STABLE PHARMACEUTICAL FORMULATION AND USE OF A FORMULATION

专利摘要:
stable pharmaceutical formulation, anti-cd20 antibody formulations and use of a formulation. The present invention relates to a stable, highly concentrated pharmaceutical formulation of a pharmaceutically active anti-cd20 antibody, such as, for example, rituximab, ocrelizumab or humabe<cd20>, or a mixture of such antibody molecules for subcutaneous injection. in particular, the present invention relates to formulations which comprise, in addition to a suitable amount of the anti-co20 antibody, an effective amount of at least one hyaluronidase enzyme as a combined formulation or for use in the form of a co-formulation. said formulations further comprise at least one buffering agent, such as, for example, a histidine buffer, a stabilizer or a mixture of two or more stabilizers (for example, a saccharide, such as a,a- trehalose or sucrose, and optionally methionine as a second stabilizer), a non-ionic surfactant and an effective amount of at least one hyaluronidase enzyme. methods for preparing such formulations and their uses are also provided.
公开号:BR112012005017B1
申请号:R112012005017-6
申请日:2010-09-10
公开日:2021-09-08
发明作者:Michael Adler；Hanns-Christian Mahler；Oliver Boris Stauch
申请人:F.Hoffmann-La Roche Ag；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The present invention relates to stable, highly concentrated pharmaceutical formulations of a pharmaceutically active anti-CD20 antibody or a mixture of such antibody molecules for subcutaneous injection. Such formulations comprise, in addition to high amounts of anti-CD20 antibody or mixture thereof, a buffering agent, a stabilizer or a mixture of two or more stabilizing agents, a nonionic surfactant and an effective amount of at least one hyaluronidase enzyme . The invention also relates to a process for the preparation of said formulation and the use of such formulation. BACKGROUND OF THE INVENTION
[002] The pharmaceutical use of antibodies has increased over the years. In many cases such antibodies are either injected or introduced through the intravenous (IV) route. Unfortunately, the amount of antibody that can be administered via the intravenous route is limited by the physiochemical properties of the antibody, in particular its solubility and stability in a suitable liquid formulation, and the volume of the infusion fluid. Alternative administration routes are subcutaneous or intramuscular injection. These injection pathways require high protein concentration in the final solution to be injected [Shire, S.J., Shahrokh, Z. et al, "Challenges in the development of high protein concentration formulations", J. Pharm. Sci. 2004; 93(6): 1390 to 1402; Roskos, L.K., Davis C.G. et al, "The clinical pharmacology of therapeutic antibodies", Drug Development Research 2004; 61(3): 108 to 120]. To increase the volume, and thus the therapeutic dose, which can be safely and comfortably administered subcutaneously, it has been proposed to use glycosaminoglycanase enzyme(s) to increase the interstitial space into which the antibody formulation can be injected [WO2006/091871] .
[003] Examples of stable formulations of pharmaceutically active antibodies for therapeutic use currently on the market are as follows.
[004] RITUXAN®/MABTHERA® (Rituximab) is a chimeric antibody that binds to the CD20 antigen on B cells. The commercial formulation is a preservative-free, colorless, clear, sterile liquid concentrate for intravenous (IV) administration. Rituximab is provided at a concentration of 10 mg/mL (10 mL) or in 100 mg or 500 mg (50 mL) from single-use containers. The product is formulated in 9 mg/mL sodium chloride, 7.35 mg/mL dehydrated sodium citrate, 0.7 mg/mL polysorbate 80, and water for injection. The pH is adjusted to 6.5. An alternative liquid formulation for Rituximab suitable for IV administration is disclosed in U.S. Patent No. 6,991,790.
[005] HERCEPTINTM (Trastuzumab) is a monoclonal antibody directed against the HER2 receptor (anti-HER2) that is currently marketed in Europe as a 150 mg lyophilized powder (containing the antibody, α,α-trehalose dihydrate, L- histidine and L-histidine hydrochloride and polysorbate 20) which must be reconstituted for infusions with water for injection to yield an injection dose of approximately 21 mg/mL. In the US and many other countries, a multiple dose container containing 440 mg of Trastuzumab is marketed.
[006] AVASTINTM (Bevacizumab) is a monoclonal antibody directed against vascular endothelial growth factor (VEGF) that is currently marketed in Europe as a liquid formulation in two types of receptacles: a) 100 mg Bevacizumab in 4 mL and b) 400 mg of Bevacizumab in 16 mL, respectively, providing a final concentration of 25 mg/mL in water for injection containing the following excipients: trehalose dihydrate, sodium phosphate and polysorbate 20.
[007] Although the above antibody formulations have been found suitable for use in intravenous administration, there is a desire to provide stable, highly concentrated pharmaceutical formulations of therapeutically active antibodies for subcutaneous injection. The advantage of subcutaneous injections is that they allow a medical professional to perform them in a very short intervention with the patient. In addition, the patient can be trained to perform the subcutaneous injection alone. Typically, injections via the subcutaneous route are limited to approximately 2 mL. For patients requiring multiple doses, multiple unit dose formulations can be injected into multiple locations on the body surface.
[008] The following two antibody products for subcutaneous administration are ready on the market.
[009] HUMIRATM (Adalimumab) is a monoclonal antibody directed against tumor necrosis factor alpha (TNF alpha) that is currently marketed in Europe as a dose of 40 mg in 0.8 mL injection volume for subcutaneous application ( concentration: 50 mg antibody/ml injection volume).
[010] XOLAIRTM (Omalizumab) a monoclonal antibody directed against immunoglobulin E (anti-IgE) that is currently marketed as a 150 mg lyophilized powder (containing the antibody, sucrose, histidine and histidine hydrochloride monohydrate and polysorbate 20) which must be reconstituted with water for subcutaneous injection to yield a 125 mg/mL injection dose.
[011] No suitable highly concentrated stable pharmaceutical anti-CD20 antibody formulation for subcutaneous administration is currently available on the market. There is then a desire to provide such stable, highly concentrated pharmaceutical formulations of therapeutically active antibodies for subcutaneous injection.
[012] Injection of parenteral drugs into the hypodermis is generally limited to volumes of less than 2 mL due to this viscoelastic resistance to hydraulic conduction in the subcutaneous tissue (SC) and back pressure generated upon injection [Aukland K. and Reed R. , "Interstitial-Lymphatic Mechanisms of the Control of Extracellular Fluid Volume", Physiology Reviews", 1993;73:1 to 78] as well as due to pain perceptions.
[013] The preparation of high concentration protein formulations is very challenging and there is a need to adapt each formulation to the particular proteins used because each protein has different aggregation behavior. Aggregates are suspected of causing immunogenicity of therapeutic proteins in at least some cases. Immunogenic reaction against protein or antibody aggregates can lead to neutralizing antibodies that can render the therapeutic protein or antibody ineffective. It appears that aggregated protein immunogenicity is more problematic in connection with subcutaneous injections, as repeated administration increases the risk of an immune response.
[014] Although antibodies have a very similar general structure, such antibodies differ in amino acid composition (in particular, in the CDR regions responsible for antigen binding) and the pattern of glycosylation. Furthermore, there may additionally be post-translational modifications such as alteration and glycosylation variants. BRIEF DESCRIPTION OF THE INVENTION
[015] The present invention provides a stable, highly concentrated pharmaceutical formulation of a pharmaceutically active anti-CD20 antibody or a mixture of such antibody molecules, preferably for subcutaneous injection.
[016] More particularly, the stable, highly concentrated pharmaceutical formulation of a pharmaceutically active anti-CD20 antibody formulation of the present invention comprises: - about 20 to 350 mg/ml of anti-CD20 antibody; - about 1 to 100 mM of a buffering agent that provides a pH of 5.5 ± 2.0; - about 1 to 500 mM of a stabilizer or a mixture of two or more stabilizers, in which methionine is optionally used as a secondary stabilizer, preferably, in a concentration of 5 to 25 mM; - 0.01 to 0.1% of a non-ionic surfactant; and - Preferably, an effective amount of at least one hyaluronidase enzyme. DETAILED DESCRIPTION OF THE INVENTION
[017] The term "antibody" in this document is used in the broad sense and specifically covers full length antibodies, genetically engineered antibodies such as monoclonal antibodies, or recombinant antibodies, polyclonal antibodies, multispecific antibodies (eg bispecific antibodies) formed from at least two full length antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, and as well as fragments of such antibodies as long as they exhibit the desired biological activity.
[018] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for variants possible that may appear during monoclonal antibody production, such variants are usually present in smaller amounts. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous as they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring antibody production by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by the first hybridoma method described by Kohler et al, Nature, 256:495 (1975), or can be made by recombinant DNA methods (see, for example, US Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clarkson et al, Nature, 352:624 to 628 (1991) and Marks et al, J. Mol. Biol, 222:581 to 597 (1991). The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of a single amino acid composition. Accordingly, the term "human monoclonal antibody" refers to antibodies that exhibit a single binding specificity that have variable constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, which has a genome comprising a human heavy chain transgene and a chain transgene. light human fused into an immortalized cell. The term "monoclonal antibodies" herein specifically includes so-called chimeric antibodies in which a portion of the light and/or heavy chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass , while the chain residue(s) is identical or homologous to corresponding sequences in antibodies derived from other species or belonging to other classes or subclasses of antibody, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (Patent No. US 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851 to 6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies which comprise variable domain antigen binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc) and human constant region sequences.
[019] "Antibody fragments" comprise a portion of a full length antibody, generally at least the antigen binding portion or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies, single chain antibody molecules, immunotoxins, and multispecific antibodies formed from antibody fragments. Furthermore, antibody fragments comprise single-chain polypeptides that have the characteristics of a VH chain, that is, being able to join with a VL chain that binds to the CD20 antigen. "Antibody fragments" also comprise such fragments which by themselves are not capable of providing effector functions (ADCC/CDC), but provide that function in a manner according to the invention after being combined with antibody constant domain(s) appropriate.
[020] A "full-length antibody" is one that comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains can be native sequence constant domains (for example, human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the full-length antibody has one or more effector functions.
[021] An "amino acid sequence variant" antibody in this document is an antibody with an amino acid sequence that differs from a major species antibody. Generally, amino acid sequence variants will process at least about 70% homology with the main species antibody, and preferably, they will be at least about 80%, more preferably at least about 90% homologous with the species antibody main. Amino acid sequence variants have substitutions, deletions, and/or additions at certain positions in or near the main species antibody amino acid sequence. Examples of amino acid sequence variants in this document include acid variant (for example, deamidated antibody variant), basic variant, the antibody with an amino terminal leader extension (for example, HSV-) in one or two light chains thereof. , antibody with a C-terminal lysine residue in one or two heavy chains thereof, etc., and include combinations of variations in light and/or heavy chain amino acid sequences. The antibody variant of particular interest herein is the antibody which comprises an amino-terminal leader extension on one or two light chains thereof, optionally further comprising another amino acid sequence and/or glycosylation differences relative to the main species antibody.
[022] A "glycosylation variant" antibody herein is an antibody with one or more carbohydrate moieties attached thereto that differs from one or more carbohydrate moieties attached to a major species antibody. Examples of glycosylation variants herein include antibody with a G1 and G2 oligosaccharide backbone, rather than G0 oligosaccharide backbone, attached to an Fc region thereof, antibody with one or two carbohydrate moieties attached to one or two light chains of the same, antibody with no carbohydrate attached to one or two antibody heavy chains, etc., and combinations of glycosylation changes. Furthermore, the term "glycosylation variant" also includes glycoengineered antibodies such as those described in WO 1 331 266 and USP 7517670.
[023] Antibody "Effector Functions" refers to such biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity (CDC); Fc receiver connection; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; low setting of cell surface receptors (eg B cell receptor; BCR), etc.
[024] Depending on the amino acid sequence of the constant domain of their heavy chains, full-length antibodies can be assigned to different "classes". There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into "subclasses" (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called α [alpha], δ [delta], ε [epsilon], Y [gamma], and μ [mi], respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
[025] In this document, "biological activity" of a monoclonal antibody refers to the ability of the antibody to bind an antigen and result in a mild biological response that can be measured in vitro or in vivo. Such activity can be antagonistic (eg where the antibody is a CD20 antibody) or agonistic.
[026] The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to the parent immunoglobulin. In a preferred embodiment, a murine CDR is mounted in the frame region of a human antibody to prepare the "humanized antibody." Particularly, preferred CDRs correspond to those representing sequences that recognize the antigens noted below for bifunctional and chimeric antibodies. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) in which residues from a hypervariable region of the receptor are replaced by residues from a hypervariable region from a non-human species (donor antibody) such as a mouse, rat, rabbit or non-primate human that has the desired specificity, affinity, and capacity. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin and all or substantially all of the FRs are those of a sequence of human immunoglobulin. The optionally humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (see, for example, Riechmann, L., et al, Nature 332 (1988) 323 to 327 and Neuberger, MS, et al, Nature 314 (1985) 268 to 270).
[027] The term "chimeric antibody" refers to a monoclonal antibody that comprises a variable region, i.e. binding region, from a source or species and at least a portion of a constant region derived from a different species or source, normally prepared by recombinant DNA techniques. Chimeric antibodies that comprise a murine variable region and a human constant region are especially preferred. Such chimeric human/murine antibodies are the product of expressed immunoglobulin genes which comprise DNA segments encoding murine immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of "chimeric antibodies" encompassed by the present invention are those in which the class or subclass has been modified or altered from the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies". Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art (see, for example, Morrison, SL, et al, Proc. Natl. Acad Sci. USA 81 (1984) 6,851 to 6,855; US 5,202,238 and US 5,204,244).
[028] The term "human antibody", as used herein, is intended to include antibodies that have variable constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Pharmacol. 5 (2001) 368 to 374). Based on such technology, human antibodies against a wide variety of targets can be produced. Examples of human antibodies are, for example, described in Kellermann, S.A., et al, Curr Opin Biotechnol. 13 (2002) 593 to 597.
[029] The term "recombinant human antibody" as used herein is intended to include all human antibodies that are prepared, expressed, raised or isolated by recombinant means, such as antibodies isolated from a host cell such as a cell NS0 or CHO or from an animal (eg, a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable constant regions derived from human germline immunoglobulin sequences in a rearranged form. Recombinant human antibodies according to the invention have been subjected to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist in the germline human antibody repertoire in vivo.
[030] As used herein, "specifically binding" or "specifically binding to" refers to an antibody binding specifically to the CD20 antigen. Preferably, the binding affinity is a Kd value of 10-9 mol/l or less (eg 10-10 mol/l), preferably with a Kd value of 10-10 mol/l or less (per example, 10-12 mol/l). Binding affinity is determined with a standard binding assay, such as surface plasma resonance technique (BIACORE®).
[031] The term "nucleic acid molecule", as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but preferably it is double-stranded DNA.
[032] The "constant domains" are not directly involved in binding the antibody to an antigen, but are involved in effector functions (ADCC, complement binding, and CDC).
[033] The "variable region" (variable region of a light chain (VL), variable region of a heavy chain (VH)) as used herein, denotes each of the pair of heavy and light chains that are directly involved in binding the antibody to the antigen. The variable human heavy and light chain domains have the same general structure and each domain comprises four framework regions (FR) whose sequences are largely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The frame regions adopt a β-sheet conformation and the CDRs can form loops that connect the b-sheet structure. The CDRs in each chain are contained in their three-dimensional structure by the framework regions and form, together with the CDRs from the other chains, the antigen-binding site. Antibody light and heavy chain CDR3 regions play a particularly important role in the binding specificity/affinity of antibodies according to the invention and thus provide a further object of the invention.
[034] The terms "hypervariable region" or "antigen-binding portion of an antibody" when used herein, refer to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs". "Frame" or "FR" regions are those variable domain regions other than the hypervariable region residues as defined herein. Thus, the heavy and light chains of an antibody comprise the C- to N-terminus of the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. Especially, heavy chain CDR3 is the region that contributes the most to antigen binding. CDR and FR regions are determined according to a standard definition of Kabat, et al, Sequences of Proteins of Immunological Interest, 5th Edition. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) and/or those residues of a "hypervariable loop".
[035] The terms "CD20" and "CD20 antigen" are used interchangeably in this document, and include any variants, isoforms and homologous species of human CD20 that are naturally expressed by cells or are expressed in cells transfected with the CD20 gene. Binding of an antibody of the invention to the CD20 antigen mediates the killing of cells expressing CD20 (eg, a tumor cell) by inactivating CD20. The killing of cells expressing CD20 can occur by one or more of the following mechanisms: antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), inducing cell death and/or apoptosis, homotypic aggregation, etc.
[036] Synonyms of CD20, as recognized in the art, include CD20 B lymphocyte antigen, B lymphocyte surface antigen B1, Leu-16, and Bp35.
[037] The term "anti-CD20 antibody" according to the invention is an antibody that specifically binds a CD20 antigen. Depending on the binding properties and biological activities of anti-CD20 antibodies to the CD20 antigen, two types of anti-CD20 antibodies (anti-CD20 antibodies type I and type II) can be distinguished according to Cragg, MS, et al , Blood 103 (2004) 2,738 to 2,743; and Cragg, MS, et al Blood 101 (2003) 1,045 to 1,051, see Table 1. TABLE 1: PROPERTIES OF TYPE I AND TYPE II ANTI-CD20 ANTIBODIES

