of two animals.

[0528] Table 10: Experimental Design. a: Animals in Groups 1 and 2 were euthanized 48 hours after administration of the blood tracer and perfused at the time of necropsy; b: Animals in Group 6 were euthanized 120 hours after administration of the blood tracer and perfused at the time of necropsy.

[0529]

[0530] Experimental Design:

[0531] After an acclimatization period of at least 4 weeks, the animals underwent cerebellomedullary cistern cannulation surgery for CSF sampling according to standard operating procedures (SOP). Each animal was pre-anesthetized by intramuscular injection of glycopyrronium bromide, ketamine, and toluenethiazide to achieve adequate sedation required for preoperative preparation. Anesthesia was maintained using isoflurane during the procedure. CSF aspiration during the procedure confirmed catheter placement in the cerebellomedullary cistern.

[0532] On day 1, RG6035 was administered as a single intravenous (IV) bolus via an appropriate peripheral vein at doses of 0.3 mg / kg (Group 3), 1 mg / kg (Group 4), and 10 mg / kg (Groups 2, 5, and 6). The control group (Group 1) received a single IV injection of the medium (20 mM histidine, 140 mM sodium chloride, pH 6.0).

[0533] For PK and PD analysis, blood and CSF samples were collected periodically from all animals. Animals assigned to groups 1, 2, and 6 received an IV injection of 10 mg / kg of blood tracer 15 minutes prior to scheduled sacrifice (see WO 2021 / 136772); groups 3, 4, and 5 were not scheduled for sacrifice. At necropsy on day 3 (groups 1 and 2) or day 6 (group 6), various tissues were harvested to assess RG6035 concentrations and B cell depletion in the brain, spleen, lymph nodes, tonsils, bone marrow, and liver.

[0534] PK sampling:

[0535] Blood samples were collected from appropriate peripheral veins of each animal to assess B cell depletion over time. The blood samples were gently mixed and stored at ambient conditions until centrifuged at 2,400 g for 10 minutes at 4°C. The resulting serum was separated, transferred to clear polypropylene tubes (target 2 × 100 µL), and frozen (-80°C) until further analysis.

[0536] Over two weeks, PK CSF samples were collected via lumbar puncture (targeting the L5–L6 space, and L4–L5 if necessary) and from cerebellomedullary cistern catheters in all treatment groups. Animals were anesthetized with ketamine, dexmedetomidine, and glycopyrronium bromide. Isoflurane was administered as needed if sedation was deemed insufficient. Fifteen minutes prior to termination, the blood tracer was administered intravenously (via the saphenous or brachial vein) to animals in groups 1, 2, and 6. Ten minutes after the blood tracer injection, 1 mL of blood was aspirated from a vein in the arm for serum preparation.

[0537] Brain perfusion sampling was performed on five brain regions in groups 1, 2, and 6. Frozen 300 mg cynomolgus brain tissue samples were thawed at room temperature for 2 hours. 800 μL of lysis buffer dissolved in 50 mL of Tissue Extraction Reagent I (Invitrogen, Waltham, MIT, USA) and one tablet of a complete protease inhibitor mixture (Roche Diagnostics GmbH, Mannheim, Germany) were added to the thawed brain tissue. The samples were homogenized at 6,500 rpm for 20 seconds, and then the tissue homogenate was centrifuged at 12,000 g for 10 minutes. Finally, the supernatant was transferred to 1.5 mL vials for further analysis or stored at -80°C.

[0538] Quantification of RG6035 in CSF / serum:

[0539] Test samples, calibration samples, and quality controls were diluted to 1:100 in Roche universal buffer (Roche Diagnostics GmbH, Mannheim, Germany). The diluted samples and controls were added to a streptavidin-coated Gyrolab Bioaffy 1000 gyroscope disk (Gyros Protein Technologies AB) using a Gyrolab xPlore instrument, along with 100 µg / mL biotinylated anti-idiotype capture antibody anti-ID CD20 (Roche Diagnostics GmbH) and 5 µg / mL Alexa-647-labeled anti-idiotype detection antibody anti-ID TfR (Roche Diagnostics GmbH). A 5% photomultiplier tube (PMT) setting was used, with the response signal proportional to the analyte concentration in the test sample. All study and control samples were analyzed in duplicate. The limit of quantitation (LOQ) was 0.1 ng / mL in CSF and 10 ng / mL in serum samples.