[038] An essential property of type I and type II anti-CD20 antibody is its mode of binding. Thus, type I and type II anti-CD20 antibody can be classified by reason of the CD20 binding capabilities on Raji cells (ATCC-No. CCL-86) of said anti-CD20 antibody compared to Rituximab.
[039] As used herein, the "anti-CD20 antibody" can be either a type I or a type II antibody. Preferably it is a type I antibody, most preferred is Rituximab.
[040] Type I anti-CD20 antibodies have a ratio of CD20 binding capacities on Raji cells (ATCC No. CCL-86) of said anti-CD20 antibody compared to Rituximab of 0.8 to 1.2, preferably from 0.9 to 1.1. Examples of such type I anti-CD20 antibodies include, for example, Rituximab, in EP2000149B1 (Anderson et al, see, for example, Figures 4 and 5), 1F5 IgG2a (ECACC, hybridoma; Press et al, Blood 69 /2:584 to 591 (1987)), HI47 IgG3 (ECACC, hybridoma), IgG1 2C6 (as disclosed in WO2005/103081), 2F2 IgG1 or ofatumumab (as disclosed in WO 2004/035607 and WO2005/103081) and 2H7 IgG1 (as disclosed in WO 2004/056312) and WO 2006/084264 (for example the variants disclosed in tables 1 and 2). Preferably, said type I anti-CD20 antibody is a monoclonal antibody that binds to the same epitope as Rituximab.
[041] Type II anti-CD20 antibodies have a ratio of CD20 binding capacities on Raji cells (ATCC No. CCL-86) of said anti-CD20 antibody compared to Rituximab of 0.3 to 0.6, preferably from 0.35 to 0.55, more preferably 0.4 to 0.5. Examples of such type II anti-CD20 antibodies include, for example, tositumomab (B1 IgG2a), humanized B-Ly1 IgG1 antibody (a chimeric humanized IgG1 antibody as disclosed in WO2005/044859), IgG1 11B8 (as disclosed in WO 2004 /035607), and AT80 IgG1. Preferably, said type II anti-CD20 antibody is a monoclonal antibody which binds to the same epitope as humanized B-Ly1 antibody (as disclosed in WO2005/044859).
[042] The "ratio of CD20 binding capacities on Raji cells (ATCC-No. CCL-86) of anti-CD20 antibodies compared to Rituximab" is determined by direct immunofluorescence measurement (mean fluorescent intensities (MFI) are measured ) using said Cy5-conjugated anti-CD20 antibody and Cy5-conjugated Rituximab on a FACSArray (Becton Dickinson) with Raji cells (ATCC-No. CCL-86), and calculated as follows: Ratio of CD20 binding abilities on Raji cells (ATCC-No. CCL-86) =

[043] MFI is the mean fluorescent intensity. The "Cy5-identification ratio" as used herein, means number of Cy-5-identifying molecules per antibody molecule.
[044] Typically, said type I anti-CD20 antibody has a ratio of CD20 binding capacities on Raji cells (ATCC-No. CCL-86) of said first anti-CD20 antibody compared to Rituximab of 0.8 to 1.2, preferably 0.9 to 1.1.
Typically, said type II anti-CD20 antibody has a ratio of CD20 binding capacities on Raji cells (ATCC-No. CCL-86) of said second anti-CD20 antibody compared to Rituximab of 0.3 to 0.6, preferably 0.35 to 0.55, more preferably 0.4 to 0.5.
[046] In a preferred embodiment, said type II anti-CD20 antibody, preferably a humanized B-Ly1 antibody, increased antibody-dependent cellular cytotoxicity (ADCC).
[047] By "antibody that has increased antibody-dependent cellular cytotoxicity (ADCC)" is meant an antibody, as the term is defined herein, that has increased ADCC as determined by any suitable method known to one of ordinary skill in the art. An accepted in vitro ADCC assay is as follows: 1) the assay uses target cells that are known to express the target antigen recognized by the antibody's antigen binding region; 2) the assay uses human peripheral blood mononuclear cells (PBMCs), isolated from the blood of a randomly chosen healthy donor, as effector cells; 3) the assay is performed according to the following protocol: i) PBMCs are isolated using standard density centrifugation procedures and are suspended at 5 X 106 cells/ml in RPMI cell culture medium; ii) target cells are grown by standard tissue culture methods, harvested from the exponential growth phase with a viability greater than 90%, washed in RPMI cell culture medium, identified with 100 microcuries of 51CI washed twice with medium of cell culture, and resuspended in cell culture medium at a density of 105 cells/ml; iii) 100 microliters of the above final target cell suspension is transferred to each well of a 96-well microtiter plate; iv) the antibody is seriously diluted from 4000 ng/ml to 0.04 ng/ml in cell culture medium and 50 microliters of the resulting antibody solutions are added to target cells in the 96-well microtiter plates, testing in several tripled antibody concentrations covering the entire above concentration range; v) for the maximum release (MR) controls, 3 additional wells in the paca containing the identified target cells, receive 50 microliters of a 2% aqueous solution (VN) of non-ionic detergent (Nonidet, Sigma, St. Louis), instead of the antibody solution (point iv above); vi) for the spontaneous release (SR) controls, 3 additional wells in the paka containing the identified target cells, receive 50 microliters of RPMI cell culture medium instead of the antibody solution (point iv above); vii) the 96-well microtiter plate is then centrifuged at 50 x g for 1 minute and incubated for 1 hour at 4°C; viii) 50 microliters of the PBMC suspension (point i above) is added to each well to yield an effector to target cell ratio of 25:1 and the plates are installed in an incubator under a 5% CO2 atmosphere at 37°C for 4 hours; ix) the free cell supernatant from each well is harvested and the experimentally released radioactivity (ER) is quantified using a gamma counter; x) the percentage of specific lysis is calculated for each antibody concentration according to the formula (ER-MR)/(MR-SR) x 100, where ER is the mean quantified radioactivity (see, point ix above) for such antibody concentration, MR is the mean quantified radioactivity (see point ix above) for the MR Controls (see V above), and SR is the mean quantified radioactivity (see point ix above) for the SR Controls (see the point seen above); 4) "Increased ADCC" is defined as either an increase in the maximum percentage of specific lysis observed in the antibody concentration range tested above, and/or a reduction in the antibody concentration required to achieve one-half the maximum percentage of specific lysis observed in the antibody concentration range tested above. The increase in ADCC relative to ADCC, measured with the above assay, mediated by the same antibody, produced by the same type of host cell, using the same standard production, purification, formulation and storage methods, which are known to those skilled in the art, but which were not produced by host cells designed to overexpress GnTIII.
[048] Said "increased ADCC" can be obtained by subjecting the glycoengineering of said antibodies, which means the so-called natural elevation, effector functions of monoclonal antibody-mediated cells by designing its oligosaccharide component as described in Umana, P. et al. , Nature Biotechnol. 17:176 to 180 (1999) and U.S. Patent No. 6,602,684.
[049] The term "complement dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells by the antibody according to the invention in the presence of complement. CDC is preferably measured by treating a cell-expressing CD20 preparation with an anti-CD20 antibody according to the invention in the presence of complement. CDC is found if the antibody induces, at a concentration of 100 nM, the lysis (cell death) of 20% or more of the tumor cells after 4 hours. The assay is preferably performed with identified 51Cr or Eu tumor cells and measurements of 51Cr or Eu released. Controls include incubation of target tumor cells with complement but without antibody.
[050] Typically, type I and type II anti-CD20 antibodies of the IgG1 isotype show characteristic CDC properties. Type I anti-CD20 antibodies have an increased CDC (if IgG1 isotype) and type II anti-CD20 antibodies have a decreased CDC (if IgG1 isotype) compared to each other. Preferably, both type I and type II anti-CD20 antibodies are IgG1 isotype antibodies.
[051] The antibody "Rituximab" is a genetically engineered chimeric human gamma 1 murine constant domain containing monoclonal antibody directed against the human CD20 antigen. Such a chimeric antibody contains human gamma 1 constant domains and is identified by the name "C2B8" in EP2000149B1 (Anderson et. al, see, for example, Figures 4 and 5). Rituximab is approved to treat patients with relapsing or low-grade refraction or follicular, CD20 positive, B-cell non-Hodgkin's lymphoma. In vitro mechanism of action studies have been shown to show Rituximab to exhibit human complement-dependent cytotoxicity (CDC) (Reff et. al, Blood 83(2): 435 to 445 (1994)). Additionally, it exhibits significant activity in assays that measure antibody-dependent cellular cytotoxicity (ADCC).
[052] The term "humanized B-Ly1 antibody" refers to humanized B-Ly1 antibody as disclosed in WO2005/044859, which was obtained from murine monoclonal anti-CD20 antibody B-Ly1 (murine heavy chain variable region (VH) : SEQ ID NO: 1; murine light chain variable region (VL): SEQ ID NO: 2; see Poppema, S. and Visser, L., Biotest Bulletin 3: 131 to 139 (1987)) by chimerization with a human IgG1 constant domain and following humanization (see, WO2005/044859). These "humanized B-Ly1 antibodies" are disclosed in detail in WO2005/044859.
[053] Preferably the "humanized B-Ly1 antibody" has heavy chain variable region (VH) selected from the group of SEQ ID No: 3 to SEQ ID No: 20 (B-HH2 to B-HH9 and B-HL8 to B-HL17 of WO2005/044859). Especially preferred are Seq. ID Nos: 3, 4, 7, 9, 11, 13 and 15 (B-HH2, BHH-3, B-HH6, B-HH8, B-HL8, B-HL11 and B-HL13 of WO2005/044859) . Most preferably, said VH is BHH6. Preferably the "humanized B-Ly1 antibody" has the light chain variable region (VL) of SEQ ID No: 20 (B-KV1) of WO2005/044859. Furthermore, the humanized B-Ly1 antibody is preferably an IgG1 antibody. Preferably, such humanized B-Ly1 antibodies are glycoengineered (GE) in the Fc region, according to the procedures described in WO2005/044859, WO 2004/065540, Umana, P. et al, Nature Biotechnol. 17:176 to 180 (1999) and WO 99/154342. Most glycoengineered humanized B-Ly1 antibodies have an altered pattern of glycosylation in the Fc region, preferably with a reduced level of fucose residues. Preferably, at least 40% or more (in one embodiment between 40% and 60% in another embodiment, at least 50% and in still another embodiment at least 70% or more) of the Fc region oligosaccharides are non-fucosylated. In addition, Fc region oligosaccharides are preferably bisected. Most preferably, the "humanized B-Ly1 antibody" comprises VH B-HH6 and VL B-KV1 of WO2005/044859. As used herein, said antibody is also called "HuMabe<CD20>". Said antibody was designated as the Afutuzumab INN. In another most preferred embodiment, said antibody has a reduced level of fucose residues as defined above and/or the Fc region oligosaccharides are most preferably bisected. In yet another most preferred embodiment, said antibody exhibits increased ADCC as defined herein.
[054] The oligosaccharide component can significantly affect properties relevant to the efficacy of a therapeutic glycoprotein, including physical stability, resistance to protease attack, interactions with the immune system, pharmacokinetics, and specific biological activity. Such properties may depend not only on the presence or absence, but also on the specific structures, of oligosaccharides. Some generalizations between oligosaccharide structure and glycoprotein function can be made. For example, certain oligosaccharide structures mediate the rapid removal of glycoprotein from the bloodstream through interactions with proteins that bind specific carbohydrates while others can be bound by antibodies and trigger unwanted immune reactions. (Jenkins et al, Nature Biotechnol. 14:975 to 81 (1996)).
[055] Mammalian cells are the preferred hosts for the production of therapeutic glycoproteins, due to their ability to glycosylate proteins in the most compatible way for human application. (Cumming et al, Glicobiology 1:115 to 30 (1991); Jenkins et al, Nature Biotechnol. 14:975 to 81 (1996)). Bacteria very rarely glycosylate proteinase, like other common host types such as yeasts, filamentous fungi, plant cells and insects, yield glycosylation patterns associated with rapid removal from the bloodstream, unwanted immunological interactions and, in some specific cases, biological activity reduced. Among mammalian cells, Chinese hamster ovary (CHO) cells have been most commonly used during the past two decades. In addition to obtaining proper glycosylation patterns, these cells allow for the consistent generation of highly productive, genetically stable clonal cell lines. They can be grown at high densities in simple bioreactors using serum-free media and allow the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NS0 and SP2/0 mouse myeloma cells. More recently, production from transgenic animals has also been tested. (Jenkins et al, Nature Biotechnol. 14:975-981 (1996)).
[056] All antibodies contain carbohydrate structures at conserved positions in the constant regions of the heavy chain, with each isotype having a distinct set of N-linked carbohydrate structures, which variably affect protein assembly, secretion, or functional activity (Wright , A. and Morrison, SL, Trends Biotech. 15: 26 to 32 (1997)). The structure of the fixed N-linked carbohydrate varies considerably depending on the degree of processing and can include complex biantennary, high-mannose, as well as multiple branched oligosaccharides. Typically, there is heterogeneous processing of core oligosaccharide structures fixed at a particular glycosylation site so that even monoclonal antibodies exist as multiple glycoforms. Likewise, it has been shown that large differences in antibody glycosylation occur between cell lines and even small differences are observed for a given cell line grown under different culture conditions (Lifely, MR et al, Glicobiology 5(8): 813 to 22 (1995)).
[057] One way to achieve large potency increases while maintaining a simple production process and potentially avoiding significant unwanted side effects is to elevate natural effector functions, mediated by monoclonal antibody cells by engineering the oligosaccharide component of the same as described in Umana, P. et al, Nature Biotechnol. 17:176 to 180 (1999) and U.S. Patent 6,602,684. IgG1 type antibodies, the most commonly used antibodies in cancer immunotherapy, are glycoproteins that have a conserved N-linked glycosylation site at Asn297 in each CH2 domain. The two biantennary complex oligosaccharides fixed on Asn297 are hidden between the CH2 domains, which form extensive contacts with the main polypeptide chain, and their presence is essential for the antibody to mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, MR et al, Glicobiology 5: 813 to 822 (1995); Jefferis, R. et al, Immunol. Rev. 163: 59 to 76 (1998); Wright, A. and Morrison, SL, Trends Biotechnol. 32 (1997)).
[058] It was previously shown that overexpression in Chinese hamster ovary (CHO) cells of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), the glycosyltransferase that catalyzes the formation of bisected oligosaccharides, significantly increases ADCC activity vitro of a chimeric anti-neuroblastoma monoclonal antibody (chCE7) produced by engineered CHO cells. (See Umana, P. et al, Nature Biotechnol. 17:176 to 180 (1999); and WO 99/154342, the contents of which are fully incorporated herein by reference). The chCE7 antibody belongs to a large class of unconjugated monoclonal antibodies that have high affinity and tumor specificity, but have too little potency to be clinically useful when produced in industry standard cell lines that lack the GnTIII enzyme (Umana, P. et al, Nature Biotechnol. 17:176 to 180 (1999)). This was the first to show that large increases in ADCC activity could be achieved by engineering cells that produce antibodies to express GnTIII, which also led to an increase in the proportion of bisected oligosaccharides associated with the constant Fc region, including bisected, non-fucosylated oligosaccharides , above the levels found in naturally occurring antibodies.
[059] The term "CD20 antigen expression" is intended to indicate a significant level of expression of the CD20 antigen in a cell, preferably on the cell surface of a B cell, more preferably a B cell, from a tumor or cancer, respectively, preferably a non-solid tumor. Patients with a "cancer expressing CD20" can be determined by standard assays known in the art. "CD20 antigen expression" is also preferably intended to indicate a significant level of antigen expression CD20 on a cell, preferably on the cell surface of a B Cell, more preferably a B Cell, in an autoimmune disease. The expression of the CD20 antigen is measured using, for example, immunohistochemical detection (ICH), FACS or by PCR-based detection of the corresponding mRNA.
[060] The "patients" or "subjects" are any mammals that suffer from the conditions or diseases, according to the invention and, preferably, are human.
[061] The term "cancer expressing CD20", as used herein, preferably refers to lymphomas (preferably B-cell non-Hodgkin's lymphomas (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example: (a) follicular lymphomas, (b) Small Non-Cleaved Cell Lymphomas/Burkitt's lymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma and non-Burkitt's lymphoma), (c) marginal zone lymphomas (including extranodal marginal zone B cell lymphoma (Lymphomas of mucosa-associated lymphatic tissue, MALT), nodal marginal zone and splenic marginal zone B cell lymphoma), (d) Mantle cell lymphoma ( MCL), (e) Large Cell Lymphoma (including Diffuse Large B Cell Lymphoma (DLBCL), Diffuse Mixed Cell Lymphoma, Immunoblastic Lymphoma, Primary Mediastinal B Cell Lymphoma, Angiocentric Lymphoma - Pulmonary B Cell Lymphoma), (f) hairy cell leukemia, (g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia, (h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, (i ) neoplasm of and plasma cell, plasma cell myeloma, multiple myeloma, plasmacytoma, and (j) Hodgkin's disease.
[062] Preferably the cancer expressing CD20 is a B-cell non-Hodgkin's lymphoma (NHL). Other examples of CD20-expressing cancers include: Mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell diffuse large cell lymphoma (DLCL), Burkitt's lymphoma, lymphocytic leukemia hairy cell, follicular lymphoma, multiple myeloma, marginal zone lymphoma, post-transplant lymphoproliferative disorder (PTLD), HIV-associated lymphoma, Waldenstrom's macroglobulinemia, or primary CNS lymphoma.
[063] As used herein, "autoimmune disease" refers to a disease or disorder that arises from and is targeted against the individual's own tissue. Examples of autoimmune diseases or disorders include, but are not limited to arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and inflammatory sclerosis, disease-associated responses disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving T-cell infiltration and responses inflammatory disorders, arteriosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), juvenile diabetes, multiple sclerosis, allergic encephalomyelitis, immune responses associated with acute and late cytokine and T lymphocyte-mediated hypersensitivity, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis, agranulocytosis. cough, vasculitis (including ANCA), aplastic anemia, Diamond-Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia , pancytopenia, leukopenia, diseases involving leukocyte diapedesis, central nervous system (CNS) inflammatory disorders, multiple organ dysfunction syndrome, myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bechet's disease, Castleman's syndrome, Goodpasture's syndrome, Lambert-Eaton's myasthenic syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens Johnson's syndrome, bullous pemphigoid, pemphigus, autoimmune polyendocrinopathies, nephropathy, IgM polyneuropathies or neuropathy IgM-mediated, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), tr autoimmune ombocytopenia, autoimmune testis and ovary disease including autoimmune orchitis and ophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Grave's disease, polyglandular autoimmune syndromes (or polyglandular I endocrinopathy syndromes), type I diabetes mellitus also called diabetes mellitus insulin dependent (IDDM) and Sheehan syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs NSIP (non-specific interstitial pneumonia), Guillain-Barre syndrome, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), middle vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), ankylosing spondylitis, Berger disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac disease (gluten enteropathy), cryoglobulinaemia, amyotrophic lateral sclerosis (ALS) , coronary artery disease, etc.
[064] A "growth inhibitory agent", when used herein, refers to a compound or composition that inhibits the growth of a cell especially a cancer expressing CD20 cell in vitro or in vivo. Thus, the growth inhibitory agent may be one that significantly reduces the percentage of cells that express CD20 in the S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (in a place other than the S phase), such as agents that induce M-phase capture and G1 capture. Classic M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topo II inhibitors such as epirubicin doxorubicin, etoposide daunorubicin, and bleomycin. Those agents that capture G1 also infiltrate S-phase capture, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. Additional information can be found in "The Molecular Basis of Cancer", Mendelsohn and Israel eds., Chapter 1 entitled "Cell cycle regulation, oncogenes and anti-tinaoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995) especially p. 13.
[065] "Treatment" refers to therapeutic treatment and prophylactic or preventive measures. Those in need of treatment include those already with the disease as well as those in whom the disease must be prevented. Therefore, the patient to be treated herein may have been diagnosed as having the disease or may be predisposed or susceptible to the disease.
[066] The term "cytotoxic agent", as used herein, refers to a substance that inhibits or prevents the function of cells and/or causes the destruction of cells. The term is intended to include radioactive isotopes (eg, At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
[067] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and (CYTOXAN™) cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARENOL™); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including synthetic analogue topotecan (HYCAMTINTM), CPT-11 (irinotecan, CAMPTOSARTM), acetylcamptothecin, scopoletin and 9-amino camptothecin); bryostatin; calistatin; CC-1065 (including the synthetic analogues adozelesin, carzelesin and bizelesin); podophyllotoxin; podophylinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleuterobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the antibiotic enediin (for example, calicheamicin, especially calicheamicin gamma-1 and calicheamicin omega 11 (see, for example, Angew, Chemie Intl. Ed. Engl, 33: 183 to 186 (1994)); Esperamycin; as well as neocarzinostatin chromophores and chromoprotein-related antibiotic enediin chromophores), aclacinomisins, actinomycin, autramycin, azasserin, bleomycins, cactinomycins, carabicin, carminomycin, carzinophilin, chromomicinia, dactinomycin, 6-dianorubin -L-norleucine, doxorubicin (including ADRIAMICINATM, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXILTM), liposomal doxorubicin TLC D-99 (MYYOCETTM) liposomal liposomal doxorubicin (MYOXETTM) and deoxidoxorubicin) epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamicin a, olivomycins, peplomycin, potfiromycin, puromycin, chelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZARTM), tegafur (UFTORALTM), capecitabine (XELODATM), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoxide; aminolevulinic acid; enyluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamin; demecolcine; diaziquone; elfornithine; elliptinium acetate; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pyrarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSKLTM polysaccharide complex (JHS Natural Products eugene, OR); razoxan; rhizoxin; sizofran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridine A, and anguidine); urethane; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacitosine; arabinoside ("Ara-C"); thiotepa; taxoids, eg, paclitaxel (TAXOLTM), engineered albumin nanoparticle paclitaxel formulation (ABRAXANETM) and docetaxel (TAXOTERETM); chloranbucil; 6-thioguanine; mercaptopurine; methliotrexate; platinum agents such as cisplatin, oxaliplatin, and platinum agents such as cisplatin, oxaliplatin ; vincas, which prevent the polymerization of tubulin from forming microtubules, including vinblastine (VELBANTM), vincristine (ONCOVINTM), vindesine (ELDISINATM), FILDESINTM) and vinorelbine (NAVELBINATM)); etoposide (VP-16); ifosfamide leuco; ; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETINTM); bisphosphonates such as clodronate (p for example, BONEFOSTM or OSTACTM) etidronate (DIDROCALTM), NE-58095, zoledronic acid/zoledronate (ZOMETATM), alendronate (FOSAMAJXTM), pamidronate (AREDIATM), tiludronate (SKELIDTM), or risedronate (ACTONELTM); troxacitabine (a 1,3-dioxolane cytosine nucleoside analogue); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPETM vaccine and gene therapy vaccines, for example, the ALLOVECTINTM vaccine, the LEUVECTINTM vaccine, and the VAXIDTM vaccine; topoisomerase 1 inhibitor (for example, LURTOTECANTM); rmRH (for example, ABARELIXTM); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitor (for example celecoxib or etoricoxib), proteosome inhibitor (for example PS341); bortezomib (VELCADETM); CCI-779; tipifarnib (R1 1577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSETM); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone (which optionally further comprises interferon-V (CHVP/interferon-V), FOLFOX, an abbreviation for a regimen of treatment with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovovin, CVP (cyclophosphamide, vincristine and prednisolone), MCP (mitozantron, chlorambucil and prednisolone), FC (fludarabine and cyclophosphamide), ICE (ifosfamide, carboplatin and etoposide), and dexamethasone cytarabine and cisplatin (DHAP), dexamethasone, liposomal doxorubicin and vincristine (DVD) etc.
[068] An "anti-angiogenic agent" refers to a compound that blocks, or interferes to some degree, the development of blood vessels. The anti-angiogenic factor can, for example, be a small molecule or antibody that binds to growth factor or growth factor receptor involved in promoting angiogenesis. The preferred anti-angiogenic factor herein is an antibody that binds to Vascular Endothelial Growth Factor (VEGF), such as Bevacizumab (AVASINTM).
[069] The term "cytokine" is a generic term for proteins released by one cell population that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicular stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH), liver growth factor; fibroblast growth factor; prolactin; placental lactogen, tumor necrosis factor α and β; mullerian inhibitory substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); neural growth factors such as NGF-β, platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-beta; growth factor I and II such as insulin; erythropoietin (EPO), osteoinductive factors; interferons such as interferon-α, -β and -y, colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL -11, IL-12, a tumor necrosis factor such as TNF-α or TNF-β; and other polypeptide factors including LIF and kit linker (KL). As used herein, the term cytokine includes proteins of natural origin or recombinant cell culture and biologically active equivalents of the native sequence cytokines.
[070] The term "effective amount" refers to an amount that provides the desired effect. In the case of a formulation ingredient such as the hyaluronidase enzyme, according to the present invention, an effective amount is the amount necessary to increase the dispersion and absorption of the co-administered anti-CD20 antibody such that the anti-CD20 antibody can act to a therapeutically effective way as mentioned above. In the case of a pharmaceutical drug substance it is the amount of active ingredient effective to treat a disease in the patient. Where the disease is cancer, the effective amount of the drug may reduce the number of cancer cells; reduce tumor size; inhibit (i.e. reduce to some extent and, preferably, stop) cancer cell infiltration into peripheral organs; inhibit (ie, reduce to some extent, and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with cancer. To the extent that the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. The effective amount can extend survival free progression, result in an objective response (including a partial response, PR, or complete response, CR), increase total survival time, and/or improve one or more symptoms of cancer.