[0540] Quantification of RG6035 in brain lysate:

[0541] 500 ng / mL biotinylated anti-ID CD20 antibody was transferred to the surface of a streptavidin-coated 96-well microtiter plate and incubated at room temperature for 1 hour. Unbound antibody was removed by washing, followed by the addition of brain lysate samples diluted 1:100 in Roche universal buffer (Roche Diagnostics GmbH) and incubation for 1 hour. After removing unbound analyte by washing, detection was performed by sequentially adding 500 ng / mL digoxigenin-conjugated anti-ID TfR antibody, incubating at room temperature for 50 minutes, washing, and adding 50 mU / mL anti-digoxigenin Fab fragment covalently bound to HRP. Following the final wash, a substrate solution of 2,2'-azobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS(R)) was added, resulting in a color change. Color intensity (absorbance at 405 nm; 490 nm reference wavelength) was determined using an enzyme-linked immunosorbent assay (ELISA) reader by luminance measurement and was proportional to the amount of RG6035 in the test sample. All study and control samples were analyzed in duplicate. The limit of quantitation was 10 ng / mL.

[0542] Quantification of blood tracers in brain lysates:

[0543] 500 ng / mL of biotinylated anti-ID DP47 1 was transferred to the surface of a streptavidin-coated 96-well microtiter plate (Microcoat GmbH, Bernier-Rid, Germany) and incubated at room temperature for 1 hour. Unbound antibody was removed by washing, followed by the addition of brain lysate sample diluted 1:100 in Roche universal buffer (Roche Diagnostics GmbH) and incubation for 1 hour. After removing unbound analyte by washing, detection was performed by sequentially adding 200 ng / mL digoxigenin-conjugated anti-ID DP47 2 antibody (Roche Diagnostics GmbH), incubating at room temperature for 50 minutes, washing, and adding 25 mU / mL of anti-digoxigenin Fab fragment covalently bound to HRP (Roche Diagnostics GmbH). ABTS substrate solution was added after the final wash. The color intensity of the reaction (absorbance at 405 nm; reference wavelength 490 nm) was determined using an ELISA reader via luminosity assay and was proportional to the amount of blood tracer in the test sample. All study and control samples were analyzed in duplicate. The limit of quantitation was 18.75 ng / mL.

[0544] Quantification of blood tracers in serum:

[0545] 500 ng / mL biotinylated anti-ID DP47 1 was transferred to the surface of a streptavidin-coated 96-well microtiter plate (Microcoat GmbH, Bernier-Rid, Germany) and incubated at room temperature for 1 hour. Unbound antibody was removed by washing, followed by the addition of serum sample diluted 1:1,000 in Roche universal buffer (Roche Diagnostics GmbH) and incubation for 1 hour. After removing unbound analyte by washing, detection was performed by sequentially adding 200 ng / mL digoxigenin-conjugated anti-ID DP47 2 antibody (Roche Diagnostics GmbH), incubating at room temperature for 50 minutes, washing, and adding 25 mU / mL anti-digoxigenin Fab fragment covalently bound to HRP (Roche Diagnostics GmbH). ABTS substrate solution was added after the final wash. The color intensity of the reaction (absorbance at 405 nm; reference wavelength 490 nm) was determined using an ELISA reader via luminosity assay and was proportional to the amount of blood tracer in the test sample. All study and control samples were analyzed in duplicate. The limit of quantitation was 187.5 ng / mL.

[0546] PK Analysis:

[0547] Phoenix WinNonlin® (Certara, Princeton, NJ, USA) was used for non-compartmental (NCA) PK analysis. PK parameters for serum RG6035 were calculated based on individual concentration data from non-terminal groups 3, 4, and 5, administered at doses of 0.3 mg / kg, 1 mg / kg, and 10 mg / kg, respectively. PK parameters for CSF were calculated based on composite concentration profiles of CSF samples collected from the cerebellomedullary cistern.