[071] The term "pharmaceutical formulation" refers to a preparation such that it allows the biological activity of the active ingredient to be effective and that it does not contain additional components that are unacceptably toxic to a subject to which the formulation was administered. Such formulations are sterile.
[072] A "sterile" formulation is aseptic or free of all living microorganisms and their spores.
[073] A "stable" formulation is one in which all proteins in it essentially retain physical stability and/or chemical stability and/or biological activity upon storage at the intended storage temperature, eg, 2 to 8 oC. Preferably, the formulation essentially retains its chemical and physical stability as well as its biological activity upon storage. The shelf life is usually selected based on the intended shelf life of the formulation. Furthermore, the formulation is preferably stable after freezing (to, for example, -20°C) and thawing of the formulation, for example, following 1 or more freeze-thaw cycles. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247 to 301, Vincent Lee Ed., Marcel Dekker, Inc., New York, New York, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29 to 90 (1993), for example. Stability can be measured at a selected temperature for a selected period of time. Stability can be assessed qualitatively and/or quantitatively in a variety of different ways, including assessment of aggregate formation (eg using size exclusion chromatography, measuring turbidity and/or by visual inspection); evaluating charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; SDS-PAGE analysis to compare reduced and intact antibody; assess the antibody's biological activity or antigen-binding function; etc. Instability can involve any one or more of: aggregation, deamidation (eg Asn deamidation), oxidation (eg Met oxidation), isomerization (eg Asp isomerization), shear/hydrolysis/fragmentation (eg , fragmentation of the joint region), formation of succinimide, mismatched cysteine(s) etc.
[074] Therapeutic formulations of the antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with pharmaceutically acceptable carriers, excipients or optional stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable excipient carriers or stabilizers are non-toxic to recipients at the dosages and concentrations employed.
[075] The term "surfactant", as used herein, denotes a pharmaceutically acceptable surface active agent. In the formulation of the invention, the amount of surfactant is described as a percentage expressed in weight/volume. The most commonly used unit weight/volume is mg/mL. Suitable examples of pharmaceutically acceptable surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyethylene-polypropylene glycols, polyoxyethylene stearates, polyoxyethylene alkyl ethers, e.g. polyoxyethylene monolauryl ether, polyoxyethylene alkylphenyl ethers (Triton-X), copolymer polyoxyethylene-polyoxypropylene (Poloxamer, Pluronic) and sodium dodecyl sulfate (SDS). The most suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20, (available under the tradename Tween 20TM) and polysorbate 80 (available under the tradename Tween 80TM). The most suitable polyethylene-polypropylene copolymers are those available under the trade names Pluronic® F68 or Poloxamer 188TM. Preferred polyoxyethylene stearates are those available under the brand name Myrj™. The most suitable polyoxyethylene alkyl ethers are those available under the brand name BrijTM. The most suitable alkylphenol polyoxyethylene ethers are available under the tradename Triton-X.
[076] The term "buffer", as used herein, denotes a pharmaceutically acceptable buffer. As used herein, the term "the buffering agent provides a pH of 5.5 to ± 2.0" refers to an agent that ensures that the solution comprising it resists changes in pH by the action of conjugated acid components /base of the same. Pharmaceutically acceptable buffers in accordance with the invention comprise, but are not limited to, histidine buffers, citrate buffers, gluconate buffers, succinate buffers, glycylglycine acetate buffers and other organic acid buffers and phosphate buffers. Preferred buffers comprise L-histidine or mixtures of L-histidine with L-histidine hydrochloride with isotonicity agents and potentially pH adjustment with an acid or base known in the art. L-histidine is most preferred.
[077] A "histidine buffer" is a buffer comprising the amino acid histidine. Examples of histidine buffers include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate. It has been found that the preferred histidine buffer identified in the examples herein is histidine chloride. In the preferred embodiment, the histidine chloride buffer is prepared by titrating L-histidine (solid, free base) with dilute hydrochloric acid or by dissolving L-histidine and L-histidine hydrochloride (for example, as a monohydrate ) in a defined ratio and quantity.
[078] By "isotonic" is meant that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations will generally have an osmolality of ~300 mOsm/kg. Isotonicity can be measured using a vapor pressure or freezing point depression type osmometer.
[079] The term "isotonicity agents", as used herein, denotes pharmaceutically acceptable isotonicity agents. Isotonicity agents are used to provide an isotonic formulation. An isotonic formulation is liquid or a liquid reconstituted from a solid form, for example, a lyophilized form, and denotes a solution that has the same tonicity as another solution to which it is compared, such as a physiological salt solution and blood serum. Suitable isotonicity agents include, but are not limited to, salts, including but not limited to sodium chloride (NaCl) or potassium chloride, sugars and sugar alcohols including but not limited to glucose, sucrose, trehalose or glycerol and any component from the group of amino acids, sugars, salts and combinations thereof. Isotonicity agents are generally used in a total amount of about 5 mM to about 350 mM.
[080] The term "liquid" as used herein in connection with the formulation according to the invention denotes a formulation that is liquid at a temperature of at least about 2 to about 8 oC. The term "lyophilized" as used herein in connection with the formulation according to the invention denotes a formulation which is freeze-dried by the formulation and subsequently sublimates the ice of the frozen content by any method of freezing known in the technique, for example, commercially available freeze drying devices.
[081] The term "salts", as used herein, denotes a salt in an amount of from about 1 mM to about 500 mM. Non-limiting examples of salts include salts of any combinations of the sodium potassium, calcium or magnesium cations with chloride, phosphate, citrate, succinate, sulfate anions or mixtures thereof.
[082] The term "amino acid", as used herein, denotes an amino acid in an amount of from about 1 to about 100 mg/mL which comprises, but is not limited to arginine, glycine, ornithine, glutamine, asparagine, lysine, histidine, glutamic acid, asparagic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine and proline.
[083] A "saccharide", herein, comprises the general composition (CH2O)n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars etc. Examples of saccharides herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran erythritol, glycerol, arabitol, silitol, sorbitol, mannitol, melibiose, melezitose, raffinose, manotriosis, maltose, stachyose lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose etc. Also included in the definition according to the invention are glucosamine, N-Methylglucosamine (called "Meglumine"), galactosamine and neuraminic acid and combinations of saccharides according to the invention. The preferred saccharide herein is a non-reducing disaccharide such as trehalose or sucrose. The most preferred saccharide in accordance with the present invention is trehalose.
[084] The term "stabilizer" refers to pharmaceutically acceptable stabilizers, such as, but not limited to, amino acids and sugars, as described in the sections above, as well as commercially available dextrans of any type and molecular weight, as known in the technique.
[085] The term "antioxidant" denotes a pharmaceutically acceptable antioxidant. May include excipients such as methionine, benzyl alcohol or any other excipient used to minimize oxidation.
[086] The term "a method of treating", or an equivalent, when applied to, for example, cancer, refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of cancer. "A method of treating" cancer or other proliferative disorder does not necessarily mean that cancer cells or another disorder will, in fact, be eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated. Often, a method of treating cancer will be carried out even with a low probability of success, but which, given a patient's medical history and expected survival, is nevertheless considered to induce an overall beneficial course of action.
[087] The problem to be solved by the present invention is therefore to provide innovative, stable, highly concentrated pharmaceutical formulations of a pharmaceutically active anti-CD20 antibody or a mixture of molecules of such an antibody for subcutaneous injection. Such formulations comprise, in addition to high amounts of anti-CD20 antibody or mixtures thereof, a buffering agent, a stabilizer or a mixture of two or more stabilizers, a nonionic surfactant and, preferably, an effective amount of at least one enzyme hyaluronidase. The preparation of highly concentrated antibody formulations is challenging because of a potential increase in viscosity at high protein concentration and a potential increase in protein aggregation, a phenomenon that is itself concentration-dependent. High viscosities negatively impact process capability (eg pumping and filtration steps) of antibody formulations and delivery (eg syringe capacity). By adding excipients, high viscosities could be reduced in some cases. Control and analysis of protein aggregation is an increasing challenge. Aggregation is potentially encountered during various steps in the manufacturing process, which include fermentation, purification, formulation and during storage. Different factors, such as temperature, protein concentration, stress on agitation, freezing and thawing, solvent and surfactant effects and chemical modifications, can influence the aggregation behavior of a therapeutic protein. During the development of a highly concentrated antibody formulation, the protein aggregation tendency has to be monitored and controlled by the addition of various excipients and surfactants [Kiese S. et al, J. Pharm. Sci., 2008; 97(10); 4347 to 4366].
[088] In a first aspect, the present invention provides a highly concentrated stable pharmaceutical formulation of a pharmaceutically active anti-CD20 antibody or a mixture of molecules of such antibody for parenteral application. Preferably, the route of application is by intravenous administration as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinal, subcutaneously, intraarticularly, intrasynovially, or intrathecally. Intravenous or subcutaneous administration of the antibodies is preferred; subcutaneous injection is most preferred. As shown above, it is not trivial to generate a highly concentrated stable pharmaceutical formulation of a CD20 antibody that is essentially free of particles. If said formulation is intended for subcutaneous application, then, in the preferred embodiment, said formulation is combined with the enzyme hyaluronidase.
[089] More particularly, the highly concentrated stable pharmaceutical formulation of a pharmaceutically active anti-CD20 antibody formulation of the present invention comprises: about 20 to 350 mg/ml of anti-CD20 antibody; about 1 to 100 mM of a buffering agent that provides a pH of 5.5 to ± 2.0; about 1 to 500 mM of a stabilizer or a mixture of two or more stabilizers, wherein, optionally, methionine is used as a secondary stabilizer, preferably at a concentration of 5 to 25 mM; 0.01 to 0.1% of a nonionic surfactant; and preferably, an effective amount of at least one hyaluronidase enzyme.
[090] The highly concentrated stable pharmaceutical formulation of a pharmaceutically active anti-CD20 antibody formulation can be provided in liquid form or it can be provided in lyophilized form. The antibody concentration in the reconstituted formulation can be increased by reconstituting a lyophilized formulation to provide a protein concentration in the reconstituted formulation that is about 2 to 40 times greater than the protein concentration in the mixture prior to the lyophilization step.
[091] The preferred concentration of anti-CD20 antibody is 50 to 150 mg/ml, more preferably it is 75 to 150 mg/ml, even more preferred it is 120 to ± 20 mg/ml, most preferably , is about 120 mg/ml.
[092] The preferred concentration of the buffering agent is from 1 to 50 mM, more preferably from 10 to 30 mM; the concentration is most preferably about 20 mM. Various buffering agents are known to the person skilled in the art as outlined above. The preferred buffering agent is selected from the group consisting of a histidine buffer, acetic acid buffer, and citric acid buffer, most preferably an L-histidine/HCl buffer. The histidine buffer according to the invention is used in an amount of from about 1mM to about 50mM, preferably from about 10mM to about 30mM and still more preferably from about 20mM. The acetic acid buffer according to the invention is preferably from about 10 mM to about 30 mM and most preferably from about 20 mM. The citric acid buffer according to the invention is preferably from about 10 mM to about 30 mM and most preferably from about 20 mM.
[093] Regardless of the buffer used, the pH will be adjusted to a value comprising about 4.5 to about 7.0 and preferably about 5.5 to about 6.5, also preferably it is selected to starting from the group consisting of 5.5, 6.0, 6.1 and 65. This pH can be achieved by adjusting with an acid or base known in the art or by using suitable mixtures of buffer components or both.
[094] The stabilizer(s) (used synonymously with the term "stabilizing agent" in this patent specification) is/are preferably selected from the group consisting of a salt, a carbohydrate, saccharide and amino acid( s), most preferably a carbohydrate or saccharide, most preferably a sugar admitted by the authorities as a suitable additive or excipient in pharmaceutical formulations, most preferably selected from the group consisting of α,α-trehalose, NaCl and methionine dihydrate . The preferred concentration of the stabilizer is from 15 to 250 mM, or more preferably from 150 to 250 mM. Most preferred is a concentration of about 210 mM. The formulation may contain a secondary stabilizer, wherein such secondary stabilizer is preferably methionine, preferably at a concentration of 5 to 25 mM, more preferably at a concentration of 5 to 15 mM. And most preferably the methionine concentration is about 10 mM.
[095] The nonionic surfactant is preferably a polysorbate, more preferably it is selected from the group of polysorbate 20, polysorbate 80 and polyethylene-polypropylene copolymer. The concentration of the nonionic surfactant is from 0.01 to 0.1% (p/v), or from 0.02 to 0.08% (p/v) and preferably from 0.02 to 0.06% (p /v), and most preferably about 0.06% (w/v).
[096] The term "sugar" as used herein denotes a pharmaceutically acceptable sugar used in an amount of from about 25 mM to about 500 mM. It is preferably from 100 to 300 mM. It is most preferably from 180 to 240 mM. And most preferably 210 mM.
[097] The concentration of the hyaluronidase enzyme depends on the hyaluronidase enzyme currently used in the preparation of the formulation according to the invention. An effective amount of the hyaluronidase enzyme can easily be determined by the person skilled in the art based on the additional presentation below. It should be provided in sufficient quantity so that an increase in dispersion and absorption of the co-administered anti-CD20 antibody is possible. The effective amount of the hyaluronidase enzyme is preferably about 1000 to 16000 U/ml, wherein said amount corresponds to about 0.01 mg to 0.15 mg of protein based on a presumed specific activity of 100,000 U/mg. The preferred concentration of the hyaluronidase enzyme is about 1500 to 12000 U/ml. Most preferably, a concentration of about 2000 U/ml or about 12000 U/ml. The amounts specified herein above correspond to the amount of hyaluronidase enzyme initially added to the formulation. The hyaluronidase enzyme is present either as a final combined formulation or for use in co-administration, for example, as a co-formulation as further outlined below. The important issue for the claimed formulation is that at the moment it is ready to use and/or is injected as in the claimed composition.
[098] The hyaluronidase enzyme can be derived from animals, human samples or manufactured based on recombinant DNA technology as further described below.
[099] More particularly the highly concentrated stable pharmaceutical formulations according to the present invention have one of the following preferred compositions: a) from 100 to 150 mg/ml of anti-CD20 antibody, wherein such antibody is preferably Rituximab, Ocrelizumab or HuMabe <CD20>; 1 to 50 mM of a histidine buffer, preferably L-histidine/HCl at a pH of about 5.5; from 15 to 250 mM of a stabilizer which is preferably α,α-trehalose dihydrate and optionally methionine as a second stabilizer at a concentration of 5 to 25 mM; a nonionic surfactant selected from the group of polysorbate 20 and polysorbate 80, preferably from 0.02 to 0.06% (w/v), and optionally from 1000 to 16000 U/ml of a hyaluronidase enzyme, preferably rHuPH20, with most preferably at a concentration of 2000 U/ml or 12000 U/ml. b) 120 ± 20 mg/ml of anti-CD20 antibody, which antibody is preferably Rituximab, Ocrelizumab or HuMabe<CD20>; from 10 to 30 mM, preferably 20 mM of a histidine buffer, preferably L-histidine/HCl at a pH of about 5.5; 150 to 250 mM, preferably 210 mM of a stabilizer which is preferably α,α-trehalose dihydrate and optionally methionine as a second stabilizer at a concentration of 5 to 25 mM, preferably 5 to 15 mM, and most preferably 10 mM; a non-ionic surfactant selected from the group of polysorbate 20 and polysorbate 80, preferably from 0.02 to 0.06% (w/v), and optionally from 1000 to 16000 U/ml, preferably from 1500 to 12000 U/ml , and most preferably 2000 U/ml or 12000 U/ml of a hyaluronidase enzyme, preferably rHuPH20. c) 120 mg/ml of anti-CD20 antibody, which antibody is preferably Rituximab, Ocrelizumab or HuMabe<CD20>; from 10 to 30 mM, preferably 20 mM of a histidine buffer, preferably L-histidine/HCl at a pH of about 5.5; 150 to 250 mM, preferably 210 mM of a stabilizer which is preferably α,α-trehalose dihydrate and optionally methionine as a second stabilizer at a concentration of 5 to 25 mM, preferably 5 to 15 mM, most preferably 10 mM; a non-ionic surfactant selected from the group of polysorbate 20 and polysorbate 80, preferably from 0.02 to 0.06% (w/v), and optionally from 1000 to 16000 U/ml, preferably from 1500 to 12000 U/ml , most preferably 2000 U/ml or 12000 U/ml of a hyaluronidase enzyme, preferably rHuPH20. d) 120 mg/ml of anti-CD20 antibody, preferably Rituximab; 20 mM of a histidine buffer, preferably L-histidine/HCl at a pH of about 5.5; 210 mM α,α-trehalose dihydrate and optionally 10 mM methionine as a second stabilizer; a non-ionic surfactant selected from the group of polysorbate 20 and polysorbate 80, preferably from 0.02 to 0.06% (w/v), and optionally 2000 U/ml or 12000 U/ml of a hyaluronidase enzyme, preferably rHuPH20 . e) A lyophilized formulation comprising 120 mg/ml of anti-CD20 antibody, preferably Rituximab; 20 mM of a histidine buffer, preferably L-histidine/HCl at a pH of about 5.5; 210 mM α,α-trehalose dihydrate and optionally 10 mM methionine as a second stabilizer; a non-ionic surfactant selected from the group of polysorbate 20 and polysorbate 80, preferably from 0.02 to 0.06% (w/v), and optionally 2000 U/ml or 12000 U/ml of a hyaluronidase enzyme, preferably rHuPH20.
[0100] A stable pharmaceutical formulation of a pharmaceutically active anti-CD20 antibody is provided which comprises about 30 mg/ml to 350 mg/ml, for example about 30 mg/ml to 100 mg/ml (including about 30mg µg/ml, about 50mg/ml or about 100mg/ml) Ocrelizumab (e.g. humanized 2H7.v16); about 1 to 100 mM of a buffering agent (eg, sodium acetate) that provides a pH of 5.5 plus or minus 2.0 (eg, pH 5.3); about 15 to 250 mM of a stabilizer or a mixture of two or more stabilizers (including trehalose, for example about 8% trehalose dihydrate); about 0.01 to 0.1% (w/v) of a nonionic surfactant; and optionally an effective amount of at least one hyaluronidase enzyme (for example rhHUPH20), preferably in an amount of from about 1,500 U/ml to about 12,000 U/ml.
[0101] Alternative compositions of preferred formulations are given in the examples.
[0102] It has been proposed to facilitate the subcutaneous injection of therapeutic proteins and antibodies the use of small amounts of soluble hyaluronidase glycoproteins (sHASEGPs); see WO2006/091871. The addition of such soluble hyaluronidase glycoproteins (either as a combined formulation or via co-administration) has been shown to facilitate the administration of the therapeutic drug into the hypodermis. Through the rapid polymerization of hyaluronan HA in the extracellular space sHASEGP reduces the viscosity of the interstitium, and thereby increases hydraulic conductance and allows larger volumes to be safely and comfortably delivered into the subcutaneous tissue. The increased hydraulic conductance induced by sHASEGP through reduced interstitial viscosity allows for greater dispersion, potentially increasing systemic bioavailability of SC-administered therapeutic drug.
[0103] The highly concentrated stable pharmaceutical formulations of the present invention which comprise a soluble hyaluronidase glycoprotein are therefore particularly suitable for subcutaneous injection. It is clearly understood by the person skilled in the art that such a formulation comprising an anti-CD20 antibody and a soluble hyaluronidase glycoprotein can be provided for administration as a single combined formulation or alternatively as two separate formulations which can be mixed moments before of subcutaneous injection. Alternatively the anti-CD20 antibody and soluble hyaluronidase glycoprotein can be administered as separate injections at different sites in the body, preferably at sites that are immediately adjacent to each other. It is also possible to inject the therapeutic agents present in the formulation according to the present invention as consecutive injections, for example, first the soluble hyaluronidase glycoprotein followed by the injection of the anti-CD20 antibody formulation. These injections can also be performed in reverse order, viz. first injecting the anti-CD20 antibody formulation followed by the injection of soluble hyaluronidase glycoprotein. In case the anti-CD20 antibody and soluble hyaluronidase glycoprotein are administered as separate injections, one or both proteins must be provided with the buffering agent, the stabilizer(s) and the nonionic surfactant at the concentrations as specified in the appended claims but excluding the hyaluronidase enzyme. The hyaluronidase enzyme can thus be supplied, for example, in an L-histidine/HC1 buffer at a pH of about 6.5, from 100 to 150 mM NaCl and from 0.01 to 0.1% (w/v) of polysorbate 20 or polysorbate 80. in a preferred embodiment the anti-CD20 antibody is provided with the buffering agent, the stabilizer(s) and the nonionic surfactant in the concentrations as specified in the attached claims.
[0104] As mentioned above the soluble hyaluronidase glycoprotein can be considered to be an additional excipient in the formulation of anti-CD20. Soluble hyaluronidase glycoprotein can be added to the anti-CD20 formulation at the time of manufacture of the anti-CD20 formulation or it can be added moments before injection. Alternatively the soluble hyaluronidase glycoprotein can be given as a separate injection. In the above case the soluble hyaluronidase glycoprotein can be supplied in a separate receptacle or in lyophilized form which needs to be reconstituted with suitable diluents before subcutaneous injection takes place, or it can be supplied as a liquid formulation by the manufacturer. The anti-CD20 formulation and the soluble hyaluronidase glycoprotein can be purchased as separate entities or can also be supplied as kits comprising both the injection components and instructions suitable for subcutaneous administration. Suitable instructions for reconstitution and/or administration of one or both formulations can also be provided.
[0105] Therefore the present invention also provides pharmaceutical compositions consisting of a highly concentrated stable pharmaceutical formulation of a pharmaceutically active anti-CD20 antibody or a mixture of such antibody and a suitable amount of at least one hyaluronidase enzyme in the form of a kit which comprises both the injection components and the appropriate instructions for their subcutaneous administration.
[0106] A further aspect of the present invention relates to injection devices comprising a highly concentrated stable pharmaceutical formulation according to the present invention. Such formulation may consist of a pharmaceutically active anti-CD20 antibody or a mixture of such antibody molecules and suitable excipients as outlined below and may further comprise a soluble hyaluronidase glycoprotein either as a combined formulation or as a separate formulation for co-administration.
[0107] A variety of anti-CD20 antibodies are known in the prior art. Such antibodies are preferably monoclonal antibodies. These can be called chimeric antibodies, humanized antibodies or fully human antibodies. These can either be full length anti-CD20 antibodies; anti-CD20 antibody fragments that have the same biological activity; including amino acid sequence variants and/or glycosylation variants of such antibodies or fragments. Examples of humanized anti-CD20 antibodies are known under the INN names Rituximab, Ocrehzumab and Afutuzumab (HuMabe<CD20>). The most successful anti-CD20 therapeutic antibody is Rituximab sold by Genentech Inc. and F. Hoffmann-La Roche Ltd under the tradename MABTHERATM or RITUXANTM.
[0108] The anti-CD20 antibody as defined herein is preferably selected from the group of Rituximab (see for example US Patent No. 7,381,560 and EP2000149B1 to Anderson et. al, see for example figures 4 and 5), Ocrehzumab (as per shown in WO 2004/056312) and in WO 2006/084264 (for example the variants shown in tables 1 and 2), preferably variant v.16 or v.114 or v.511 and Afutuzumab (HuMabe<CD20>; see document WO2005/044859). And most preferably the anti-CD20 antibody is Rituximab. The terms "Rituximab", "Ocrelizumab" and "Afutuzumab" (HuMabe<CD20>) encompass all corresponding anti-CD20 antibodies that meet the necessary requirements to obtain a marketing authorization as an identical or biosimilar product in a country or territory selected from the group of countries consisting of the USA, Europe and Japan. Rituximab has the CDR regions defined in US Patent No. 7,381,560 and in EP2000149B1.
[0109] Numerous soluble hyaluronidase glycoproteins are known in the prior art. in order to further define the function, mechanism of action and properties of such soluble hyaluronidase glycoproteins the following background information is provided.
[0110] The interstitial matrix (hypodermic) SC is comprised of a network of fibrous proteins embedded within a viscoelastic gel of glycosaminoglycans. Hyaluronan (HA), an unsulfated linear repeating disaccharide, is the prominent glycosaminoglycan of SC tissue. HA is secreted into the interstitium via fibroblasts as a viscous polymer in high molecular weight megadaltons that are subsequently degraded locally, in the lymph, and in the liver, through the action of lysosomal hyaluronidases and exoglycosidases. Approximately 50% of the hyaluronan in the body is produced by SC tissue, which is found in approximately 0.8 mg/gm wet weight tissue [Aukland K. and Reed R., "Interstitial-Lymphatic Mechanisms in the control of Extracellular Fluid Volume ", Physiology Reviews", 1993; 73: 1 to 78]. It is estimated that the average 70 kg adult contains 15 grams of HA, of which 30 percent is transformed (synthesized and degraded) daily [Laurent LB, et al, "Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver", Exp. Physiol. 1991; 76: 695 to 703] As a major constituent of the gel-like component of the hypodermic matrix, HA contributes significantly to viscosity .
[0111] Glycosaminoglycans (GAGs) are complex linear polysaccharides of the extracellular matrix (ECM). GAGs are characterized by repeating disaccharide structures of an N-substituted hexosamine and a uronic acid (in the case of hyaluronan (HA), chondroitin sulphate (CS), chondroitin (C), dermatan sulphate (DS), sulphate heparan (HS), and heparin (H)), or a galactose (in the case of keratin sulfate (KS)). Except for HA, all exist covalently agglutinated to core proteins. GAGs with their core proteins are structurally called proteoglycans (PGs).
[0112] Hyaluronan (HA) is found in mammals predominantly in connective tissues, skin, cartilage, and synovial fluid. Hyaluronan is also the main constituent of the vitreous of the eye. In connective tissue, the hydrating water associated with hyaluronan creates hydrated matrices between the tissues. Hyaluronan plays a key role in the biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis, logical embryo development, wound healing, angiogenesis, and tumorigenesis [Toole, Cell Biol. Extracell. Matrix, Hay (ed), Plenum Press, New York, 1991; pp. 1384 to 1386; Bertrand et al, Int. J. Cancer 1992; 52: 1 to 6; Knudson et al, FASEB J. 1993; 7: 1233 to 1241]. Additionally, levels of hyaluronan correlate with tumor aggressiveness [Ozello et al, Cancer Res. 1960; 20:600 to 604; Takeuchi et al, Cancer Res. 1976; 36:2133 to 2139; Kimata et al, Cancer Res. 1983; 43: 1347 to 1354].
[0113] HA is found in the extracellular matrix of many cells, especially in soft connective tissues. HA has been designed for several physiological functions, such as in water and plasma protein homeostasis [ Laurent T.C. et al, FASEB J., 1992; 6:2397 to 2404]. HA production increases in proliferating cells and may play a role in mitosis. This has also been implicated in cell locomotion and migration. HA appears to play important roles in cell regulation, development, and differentiation [Laurent et al, supra].
[0114] HA has been widely used in clinical medicine. Its protective tissue and rheological properties have proven useful in ophthalmic surgery (eg to protect the corneal endothelium during cataract surgery). Serum HA is diagnostic of liver disease and various inflammatory conditions such as rheumatoid arthritis. Interstitial edema caused by HA accumulation can cause dysfunction in various organs [Laurent et al, supra].
[0115] Hyaluronan protein interactions are also involved in the structure of the extracellular matrix or "background substance".
[0116] Hyaluronidases are a group of generally acidic or neutral active enzymes found throughout the animal kingdom. Hyaluronidases vary with respect to substrate specificity, and mechanism of action (WO 2004/078140). There are three general classes of hyaluronidases: 1. Mammalian-type hyaluronidases, (EC 3.2.1.35) which are endo-beta-N-acetylhexosamimdases with tetrasaccharides and hexasaccharides as the main end-products. These have both hydrolytic and transglycosidase activities, and can degrade hyaluronan and chondroitin sulfates (CS), usually C4-S and C6-S. 2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan and, to varying degrees, CS and DS. These are endo-beta-N-acetylhexosaminidases that operate by a beta elimination reaction that yields primarily disaccharide end products. 3. Hyaluronidases (EC 3.2.1.36) from slugs, other parasites, and crustaceans are endo-beta-glucuronidases that generate tetrasaccharide and hexasaccharide end-products through hydrolysis of the 1-3 beta bond.
[0117] The mammalian hyaluronidases can be further divided into two groups: acid active and neutral active enzymes. There are six hyaluronidase-like genes in the human genome, HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 and PH20/SPAM1. HYALP1 is a pseudogene, and HYAL3 has not been shown to possess enzyme activity towards any known substrates. HYAL4 is a chondroitinase and exhibits little activity towards hyaluronan. HYAL1 is the prototypic acid active enzyme and PH20 is the prototypic neutral active enzyme. Acidic active hyaluronidases such as HYAL1 and HYAL2 generally lack catalytic activity at neutral pH (ie, pH 7). For example, HYAL1 has little in vitro catalytic activity over pH 4.5 [Frost I.G. and Stern, R., "A microtiter-based assay for hyaluronidase activity not requiring specialized reagents", Anal. Biochemistry, 1997; 251:263 to 269]. HYAL2 is an active acidic enzyme with very low specific activity in vitro.
[0118] Hyaluronidase-like enzymes can also be characterized by those that are generally tethered to the plasma membrane via a glycosylphosphatidyl inositol anchor such as human HYAL2 and human PH20 [Danilkovitch-Miagkova et al, Proc. Natl. Academic Sci. USA, 2003; 100(8):4580 to 4585; Phelps et al, Science 1988; 240(4860): 1780 to 1782], and those that are generally soluble such as human HYAL1 [Frost, I.G. et al, "Purification, cloning, and expression of human plasma hyaluronidase", Biochem. Biophysis. Common Res. 1997; 236(1): 10 to 15]. However, there are species-to-species variations: bovine PH20, for example, is very weakly attached to the plasma membrane and is not anchored via a phospholipase-sensitive anchor [Lalancette et al, Biol Reprod., 2001; 65(2):628 to 36]. This unique feature of bovine hyaluronidase allowed the use of the soluble test bovine hyaluronidase enzymes as an extract for clinical use (WydaseTM, HyalaseTM). Other PH20 species are lipid-anchored enzymes that are generally not soluble without the use of detergents or lipases. For example, PH20 humanoids anchored to the plasma membrane via a GPI anchor. Attempts are made to make PH20 human DNA constructs that should not introduce a lipid anchor into the peptide resulting in either a catalytically inactive enzyme, or an insoluble enzyme [Arming et al, Eur. J. Biochem., 1997; 1; 247(3):810-4]. The naturally occurring monkey sperm hyaluronidase is found in both an agglutinated and soluble membrane form. While the 64 kDa membrane agglutinated form has enzyme activity at pH 7.0, the 54 kDa form is only active at pH 4.0 [Cherr et al, Dev. Biol, 1996;10; 175(1): 142 to 53]. Thus, soluble forms OF PH20 are generally lacking in enzyme activity under neutral conditions.
[0119] As mentioned above and in accordance with the teachings of WO2006/091871 and US Patent 7,767,429, small amounts of soluble hyaluronidase glycoproteins (sHASEGPs) can be introduced into a formulation in order to facilitate the administration of therapeutic drug in the hypodermis. Through rapid HA polymerization in the extracellular space sHASEGP reduces interstitial viscosity, thereby increasing hydraulic conductance and allows larger volumes to be safely and comfortably delivered into SC tissue. The increased hydraulic conductance induced by sHASEGP through reduced interstitial viscosity allows for greater dispersion, potentially increasing systemic bioavailability of the SC-administered therapeutic drug.
[0120] When injected into the hypodermis, the polymerization of HA by sHASEGP is localized to the injection site in SC tissue. Experimental evidence has shown that sHASEGP is locally inactivated in the interstitial space with a half-life of 13 to 20 minutes in mice, with no detectable systemic absorption into blood following a single intravenous dose in CD-I mice. Within the vascular compartment sHASEGP demonstrates a half-life of 2.3 and 5 minutes in mice and cynomolgus monkeys, respectively, at doses up to 0.5 mg/kg. The rapid removal of sHASEGP, combined with continuous synthesis of the HA substrate in SC tissue, results in an enhancement of locally active and transient permeation for other co-injected molecules, the effects are fully reversible within 24 to 48 hours after administration [ Bywaters GL, et al, "Reconstitution of the dermal barrier to dye spread after hyaluronidase injection", Br. Med. J., 1951; 2 (4741): 1178 to 1183].
[0121] In addition to its effects on local fluid dispersion, sHASEGP also acts as an absorption enhancer. Macromolecules larger than 16 kilodaltons (kDa) are largely excluded from absorption through capillaries through diffusion and are mostly absorbed through draining lymph nodes. A macromolecule administered subcutaneously such as, for example, a therapeutic antibody (with a molecular weight of approximately 150 kDa) must therefore traverse the interstitial matrix prior to reaching the draining lymphatics for subsequent absorption into the vascular compartment. by increased local dispersion, sHASEGP increases the rate (Ka) of absorption of many macromolecules. This leads to increased peak blood levels (Cmax) and potentially increased bioavailability relative to SC administration in the absence of sHASEGP [Bookbinder L.H., et al, "A recombinant human enzyme for enhanced interstitial transport of therapeutics", J. Control. Release 2006; 114: 230 to 241].
[0122] Hyaluronidase products of animal origin have been used clinically for over 60 years, primarily to increase dispersion and absorption of or co-administered drugs and for hypodermoclysis (SC injection/infusion of large volume fluid) [Frost GI , "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007; 4: 427 to 440]. Details of the mechanism of action of hyaluronidases were described in detail in the following publications: Duran-Reynolds F., "A spreading factor in certain snake venoms and its relation to their mode of action", CR Soc Biol Paris, 1938; 69 to 81; Chain E., "A mucolytic enzyme in test extracts", Nature 1939; 977 to 978; In Weissmann B., "The transglycosylative action of testicular hyaluronidase", J. Biol. Chem., 1955; 216: 783 to 94; Tammi, R., Saamanen, A.M., Maibach, H.I., Tamrni M., "Degradation of newly synthesized high molecular mass hyaluronan in the epidermal and dermal compartments of human skin in organ culture", J. Invest. Dermatol. 1991; 97: 126 to 130; Laurent, U.B.G., Dahl, L.B., Reed, R.K., "Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver", Exp. Physiol. 1991; 76: 695 to 703; Laurent, T.C. and Fraser, J.R.E., "Degradation of Active Biosubstances: Physiology and Pathophysiology", Henriksen, J.H. (Ed) CRC Press, Boca Raton, FL; 1991. pp. 249 to 265; Harris, E.N., et al, "Endocytic function, glycosaminoglycan specificity, and antibody sensitivity of the recombinant human 190-kDa hyaluronan receptor for endocytosis (HARE)", J. Biol. Chem. 2004; 279:36201 to 36209; Frost, G.I., "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007; 4: 427a440, Hyaluronidase products approved in European Union countries include Hylase® "Dessau" and Hyalase®. Approved US animal-derived hyaluronidase products include VitraseTM, HydaseTM, and AmphadaseTM.
[0123] The safety and efficacy of hyaluronidasehave products have been extensively proven. The most significant safety risk identified is hypersensitivity and/or allergenicity, which is assumed to be due to the lack of purity of animal-derived preparations [Frost, GI, "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007; 4:427 to 440]. It should be noted that there are differences with respect to approved dosages of animal-derived hyaluronidases between the UK, Germany and the US. In the UK, the usual dose as an adjuvant for subcutaneous or intramuscular injection is 1500 units, added directly to the injection. In the US, the usual dose used for this purpose is 150 units. In hypodermoclysis, hyaluronidase is used to aid the subcutaneous administration of relatively large volumes of fluid. In the UK, 1500 units of hyaluronidase are generally applied with 500 to 1000 ml of fluid for subcutaneous use. In the US, 150 units are considered adequate for each liter of hypodermoclysis solution. In Germany, 150 to 300 units are considered suitable for this purpose. In the UK, the spread of local anesthetics is accelerated by the addition of 1500 units. In Germany and the USA 150 units are considered suitable for this purpose. Differences in dosage however (the dosage in the UK is ten times higher than in the US), no apparent difference in the safety profiles of animal-derived hyaluronidase products marketed in the US and UK, respectively, have been reported.
[0124] On December 2, 2005, Halozyme Therapeutics Inc. received FDA approval for an injectable formulation of recombinant human hyaluronidase, rHuPH20 (HYLENEXTM). HYLENEXTM FDA approved at a dose of 150 units for SC administration of the following indications: - as an adjunct to increase absorption and dispersion of other injected drugs - for hypodermoclysis - as an adjunct in SC urography to improve reabsorption of agents radiopaque.
[0125] As a part of the regulatory review it was established that rHuPH20 has the same dispersion and absorption enhancing properties of other injected drugs as previously approved animal-derived hyaluronidase preparations, but with an improved safety profile. In particular, the use of recombinant human hyaluronidase (rHuPH20) compared to animal-derived hyaluronidases minimizes the potential risk of contamination with animal pathogens and transmissible spongiform encephalopathies.
[0126] Soluble hyaloronidase glycoproteins (sHASEGP), a process to prepare the same and their use in pharmaceutical compositions have been described in WO 2004/078140.
[0127] Detailed experimental work as further outlined below has shown that the claimed formulation surprisingly has favorable storage stability and meets all favorable requirements for approval by the health authorities.
[0128] It is believed that hyaluronidase enzyme in the formulation according to the present invention improves the delivery of anti-CD20 antibody to the systemic circulation, for example, by increasing the absorption of the active substance it acts as a permeation enhancer). The hyaluronidase enzyme is also believed to increase the delivery of therapeutic anti-CD20 antibody into the systemic circulation via the subcutaneous delivery route by reversible hydrolization of hyaluronan, an extracellular component of SC interstitial tissue. Hyaluronan hydrolysis in the hypodermis temporarily opens channels in the interstitial space of SC tissue and thereby improves the delivery of therapeutic anti-CD20 antibody into the systemic circulation. Additionally, administration shows reduced pain in humans and less swelling derived from SC tissue volume.
[0129] Hyaluronidase, when administered locally has its full effect locally. In other words, hyaluronidase is inactivated and metabolized locally within minutes and has not been found to have systemic or long-term effects. The rapid inactivation of hyaluronidase within minutes when it enters the bloodstream is opposed to a realistic performance ability compared to biodistribution studies between different hyaluronidase products. This property also minimizes any potential systemic safety concerns as the hyaluronidase product cannot act at distant sites.
[0130] The unifying feature of all hyaluronidase enzymes according to the present invention is their ability to polymerize hyaluronan, regardless of differences in chemical structure, species sources, tissue sources, or batches of drug products originated from of the same species and tissues. These are unusual in that their activity is the same (except for potency) despite having different structures.
[0131] The hyaluronidase enzyme according to the formulation of the present invention is characterized by having no adverse effect on the molecular integrity of the anti-CD20 antibody in the stable pharmaceutical formulation described herein. Furthermore, the hyaluronidase enzyme merely modifies the delivery of the anti-CD20 antibody into the systemic circulation but it does not have any properties that could provide or contribute to the therapeutic effects of the systemically absorbed anti-CD20 antibody. The hyaluronidase enzyme is not bioavailable systemically and does not adversely affect the molecular integrity of the anti-CD20 antibody under recommended storage conditions of the stable pharmaceutical formulation according to the invention. It should therefore be considered as an excipient in the anti-CD20 antibody formulation according to this invention. As it has no therapeutic effect, it represents a constituent of the pharmaceutical form in addition to the therapeutically active anti-CD20 antibody.
[0132] Numerous hyaluronidase enzymes suitable in accordance with the present invention are known from the prior art. The preferred enzyme is a human hyaluronidase enzyme, with the greatest preference for the enzyme known as rHuPH20. rHuPH20 is a member of the family of neutral and acid-active β-1.4 glycosyl hydrolases that polymerize hyaluronan by hydrolysis of the β-1.4 bond between the C1 position of N-acetyl glucosamine and the C4 position of glucuronic acid. Hyaluronan is a polysaccharide found in the intracellular base substance of connective tissue, such as subcutaneous interstitial tissue, and of certain specialized tissues, such as the umbilical cord and the vitreous humor. Hyaluronan hydrolysis temporarily reduces the viscosity of the interstitial tissue and promotes the dispersion of injected fluids or localized transudates or exudates, thus facilitating absorption. The effects of hyaluronidase are local and reversible with complete reconstitution of hyaluronan tissue occurring within 24 to 48 hours [Frost, GI, "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery , 2007; 4:427 to 440]. The increase in connective tissue permeability through hyaluronan hydrolysis correlates with the effectiveness of hyaluronidase for the ability to increase the dispersion and absorption of co-administered molecules.
[0133] The human genome contains several hyaluronidase genes. Only the PH20 gene product has effective hyaluronidase activity under extracellular conditions and acts as a dispersing agent, whereas acid-active hyaluronidases do not have this property.
[0134] The rHuPH20 enzyme is the first and only recombinant human hyaluronidase enzyme currently available for therapeutic use. The human genome contains several hyaluronidase genes; only the PH20 gene product has effective hyaluronidase activity under physiological extracellular conditions and acts as a dispersing agent. The naturally-occurring human PH20 protein has a lipid anchor attached to the carboxy-terminal amino acid that anchors it to the plasma membrane. The rHuPH20 enzyme developed by Halozyme is a truncated deletion variant that lacks such amino acids at the carboxy terminus responsible for lipid fixation. This gives rise to an enzyme active at neutral and soluble pH similar to the protein found in bovine testis preparations. The rHuPH20 protein is synthesized with a 35-amino acid signal peptide that is removed from the N-terminus during the secretion process. The mature rHuPH20 protein contains an N-terminal amino acid sequence orthologous to that found in some bovine hyaluronidase preparations.
[0135] Hyaluronidases PH20, including animal-derived PH20 and recombinant human rHuPH20, depolymerize hyaluronan by hydrolysis of the β-1,4 bond between the C1 position of N-acetyl glucosamine and the C4 position of glucuronic acid. Tetrasaccharide is the smallest digestion product [Weissmann, B., "The transglycosylative action of testicular hyaluronidase", J. Biol. Chem., 1955; 216:783-94]. This N-acetyl glucosamine/glucuronic acid structure is not found in N-linked glycans of recombinant biological products and therefore rHuPH20 will not affect antibody glycosylation it is formulated with, for example, Rituximab. The rHuPH20 enzyme itself has six N-linked glycans per molecule with core structures similar to those found in monoclonal antibodies. As anticipated, these N-linked structures do not change over time, confirming the lack of enzymatic activity of rHuPH20 in these N-linked glycan structures. rHuPH20's short half-life and constant hyaluronan synthesis lead to short, local action of the enzyme in tissues.
[0136] The hyaluronidase enzyme which is an excipient in the subcutaneous formulation in accordance with the present invention is preferably prepared using recombinant DNA technology. This ensures that the same protein (identical amino acid sequence) is obtained every time and that allergic reactions caused by contaminating proteins co-purified during tissue extraction are avoided. The hyaluronidase enzyme used in the formulation in accordance with the present invention is preferably a human enzyme, more preferably rHuPH20,
[0137] The amino acid sequence of rHuPH20 (HYLENEXTM) is well known and available under CAS Registry No. 75971-58-7. The approximate molecular weight is 61 kDa (see also US Patent 7,767,429).
[0138] Multiple functional and structural comparisons were performed between naturally occurring mammalian hyaluronidase and human and other mammalian PH-20 cDNA clones. The PH-20 gene is the gene used for the rHuPH20 recombinant product; however, the recombinant drug product is a truncated 447 amino acid version of the entire protein encoded by the PH-20 gene. Structural similarities with respect to amino acid sequence rarely exceed 60% in any comparison. Functional comparisons show that the activity of rHuPH20 is very similar to that of previously approved hyaluronidase products. This information is consistent with clinical findings over the past 50 years that, regardless of the source of the hyaluronidase, the clinical efficacy and safety of hyaluronidase units are equivalent.
[0139] The use of rHuPH20 in the SC anti-CD20 antibody formulation in accordance with the present invention allows the administration of larger volumes of drug product and potentially improve the absorption of subcutaneously administered CD20 antibody, preferably Rituximab in the systemic circulation.
[0140] The osmolality of the stable pharmaceutical formulation in accordance with the invention is 350 ± 50 mOsm/kg.
[0141] The stable pharmaceutical formulation in accordance with the invention is essentially free of visible particles (the human eye inspection). Subvisible particles (as measured by obscuration) should preferably meet the following criteria: - maximum number of particles > 10 μm per receptacle -> 6,000 - maximum number of particles > 25 μm per receptacle -> 600
[0142] In a further aspect, the present invention provides a use of a formulation for the preparation of a medicament useful for treating a disease or disorder responsive to treatment with an anti-CD20 antibody such as, preferably, cancer or a non-malignant disease. in an individual comprising administering the formulation described in the present invention to a subject in an amount effective to treat said disease or disorder. Preferably, the anti-CD20 antibody is co-administered concomitantly or sequentially with a chemotherapeutic agent.
[0143] In a further aspect the present invention provides a method of treating a disease or disorder that is responsive to treatment with an anti-CD20 antibody (e.g. cancer (preferred) or a non-malignant disease) in a subject comprising administering the formulation described in the present invention to a subject in an amount effective to treat said disease or disorder. Cancer or a non-malignant disease will generally involve cells expressing CD20, such that the CD20 antibody in the therapeutic SC pharmaceutical formulation in accordance with the present invention is capable of binding to the affected cells. The cancer is preferably a cancer expressing CD20. The non-malignant disease which can be treated with the composition in accordance with the present invention is preferably an autoimmune disease as defined in the present invention. Preferably, the anti-CD20 antibody is co-administered concomitantly with a chemotherapeutic agent.