[0548] Immunophenotyping:

[0549] For blood PD assessment, a 2 mL blood sample is used to assess B cell depletion over time and other lymphocyte subsets.

[0550] Immunophenotyping was used over 6 weeks to determine whether peripheral B cells were activated (CD69 and CD86 surface markers) and whether specific subsets of B cells were preferentially depleted. Blood immunophenotyping and activation markers used were CD3, CD4, CD8, CD16, and CD19. The B cell subsets used (blood and lymph nodes) were CD45 / CD1, and the activation markers used were CD69 and CD86.

[0551] Immunophenotyping:

[0552] Samples from the spleen, mesentery, and mandibular lymph nodes (LNs), as well as tonsils, were collected from all animals at the scheduled necropsy. B cell exhaustion was assessed using a clean removal technique. A mid-spleen section (approximately 1 cm thick) was taken from the spleen and preserved in a conical tube containing 10 mL of assay medium (RPMI-1640 containing 5% (v / v) fetal bovine solution (FBS)) to prepare a single-cell suspension. A portion of each tonsil and LN was also taken and preserved in a conical tube containing 5 mL of assay medium (RPMI-1640 containing 5% (v / v) FBS) to prepare a single-cell suspension.

[0553] Cellular antigens and cell populations (Table 11) were quantified and reported as relative percentages of spleen, lymph nodes and tonsils, as well as absolute spleen counts (cells / organ) (hematological assessment (ADVIA® system, Siemens Healthineers, Erlangen, Germany)).

[0554] Table 11: Tissue biomarkers; a: Absolute counts and parental percentages will be calculated and reported from the specified parental populations; b: The median fluorescence intensity of CD69 will be reported as the activation marker.

[0555]

[0556] Immunofluorescence staining:

[0557] Formalin-fixed paraffin-embedded (FFPE) samples from the tonsils, spleen, cervical lymph nodes, and mandibular lymph nodes of all animals treated with RG6035 or the medium were cut into 2 µm sections and mounted on Superfrost Plus slides. The tissue sections were baked overnight at 37°C. Staining was performed using a Ventana Discovery Ultra automated tissue staining system (Ventana / Roche Tissue Diagnostics, Tucson, USA). Primary antibodies used for immunofluorescence assays (anti-CD19, 1:400, #ab182422, Abcam, Cambridge, UK; anti-CD20, 1:200, #M0755, Dako, Agilent, Santa Clara, California, USA) were diluted in Discovery Ab Diluent (#760-108, Ventana / Roche Tissue Diagnostics). Primary antibodies were detected using anti-species secondary antibodies conjugated to wasabi peroxidase (HRP; RocheDiagnostics GmbH) and subsequently enhanced with a tyrosine signal amplification (TSA) fluorescence kit (FAM or Cy5). Tissues were counterstained with DAPI (Roche Tissue Diagnostics) and mounted.

[0558] Image acquisition and analysis:

[0559] Slides were digitized using a Zeiss Axio Z.1 (20x magnification full slide scanner; Zeiss, Oberkochen, Germany) to produce single images and overlays in FAM, Cy5, and DAPI channels. Raw image data were saved in .czi format. Slides were viewed using ZEN blue software (Zeiss). All images had a pixel size of 0.27 pixels / µm. Image analysis was performed using HALO AI 3.2 (IndicaLab, Albuquerque, New Mexico, USA). A random forest classifier was used to detect tissue and eliminate artifacts. Cell segmentation was performed using the NucleiSeg Artificial Intelligence (AI) module. For subsequent quantification of B cells, the expression of previously segmented intracellular immunofluorescence markers was dynamically measured using the HighPlexFL v4.03 module, with AI-preset nuclear segmentation (nuclear contrast threshold = 0.5; minimum nuclear intensity = 0.005; nuclear segmentation aggressiveness = 0.2; maximum cytoplasmic radius = 1µm; cell size = 6–600 µm). 2 Positive and negative signals for each marker in each organ were determined using control staining (vector animals; Alexa Fluor 488 (CD19) cytoplasmic positivity threshold = 1400; 0% integrity; Figure 27). The total cell number, the number of positive cells for each marker, and the number of positive cells for each phenotype were normalized by tissue area detected by the random forest tissue classifier.

[0560] Assay for anti-drug antibodies against immune complexes (ADA):

[0561] To qualitatively detect antibodies against RG6035, a biotinylated capture antibody mAb targeting the human Fc region of RG6035 was prepared. <hu-igg>M-R10Z8E9 (Roche Diagnostics GmbH, Mannheim, Germany) was bound to streptavidin-coated microtiter plates at 0.5 pg / mL and incubated for 1 hour at room temperature. After washing, samples and standards were diluted 1 : 100 in LowCross buffer (Candor Bioscience GmbH, Wangen im Allgaeu, Germany) spiked with 1 pg / mL RG6035. 100 pL were added to each well of the streptavidin-coated microtiter plates and incubated for 1 hour at room temperature. After washing, the digoxinylated anti-cynomolgus IgG detection antibody (Roche Diagnostics GmbH) was added to the wells of the microtiter plates at 0.5 pg / mL and incubated for 1 hour. After washing, 25 mU / mL of a polyclonal anti-digoxigenin HRP conjugate was added and incubated for 1 hour. After adding the substrate solution ABTS to the plates, the peroxidase catalyzed color reaction of the antibody-enzyme conjugate. The absorbance was measured at a wavelength of 405 nm (reference wavelength: 490 nm) by an ELISA reader. The two-fold absorbance signal value of the pre-dose sample of each cynomolgus animal was set as the cut-off to assess ADAs in the post-dose samples.

[0562] PK / PD modeling in cynomolgus monkeys:

[0563] PK / PD modeling was performed on the PK in serum, PK in brain and CSF, and B-cell dynamics in blood of cynomolgus monkeys using the non-linear mixed effects approach with the Monolix software (Monolix version 2019R2, http: / / lixoft.com / products / monolix / , Lixoft, Antony, France). MATLAB (MATLAB version R2020a, https: / / ch.mathworks.com / products / matlab.html, MathWorks, Natick, MA, USA) was used for simulation of the human prediction model.

[0564] In more detail, the PK of RG6035 was modeled using a two-compartment model, including a quasi-equilibrium approximation of target-mediated drug disposition with a constant target pool.

[24] Without being bound by this theory, it was assumed that this target-mediated disposition is likely driven by binding to CD20. An accelerated clearance term related to ADA generated due to the immune response against RG6035 was also added to the model.

[28] Parameters were estimated using nonlinear mixed effect modeling (NLME) and further using individual (post-hoc) PK parameters to calculate PK inputs to analyze the distribution of RG6035 in CSF and brain and the PD of RG6035 on B cells in blood.

[0565] The distribution of RG6035 in brain tissue and CSF was modeled by separate compartments for each brain region or CSF compartment. It was assumed that the outflow rate is equal for all these compartments. The inflow rate (multiplied by serum concentration) was estimated as the product of the outflow rate parameter and the equilibrium ratio (Kp) relative to serum concentration (similar to the concept of a partition coefficient), which was estimated for each brain tissue and CSF compartment, respectively. A simple summation approach was taken to estimate the brain distribution parameters.

[0566] The complex kinetic model for blood B cells was built on the concept of a steady-state balance between first-order cell loss and replacement at a constant rate. It was assumed that RG6035 stimulates the loss of cells from the main B cell pool, the rate of which is proportional to its concentration.[27,28] To reconcile the relatively fast depletion observed in vitro and the long-lasting effect in vivo, it was further assumed that B cells in blood are not directly represented by the main B cell pool, but only reflect the depletion in the main pool with a certain delay. This was achieved by modeling blood B cells by another pool that slowly exchanges with the main pool.

[0567] Immediately after dosing, blood B cells show a fast and transient depletion, but this seems to be almost independent of the dose. The model was extended by a time-dependent (exponential) function that is triggered by the dosing event, but otherwise independent of the dose and exposure of RG6035, to capture the first phase.

[0568] All parameter estimates were performed using Monolix version 2020R1 (Lyxsoft).