[0144] The addition of hyaluronidase to the formulation allows for an increase in the injection volume that can be safely and comfortably administered subcutaneously. The preferred injection volume is 1 to 15 mL. It has been observed that administration of the formulation in accordance with the present invention increases therapeutic antibody dispersion, absorption and bioavailability. Large molecules (ie, >16 kDa) that are delivered via the SC pathway are preferentially absorbed into the vascular compartment via draining lymph fluids [Supersaxo, A., et al, "Effect of Molecular Weight on the Lymphatic Absorption of WaterSoluble Compounds Following Subcutaneous Administration”, 1990; 2: 167 to 169; Swartz, MA, "Advanced Drug Delivery Review, The physiology of the lymphatic system", 2001; 50: 3 to 20]. The rate of introduction of these large molecules into the circulation Systemic is thus related to intravenous infusion, therefore potentially resulting in reduced intensity/frequency of infusion-related reactions.
[0145] For the production of subcutaneous CD20 antibody (preferably Rituximab) the formulation in accordance with the invention requires high concentrations of antibodies (approximately 120 mg/mL) in the final purification step of the manufacturing process. Therefore, an additional process step (ultrafiltration/diafiltration) is added to the conventional manufacturing process for the CD20 antibody, preferably Rituximab. The stable and highly concentrated pharmaceutical anti-CD20 antibody formulation in accordance with the present invention may also be provided as a stabilized protein formulation which can be reconstituted with a suitable diluent to generate a reconstituted formulation of high anti-CD20 antibody concentration.
[0146] The SC formulation of CD20 antibody in accordance with this invention is preferably used to treat cancer, preferably a cancer expressing CD20,
[0147] The term "about" as used in this patent specification shall specify that the specific value provided may vary to a certain extent, such as, for example, means variations in the range of ± 10%, preferably ± 5 %, more preferably ± 2% are included in the given value.
[0148] With the exception of the above assays, several in vivo assays are available to one of ordinary skill in the art. For example, an expert can expose cells within the patient's body to an antibody that is optionally labeled with a detectable label, eg a radioactive isotope, and the binding of the antibody to cells in the patient can be assessed, eg, by scanning by radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.
[0149] It is contemplated that the SC formulation of CD20 antibody in accordance with this invention may also be used to treat various non-malignant disorders or diseases, such as the autoimmune disease as defined in the present invention; endometriosis; scleroderma; restenosis; polyps such as colon polyps, nasal polyps or gastrointestinal polyps; fibroadenoma, respiratory disease; cholecystitis; neurofibromatosis; polycystic kidney disease, inflammatory diseases, skin diseases including psoriasis and dermatitis, vascular disease; conditions involving abnormal proliferation of vascular epithelial cells; gastrointestinal ulcers, Ménétrier's disease, secretory adenomas or protein loss syndrome, kidney disorders, angiogenic disorders; ocular disease such as age-related macular degeneration, presumed ocular histoplasmosis syndrome, retinal neovascularization of proliferative diabetic retinopathy, retinal vascularization, diabetic retinopathy or age-related macular degeneration; bone-associated conditions such as osteoarthritis, osteoporosis and rickets; damage following an ischemic brain event; fibrotic or endemic diseases such as liver cirrhosis, pulmonary fibrosis, carcoidosis, thyroiditis, systemic hyperviscosity syndrome, Osler-Weber Rendu disease, chronic obstructive pulmonary disease or edema following burns, trauma, hypoxia, radiation, stroke or ischemia; skin hypersensitivity reaction; diabetic retinopathy and diabetic nephropathy, Guillain-Barre syndrome; graft versus host disease, transplant rejection, Paget's disease; inflammation of joints or bones; photoaging (eg caused by UV radiation from human skin); benign prostatic hypertrophy, certain microbial infections, including microbial pathogens selected from adenoviruses, hantaviruses, Borrelia burgdorferi, Yersinia spp. and Bordetella pertussis; thrombi caused by platelet aggregation; reproductive conditions such as endometriosis, ovarian hyperstimulation syndrome, pre-eclampsia, dysfunctional uterine bleeding, or menometrorrhagia; synovitis; atheroma; acute and chronic nephropathies (including proliferative glomerulonephritis and kidney disease-induced diabetes); eczema; hypertrophic scar formation endotoxic shock and fungal infection; familial adenomatous polyposis, neurodegenerative diseases (eg, Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy, and cerebellar degeneration); myelodysplastic syndromes; aplastic anemia: ischemic injury, lung, kidney, or liver fibrosis; T-cell mediated hypersensitivity disease, hypertrophic stenosis of infantile pylorus, urinary obstructive syndrome, psoriatic arthritis; and Hasimoto's thyroiditis. Preferred non-malignant indications for therapy are as defined in the present invention.
[0150] When the indication is cancer, the patient can be treated with a combination of the antibody formulation and a chemotherapeutic agent. Combined administration includes co-administration or concurrent administration, with the use of separate formulations or a single pharmaceutical formulation and consecutive administration in any order, where preferably there is a period of time while both (or all) of the active agents simultaneously exert their biological activities. Thus, the chemotherapeutic agent can be administered prior to or after administration of the antibody formulation in accordance with the present invention. In this embodiment, the timing between at least one administration of the chemotherapeutic agent and at least one administration of the antibody formulation in accordance with the present invention is preferably about 1 month or less, and more preferably about 2 weeks or less. Alternatively, the chemotherapeutic agent and antibody formulation in accordance with the present invention are administered to the patient at the same time, in a single formulation or separate formulations.
[0151] Treatment with said antibody formulation will result in an improvement in the signs and symptoms of cancer or disease. For example, when the disease being treated is cancer, such therapy may result in an improvement in survival (overall survival and/or progression-free survival) and/or may result in an objective clinical response (partial or complete). Furthermore, treatment with the combination of the chemotherapeutic agent and the antibody formulation may result in a therapeutic benefit greater than additive or synergistic to the patient.
[0152] Preferably, the antibody in the administered formulation is a recombinant antibody. However, the antibody administered can be conjugated to a cytotoxic agent. Preferably, the immunoconjugate and/or antigen to which it is bound is internalized by the cell, resulting in increased therapeutic efficacy of the immunoconjugate in killing the cancer cell to which it binds. In a preferred embodiment, the cytotoxic agent targets or interferes with nucleic acid in the cancer cell. Examples of such cytotoxic agents include maytansinoids, calicheamicins, ribonucleases and DNA endonucleases. Preferred immunoconjugates are Rituximab-maytansinoid immunoconjugate similarly to Trastuzumab-DM1 (T-DM1) as described in WO 2003/037992, most preferably the T-MCC-DM1 immunoconjugate.
[0153] For subcutaneous delivery, the formulation can be administered through a suitable device, such as (but not limited to) a syringe; an injection device (for example, the INJECT-EASETM and GENJECTTM device); an infusion ramp (such as, for example, Accu-ChekTM); an injector pen (such as the GNPENTM); a needleless device (for example, MEDDECTORTM and BIOJECTORTM); or through a subcutaneous batch delivery system.
[0154] The administration amount of said anti-CD20 antibody formulation for the prevention or treatment of the disease and the timing of administration will depend on the type (species, gender, age, weight, etc.) and the condition of the patient being treated and the severity of the disease or condition being treated. Also important in determining the appropriate dose are the course of the disease, whether the antibody is administered for therapeutic or preventive purposes, prior therapy, the patient's medical history, and the patient's response to the antibody. Determination of the final dose is at the discretion of the treating physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 µg/kg to 50 mg/kg (for example, 0.1 to 20 mg/kg) of said anti-CD20 antibody is an initial candidate dosage for patient administration.
The preferred dosage of said anti-CD20 antibody will range from about 0.05mg/kg to about 30mg/kg of body weight. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, 10mg/kg or 30mg/kg (or any combination thereof) can be administered to the patient. Depending on the type (species, gender, age, weight, etc.) and the condition of the patient and the type of anti-CD20 antibody, the dosage of said first antibody may be different from the dosage of said second anti-CD20 antibody. Such doses can be administered daily or intermittently, for example, every third day to sixth, or even every one to three weeks. An initial larger loading dose followed by one or more smaller doses may be administered. Based on clinical studies (see also Examples 3 and 4 for non-limiting exemplification for Rituximab), the preferred dosage range is 300 mg/m2 to 900 mg/m2. More preferably, the preferred dosage range of said anti-CD20 antibody is about 375 mg/m2 to about 800 mg/m2. Preferred specific dosages of said anti-CD20 antibody are dosages of about 375 mg/m2, about 625 mg/m2 and about 800 mg/m2. Fixed doses of said anti-CD20 antibody are also preferred.
[0156] In one embodiment, fixed dosages for B-cell lymphomas, preferably non-Hodgkin's lymphoma, are as follows. About 1200 mg to about 1800 mg of said anti-CD20 antibody per dose are preferred. Dosages selected from the group of about 1300mg, about 1500mg, about 1600mg and about 1700mg of said anti-CD20 antibody per dose are most preferred. Most preferably, the fixed dosage for B-cell lymphoma patients, preferably non-Hodgkin's lymphoma patients, is about 1400 mg of said anti-CD20 antibody (eg Rituximab) per dose which can be administered in accordance with various schedules including approximately every 2 months (including approximately every 8 weeks), approximately every 3 months (including approximately every 12 weeks), for approximately 2 years (or more), etc. (See also Examples 3 and 4 for non-limiting exemplification for Rituximab).
[0157] In another embodiment, fixed dosages for patients with leukemia, preferably patients with chronic lymphocytic leukemia (CLL), are as follows. About 1600 mg to about 2200 mg of said anti-CD20 antibody per dose are preferred. Most preferred are dosages selected from the group of about 1700 mg, about 1800 mg, about 1900 mg and about 2100 mg of said anti-CD20 antibody per dose. In one embodiment, the fixed dosage for leukemia patients, preferably CLL patients, is about 1,870 mg of said anti-CD20 antibody (e.g. Rituximab) per dose.
[0158] In yet another embodiment, fixed dosages for patients with autoimmune disease such as rheumatoid arthritis, multiple sclerosis, lupus nephritis, diabetes, ITP, and vasculitis are as follows. About 1200mg to about 2200mg of said anti-CD20 antibody per dose are preferred, for example about 1500mg of said anti-CD20 antibody (eg Rituximab) per dose.
[0159] If a chemotherapeutic agent is administered, it is usually administered at known dosages, therefore, or optionally reduced due to the combined action of the drugs or negative side effects attributable to administration of the chemotherapeutic agent. Dosage and preparation schedules for such chemotherapeutic agents can be used in accordance with the manufacturer's instructions or as determined empirically by one of ordinary skill in the art. Dosage and preparation schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992).
[0160] The stable pharmaceutical formulation of the pharmaceutically active anti-CD20 antibody in accordance with the invention is preferably administered as a subcutaneous injection, whereby administration is preferably repeated several times with time intervals of 3 weeks (q3w). More preferably the total volume of injection fluid is administered within a time period of 1 to 10 minutes, preferably 2 to 6 minutes, most preferably 3 ± 1 minutes. More preferably, 2 ml/min is administered, that is, for example, approximately 240 mg/min. For many patients when no other intravenous (IV) chemotherapeutic agents have been given, such subcutaneous administration leads to increased patient convenience with the potential for self-administration at home. This leads to improved compliance and would reduce/eliminate costs associated with IV administration (ie nursing costs for IV administration, daily bed rental, patient travel, etc.). Subcutaneous administration in accordance with the present invention will most likely be associated with a reduced frequency and/or intensity of infusion-related reactions.
[0161] In a preferred embodiment, the drug is useful to prevent or reduce metastasis or further spread in such a patient suffering from cancer that expresses CD20. The drug is useful in increasing the survival duration of such a patient, increasing the progression-free survival of that patient, increasing the response duration, resulting in a statistically significant and clinically significant improvement in the treated patient as measured by the duration of survival, free survival progression, response rate, or response duration. In a preferred embodiment, the drug is useful for increasing the response rate in a group of patients.
[0162] In the context of this invention, one or more additional growth-inhibiting, cytotoxic, chemotherapeutic, anti-angiogenic, anti-cancer agents or cytokine(s), or compounds that enhance the effects of such agents may be used in anti-CD20 antibody treatment of cancer that expresses CD20. Preferably anti-CD20 antibody treatment is used without such additional cytotoxic, chemotherapeutic or anti-cancer agents, or compounds which enhance the effects of such agents.
[0163] Such agents include, for example: alkylating agents or agents with an alkylating action, such as cyclophosphamide (CTX; for example, Cytoxan®), chlorambucil (CHL; for example, leukeran®), cisplatin (CisP; for example, platinol®) busulfan (eg, myleran®), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and the like; antimetabolites such as methotrexate (MTX), etoposide (VP 16; eg vepesid®), 6-mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU) , capecitabine (eg, Xeloda®), dacarbazine (DTIC), and the like; antibiotics such as actinomycin D, doxorubicin (DXR; e.g., adriamycin®), daunorubicin (daunomycin), bleomycin, mithramycin and the like; alkaloids such as vinca alkaloids such as vincristine (VCR), vinblastine, and the like; and other antitumor agents such as paclitaxel (eg taxol®) and paclitaxel derivatives, cytostatic agents, glucocorticoids such as dexamethasone (DEX; eg decadron®) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea , amino acid depleting enzymes such as asparaginase, leucovorin and other folic acid derivatives, and similar and diverse antitumor agents. The following agents can also be used as additional agents: arnifostin (eg ethyol®), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, lomustine (CCNU), lipo doxorubicin (eg doxil®), gemcitabine ( eg gemzar®), lipo daunorubicin (eg daunoxome®), procarbazine, mitomycin, docetaxel (eg taxotere®), aldesleucine, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy 7 -ethyl-camptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, piplicabromane teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil. Preferably anti-CD20 antibody treatment is used without additional agents.
[0164] The use of the cytotoxic and anticancer agents described above as well as targeted antiproliferative specific anticancer drugs as protein kinase inhibitors in chemotherapeutic regimens is generally well characterized in cancer therapy techniques, and their use in the present invention is under the same considerations for monitoring tolerance and effectiveness and for controlling administration routes and dosages, with some adjustments. For example, actual dosages of cytotoxic agents may vary depending on the patient's cultured cell response determined using histoculture methods. Generally, the dosage will be reduced compared to the amount used in the absence of other additional agents.
[0165] Typical dosages of an effective cytotoxic agent may be in the ranges recommended by the manufacturer, and where indicated by in vitro responses or responses in animal models, may be reduced by up to about an order of concentration or amount of magnitude. Thus, the actual dosage will depend on the judgment of the physician, the condition of the patient, and the efficacy of the therapeutic method based on the in vitro responsiveness of the primary cultured malignant cells or histocultured tissue sample, or the responses observed in the appropriate animal models.
[0166] In the context of this invention, an effective amount of ionizing radiation can be performed and/or a radiopharmaceutical can be used in addition to anti-CD20 antibody treatment of cancer expressing CD20. The radiation source can be either external or internal to the patient being treated. When the source is external to the patient, the therapy is known as external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the treatment is called brachytherapy (BT). Radioactive atoms for use in the context of this invention may be selected from the group including, but not limited to, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium -99, iodine-123, iodine-131, and indium-111. It is also possible to label the antibody with such radioactive isotopes. Preferably anti-CD20 antibody treatment is used without ionizing radiation.
[0167] Radiation therapy is standard treatment to control inoperable or unresectable tumors and/or tumor metastases. Improved results were seen when radiation therapy was combined with chemotherapy. Radiation therapy is based on the principle that high dose radiation delivered to a target area will result in the death of reproductive cells in both normal and tumor tissues. The radiation dosage regimen is generally defined in terms of absorbed radiation dose (Gy), time and fractionation and must be carefully defined by the oncologist. How much radiation a patient receives will depend on several considerations, but the two most important are the location of the tumor in relation to other critical structures or organs in the body and the extent to which the tumor has spread. A typical course of treatment for a patient undergoing radiation therapy will be a treatment schedule over a period of 1 to 6 weeks, with a total dose of between 10 and 80 Gy given to the patient in a single daily fraction of about 1, 8 to 2.0 Gy, 5 days a week. In a preferred embodiment of this invention there is synergy when tumors in human patients are treated with a combination of the treatments of the invention and radiation. In other words, tumor growth inhibition by agents comprising the CD20 antibody formulation of the invention is high when combined with radiation, optionally with anti-cancer or chemotherapeutic agents. Parameters of adjuvant radiation therapies are, for example, contained in WO 99/60023.
[0168] Other therapeutic regimens can be combined with the antibody including, but not limited to, a second (third, fourth, etc.) chemotherapeutic agent(s) (in other words, a "cocktail" of different chemotherapeutic agents) ; another monoclonal antibody; a growth inhibitory agent; a cytotoxic agent; a chemotherapeutic agent; an anti-angiogenic agent; and/or cytokine, etc.; or any suitable combination thereof.
[0169] In addition to therapeutic regimens, the patient may undergo surgical removal of cancer cells and/or radiation therapy.
[0170] In another embodiment of the invention, an article of manufacture is provided that contains the pharmaceutical formulation of the present invention and provides instructions for its use. This article of manufacture comprises a container. Suitable containers include, for example, bottles, receptacles (e.g., multi-chamber or dual-chamber receptacles), syringes (such as, multi-chamber or dual-chamber syringes) and test tubes. The container can be formed from a variety of materials such as glass and plastic. The container holds the formulation and the label on or associated with the container may indicate directions for use. The container containing the formulation may be a multi-use receptacle that allows for repeated administrations (e.g., 2 to 6 administrations) of the reconstituted formulation. The article of manufacture may additionally include other materials desirable from a user or commercial standpoint including other buffers, diluents, filters, needles, syringes and package insert inserts with instructions for use.
[0171] The antibody that is formulated in accordance with the present invention is preferably essentially pure and desirably essentially homogeneous (i.e., free of contaminating proteins, etc., whereby the hyaluronidase enzyme in the formulation accordingly with this invention is not considered a contaminating protein of the anti-CD20 monoclonal antibody in accordance with the present invention).
[0172] The invention will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the invention. All patent and literature citations are incorporated herein by reference. EXAMPLES
[0173] The anti-CD20 formulations for subcutaneous administration according to the invention were developed based on the experimental results as provided below with the use of the general analytical and preparatory assays and methods as outlined below. EXAMPLE 1: PREPARATION OF HIGHLY CONCENTRATED LIQUID FORMULATIONS
[0174] Rituximab is manufactured by techniques generally known from the production of recombinant proteins. A Chinese hamster ovary (CHO) cell line prepared as described in EP-B-2000149 is expanded in cell culture from a master cell bank. Rituximab monoclonal antibody is collected from cell culture fluid and purified using immobilized Protein A affinity chromatography, cation exchange chromatography, a filtration step to remove viral contaminations, followed by anion exchange chromatography, and an ultrafiltration/diafiltration step.
[0175] rHuPH20 is manufactured by generally known techniques of recombinant protein production. The process begins with thawing cells from the working cell bank (WCB) or main cell bank (MCB) and expansion through cell culture in a series of roller bottles. Cell culture up to 6 liters is used to provide a continuous source of cells maintained under selective pressure with methotrexate. When expanded to approximately 36 liters the culture is transferred to a 400 liter bioreactor for a final batch volume of approximately 300 liters. The production bioreactor is operated in fed-batch mode without any selection pressure and the duration of the production phase is approximately two weeks. rHuPH20 is secreted into the culture fluid. A 1,000 bioreactor can also be used for a final batch volume of 500 liters. Upon completion of the production phase, the collection is clarified by filtration and is then treated with solvent/detergent to inactivate virus. The protein is then purified by a series of four column chromatography processes to remove product and process related impurities. A viral filtration step is performed and the filtered mass is then considered, formulated in the final buffer: 10 mg/ml rHuPH20 in 20 mM L-histidine/HCl buffer, pH 6.5, 130 mM NaCl, 0 0.05% (w/v) polysorbate 80. The mass of rHuPH20 is stored below -70°C.
[0176] The other formulation excipients in accordance with the present invention are widely used in practice and known to those skilled in the art. There is therefore no need to explain them in detail here.
[0177] Liquid drug product formulations for subcutaneous administration according to the invention were developed as follows.
[0178] For the preparation of the liquid formulations, Rituximab was buffer exchanged for a diafiltration buffer containing the anticipated buffer composition and when required, concentrated by diafiltration to an antibody concentration of approximately 200 mg/mL. After completion of the diafiltration process, excipients (eg trehalose, rHuPH20, surfactant) were added as stock solutions to the antibody solution. Finally, the protein concentration was adjusted with a buffer to a final Rituximab concentration of approximately 120 mg/ml.
[0179] All formulations were sterile filtered through 0.22 μl low protein binding filters and aseptically filled into sterile 6 mL glass receptacles sealed with ETFE (Copolymer of Ethylene and Tetrafluoroethylene) coated rubber buffers and crimped aluminum covers. The filling volume was approximately 3.0 mL. These formulations were stored at different weather conditions (5°C, 25°C and 40°C) for different time intervals and stressed by agitation (1 week at a agitation frequency of 200 rpm at 5°C and 25°C ) and freeze-thaw stress methods. The samples were analyzed before and after the application of stress tests by the following analytical methods: 1) UV spectrophotometry; 2) Size Exclusion Chromatography (SEC); 3) by Ion Exchange Chromatography (IEC); 4) by turbidity of the solution; 5) for visible particles; and 6) for rHuPH20 activity. UV spectrophotometry, used for the determination of protein content, was performed on a Perkin Elmer À35 UV spectrophotometer in a wavelength range from 240 nm to 400 nm. Pure protein samples were diluted to approximately 0.5 mg/ml with the corresponding formulation buffer. Protein concentration was calculated according to Equation 1. EQUATION 1:

[0180] The UV light absorption at 280 nm was corrected for light scattering at 320 nm and multiplied with the dilution factor, which was determined from the weighted densities and masses of the pure sample and the dilution buffer. The numerator was divided by the product of the cuvette path length d and the extinction coefficient .-.
[0181] Size Exclusion Chromatography (SEC) was used to detect soluble high molecular weight species (aggregates) and low molecular weight (LMW) hydrolysis products in the formulations. The method used a suitable HPLC instrument equipped with a UV detector (detection wavelength 280nm) and a TosoHaas TSK G3000SWXL column (7.8 x 300 mm). The hydrolysis products and aggregates and intact monomer were separated by an isocratic extraction profile, using 0.2M di-potassium hydrophosphate, 025M potassium chloride, pH 7.0 with a flow rate of 0 .5 ml/min.
[0182] Ion Exchange Chromatography (IEC) was performed to detect chemical degradation products that alter the lattice charge of Rituximab in the formulations. For that purpose, Rituximab was digested with Papain. The method used a suitable HPLC instrument equipped with a UV detector (detection wavelength 280nm) and a PL-SCX 1000A analytical cation exchange column from Polymer Labs. 10 mM MES, pH 6.0 and 10 mM MES, 0.2 M potassium chloride, pH 6.0 were used as mobile phases A and B, respectively with a flow rate of 1 ml/min.
[0183] For the determination of turbidity, opalescence was measured in FTU (turbidity units) using a HACH 2100 AN turbidimeter at room temperature.
[0184] Samples were analyzed for visible particles using a Seidenader V90-T visual inspection instrument.
[0185] An in vitro assay of rHuPH20 as a hyaluronidase was used as an activity assay. The test is based on the formation of an insoluble precipitate when hyaluronan (sodium hyaluronate) binds to a cationic precipitant. Enzyme activity was measured by incubating rHuPH20 with hyaluronan substrate and then precipitation of indigestible hyaluronan with acidified albumin serum (equine serum). Turbidity was measured at a wavelength of 640 nm and the decrease in turbidity resulting from enzyme activity on the hyaluronan substrate is a measure of enzyme activity. The procedure is performed using a standard curve generated with rHuPH20 assay reference standard dilutions, and the sample activity is read from the curve.
[0186] Stability test results for Formulations A through J are given in the tables added below. COMPOSITIONS AND STABILITY DATA OF RITUXIMAB LIQUID DRUG PRODUCT FORMULATIONS ACCORDING TO THIS INVENTION
Formulation A is a liquid formulation with the composition of 120 mg/ml Rituximab, 20 mM L-histidine, 210 mM trehalose dihydrate, 10 mM methionine, 0.06% polysorbate 80, 2,000 U µg/ml of rHuPH20 at pH 5.5.