[0569] Human predictions:

[0570] PK and PD predictions in humans for RG6035 were built on the basis of the PK / PD model in cynomolgus monkeys. For PK, volume was proportional to body weight, clearance was proportional to body weight to the power of 0.85, and all rate parameters were proportional to body weight to the power of -0.15.

[23] Assuming first-order process parameter values consistent with expectations for this type of molecule (absorption rate of 0.02 h -1 and an absorption fraction of 50%), a provisional prediction of subcutaneous absorption in humans was modeled.

[22] For brain distribution, it was assumed that the brain-to-serum concentration ratio of RG6035 was equal between cynomolgus monkeys and humans, and outflow rate was allometrically scaled to the power of -0.15. Since immunogenicity in cynomolgus monkeys was not predictive for humans, its impact was not considered in the translation. Human prediction of PK / PD in blood B cells was performed using in vitro data (EC50 of 1.68 nM in cynomolgus monkeys vs. EC50 of 0.4 nM in humans) to account for species differences in efficacy.

[0571] To provisionally predict PK / PD in B cells in the brain, a corresponding B cell pool was introduced, into which B cells migrated from blood and left at a rate assumed to be equal to the exchange rate of the two pools of the blood B cell model. Without being bound by this theory, this seems to be in line with published B cell depletion in CSF after treatment with ocrelizumab, assuming that B cell depletion in CSF is a consequence of reduced inflow due to blood depletion.

[44] Local B cell killing was assumed to have the same efficacy as peripheral cells but to be driven by interstitial concentrations of RG6035 in the brain, which were calculated from expected brain tissue concentrations assuming that the compound concentrates in the extracellular space, which is assumed to be about 20% of total brain tissue.

[45] The structure of the fully assembled model is shown in Figure 28.

[0572] All model simulations for human predictions were performed using Matlab version 2020a (Mathworks).

Claims

1. RG6035, which is used as a drug to treat multiple sclerosis, wherein RG6035 is administered at doses of 140 mg to 210 mg.

2. RG6035, which is used to treat multiple sclerosis, is administered at doses of 140 mg to 210 mg.

3. The use according to any one of claims 1 to 2, wherein the multiple sclerosis is primary progressive multiple sclerosis or secondary progressive multiple sclerosis.

4. The use according to any one of claims 1 to 3, wherein the dose effectively depletes more than 95% of B cells in the CSF of the subject who has been administered RG6035.

5. The use according to claim 4, wherein the depletion is achieved within 8 weeks after the start of the application.

6. The use according to any one of claims 1 to 5, wherein the concentration of RG6035 in the CSF is 1% or more of the serum concentration of RG6035.

7. The use according to any one of claims 1 to 6, wherein the concentration of RG6035 in the CSF is at least 0.055 μg / mL.

8. The use according to any one of claims 4 to 7, wherein the B cells are CD19 positive B cells.

9. The use according to any one of claims 1 to 8, wherein RG6035 is administered in a second dose after more than 95% of B cells have been depleted from the CSF to maintain the depletion of B cells in the CSF at the stated level.

10. The use according to any one of claims 4 to 9, wherein the depletion of said B cells is determined by comparing the number of B cells prior to the first application of RG6035.

11. The use according to any one of claims 1 to 10, wherein the application is performed once every four weeks.

12. The use according to any one of claims 1 to 10, wherein the administration begins once a week and, after more than 95% of the B cells have been depleted from the CSF, is changed to once every four weeks to maintain the depletion of the B cells in the CSF at the stated level.

13. The use according to any one of claims 1 to 12, wherein the administration of RG6035 is intravenous or subcutaneous.

14. The use according to any one of claims 1 to 13, wherein the application of RG6035 is subcutaneous.

15. The use according to any one of claims 1 to 14, wherein RG6035 comprises: a first polypeptide having the amino acid sequence of SEQ ID NO: 01, a second polypeptide having the amino acid sequence of SEQ ID NO: 02, a third polypeptide having the amino acid sequence of SEQ ID NO: 03, and a fourth polypeptide having the amino acid sequence of SEQ ID NO: 05.

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