na not determined
Formulation B is a liquid formulation with the composition of 120 mg/ml Rituximab, 20 mM L-histidine, 210 mM trehalose dihydrate, 10 mM methionine, 0.06% polysorbate 80, 2,000 U µg/ml of rHuPH20 at pH 6.1.

na not determined
Formulation C is a liquid formulation with the composition of 120 mg/ml Rituximab, 20 mM L-histidine, 210 mM trehalose dihydrate, 10 mM methionine, 0.06% polysorbate 80, 12,000 U µg/ml of rHuPH20 at pH 5.5.

na not determined
[0190] Formulation D is a liquid formulation with the composition of 120 mg/ml Rituximab, 20 mM acetic acid, 210 mM trehalose dihydrate, 10 mM methionine, 0.06% polysorbate 20, 12,000 U/ ml of rHuPH20 at pH 5.5.

na not determined
Formulation E is a liquid formulation with the composition of 120 mg/ml Rituximab, 20 mM L-histidine, 210 mM trehalose dihydrate, 10 mM methionine, 0.06% polysorbate 20, 12,000 U µg/ml of rHuPH20 at pH 5.5.

na not determined
[0192] Formulation F is a liquid formulation with the composition of 120 mg/ml Rituximab, 20 mM L-histidine, 120 mM sodium chloride, 10 mM methionine, 0.02% polysorbate 80, 12,000 U µg/ml of rHuPH20 at pH 5.5.

na not determined
[0193] Formulation G is a liquid formulation with the composition of 120 mg/ml Rituximab, 20 mM citric acid, 120 mM sodium chloride, 10 mM methionine, 0.02% polysorbate 80, 12,000 U/ ml of rHuPH20 at pH 6.5.

na not determined
Formulation H is a liquid formulation with the composition of 120 mg/ml Rituximab, 20 mM citric acid, 210 mM trehalose dihydrate, 10 mM methionine, 0.06% polysorbate 80, 12,000 U/ ml of rHuPH20 at pH 6.5.
na not determined
[0195] Formulation I is a liquid formulation with the composition of 120 mg/ml Rituximab, 20 mM L-histidine, 120 mM sodium chloride, 10 mM methionine, 0.04% polysorbate 80, 12,000 U/ml rHuPH20 at pH 6.0.

na not determined
[0196] Formulation J is a liquid formulation with the composition of 25 mg/ml GAlO l (HuMabe<CD20>), 20 mM L-histidine, 240 mM trehalose dihydrate, 0.02% poloxamer 188, 2,000 U/ml of rHuPH20 at pH 6.0.

EXAMPLE 2: PREPARATION OF HUMANIZED 2H7 ANTI-CD20 LIQUID FORMULATIONS
[0197] For the preparation of the liquid formulations, the recombinant humanized anti-CD20 antibody (2H7.vl6 as disclosed in WO 2006/084264) was exchanged through buffer in relation to a diafiltration buffer containing the anticipated buffer composition and when required, concentrated at an antibody concentration of approximately 60 and 120 mg/mL. After reaching the target concentration, excipients (eg trehalose, rHuPH20, polysorbate 20) were then added as stock solutions to the antibody solution. Finally, the protein concentration was adjusted with the final formulation buffer to a humanized 2H7 concentration of approximately 30, 50, and 100 mg/ml.
[0198] All formulations were sterilized by filtration through 0.22 μm binder filters with low protein content and aseptically filled in sterile 3 mL glass receptacles buffered with butyl rubber buffers laminated with fluorine resin and terminated with seals plastic/aluminium flip-off type. The filled volume was approximately 1.2 ml. These formulations were stored at different temperatures (5°C, 25°C and 40°C) for different time intervals. Samples were analyzed at each at each stability time point by the following analytical methods: 1) UV spectrometry 2) Size Exclusion Chromatography (SEC); 3) Ion exchange chromatography (IEC); 4) Complement dependent cytotoxicity assay for humanized 2h7 activity 5) Turbidimetric assay for rHuPH20 activity 1) . Protein concentration was determined by ultraviolet absorption spectroscopy using an Agilent 8453 spectrophotometer in a wavelength range from 240 nm to 400 nm. Samples were gravimetrically diluted to approximately 0.5 mg/ml with the corresponding formulation buffer. Protein concentrations were calculated using Equation 1:
[0199] Protein concentration = ((Amax - A320) x DF) / (8(cm2/mg) xd(cm)) (Equation 1) where DF is the dilution factor, d is the path length of crucible and ε is the extinction coefficient, which is 1.75 (cm2/mg-1) for 2H7 at Amax. UV light absorption at Amax (typically 278 to 280 nm) was corrected for light scatter at 320 nm and multiplied with the dilution factor, which was determined from the weighted masses and densities of the pure sample and the dilution buffer. The numerator was divided by the product of the crucible path length d and the extinction coefficient ε. two) . Size Exclusion Chromatography (SEC) was used to detect soluble high molecular weight species (aggregates) and low molecular weight hydrolysis products (fragments) in the formulations. SEC was run on an Agilent Technologies, Inc. 1100 series HPLC equipped with a UV detector (detection wavelength 280nm) and a TSK G3000SWXL column (7.8x300 mm). Hydrolysis products, aggregates and intact monomers were separated through an isocratic elution profile using 0.20M potassium phosphate and 0.25M potassium chloride at pH 6.2 with a flow rate of 0.3 mL /min. 3) . Ion exchange chromatography (IEC) was performed to detect chemical degradation products that alter the net charge of the anti-CD20 antibody in the formulations. For this purpose, the anti-CD20 antibody is incubated with Carboxypeptidase B to catalyze the hydrolysis of basic amino acids. Ion exchange chromatography was performed on an Agilent Technologies, Inc. 1100 series HPLC with a UV detector (detection wavelength 280nm) and a Dionex ProPac WCX-10 column (4 x 250 mm). Acidic and basic variants were separated using a linear gradient of 25mM potassium phosphate at pH 6.9 (mobile phase A) and 120mM potassium chloride dissolved in 25mM potassium phosphate (mobile phase B) at one rate flow rate of 0.5 ml/min. 4) . Complement dependent cytotoxicity assay (CDC) assay was performed to determine the in vitro activity of the anti-CD20 antibody. The complement dependent cytotoxicity (CDC) potency assay is used to measure the antibody's ability to cause lysis of human B lymphoblastoid cells (WIL2-S) in the presence of human complement. The assay is performed in 96-well tissue culture microtiter plates. In this assay, varying concentrations of anti-CD20 antibody reference material, control, or sample(s) diluted in assay diluent are incubated with WIL2-S cells (50,000 cells/well) in the presence of a fixed amount of human complement. The plate is incubated at 37°C/5% CO2 in a humidified incubator for 1 to 2 hours. At the end of the incubation period, 50μl of redox dye, ALAMARBLUE™ is added to each well and the plate is incubated for 15 to 26 hours. ALAMARBLUE™ is an redox dye that fluoresces at an excitation wavelength of 530 nm and an emission wavelength of 590 nm when reduced by living cells. Therefore, changes in color and fluorescence are proportional to the number of viable cells. The results, expressed in relative fluorescence units (RFU), are plotted against anti-CD20 antibody concentrations and a parallel line program is used to estimate the activity of anti-CD20 antibody samples against the reference material. 5) . A turbidimetric assay was used to determine hyaluronidase activity and enzyme concentration. This method is based on the formation of an insoluble precipitate when hyaluronic acid is agglutinated with acidified albumin serum. Briefly, a working rhuPH20 hyaluronidase (Halozyme, Inc.) working reference standard dilution series ranging from 2.5 U/mL to 0.25 U/mL is prepared in enzyme diluent (70 mM NaCl, 25 mM PIPES, pH 5.5, 0.66 mg/ml hydrolyzed gelatin, 0.1% human serum albumin). Test samples are diluted to a final concentration of 1.5 U/mL in Enzyme Diluent. 30 μm of the standard and sample dilutions are transferred into a 96-well “black and light bottom” plate (Nunc). The plate is then covered and prewarmed for 5 minutes at 37°C. The reaction is then initiated by adding 30 μm of 0.25 mg/mL prewarmed hyaluronic acid substrate solution (70 mM NaCl, 25 mM PIPES, pH 5.5, 0.25 mg/mL hyaluronate Dry Sodium, Lifecore Biomedical). The plate is shaken briefly and incubated for 10 minutes at 37°C. After this incubation step, the reaction is stopped by adding 240 μm of working serum solution (2.5% equine serum, 500 mM potassium acetate, pH 4.25). After a 30-minute development period at room temperature, the turbidity of the reaction is measured at a wavelength of 640 nm in a microplate reader. The decrease in turbidity resulting from enzyme activity in the hyaluronic acid substrate is a measure of hyaluronidase activity. Sample activity is determined relative to the calibration curve generated with the working reference standard dilutions of rhuPH20.
[0200] The results obtained with the various humanized 2H7 antibody formulations are shown in the following tables:
[0201] Formulation K is a liquid formulation with the composition of 30 mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02% polysorbate 20.0 U/ml rhuPH20 in pH 5.3.
*ND not determined
[0202] Formulation L is a liquid formulation with the composition of 30 mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02% polysorbate 20, 1500 U/ml rhuPH20 in pH 5.3.
*ND not determined
[0203] Formulation M is a liquid formulation with the composition of 30 mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02% polysorbate 20, 12,000 U/ml rhuPH20 in pH 5.3.

*ND not determined
[0204] Formulation N is a liquid formulation with the composition of 50 mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02% polysorbate 20.0 U/ml rhuPH20 in pH 5.3.
*ND not determined
[0205] Formulation O is a liquid formulation with the composition of 50 mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02% polysorbate 20, 1500 U/ml rhuPH20 in pH 5.3.

*ND not determined
[0206] Formulation P is a liquid formulation with the composition of 50 mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02% polysorbate 20, 12,000 U/ml rhuPH20 in pH 5.3.
*ND not determined
[0207] Formulation Q is a liquid formulation with the composition of 100 mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02% polysorbate 20.0 U/ml rhuPH20 in pH 5.3.
*ND not determined
[0208] Formulation R is a liquid formulation with the composition of 100 mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02% polysorbate 20, 1500 U/ml rhuPH20 in pH 5.3.

*ND not determined
[0209] Formulation S is a liquid formulation with the composition of 100 mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02% polysorbate 20, 12,000 U/ml rhuPH20 in pH 5.3.
*ND not determined EXAMPLE 3: TREATMENT OF PATIENTS WITH THE FORMULATION
[0210] Rituximab-containing regimens have become the standard of care for patients suffering from a variety of CD20-positive B-cell malignancies. Currently, Rituximab is given as an intravenous (IV) infusion over several hours. These long infusion periods and infusion-related side effects have been cited by some patients as uncomfortable consequences of current therapeutic treatment. Additionally, the procedure required to establish intravenous access is considered invasive and can be painful, particularly in patients with malignancies that are treated repeatedly. Subcutaneous (SC) administration could significantly simplify treatment, shortening administration to less than 10 minutes and improving the patient experience. Recombinant human hyaluronidase (rHuPH20) was developed and approved to improve the dispersion and absorption of co-administered drugs. It was combined with Rituximab to allow injection volumes greater than 10 mL for safe and comfortable SC administration. The objectives of this treatment were to select the dose of the Rituximab SC formulation with rHuPH20 prepared as described in Example 1 (Formulation A) providing comparable exposure to Rituximab IV and to assess its safety and tolerability in female and male patients with follicular lymphoma (FL) during treatment maintenance.
[0211] This example provides stage 1 data from a multi-core, open-label, randomized, adaptive Phase Ib study. 124 patients were randomized to one of four Rituximab maintenance treatment groups: 16 patients on IV control, 34 patients on 1 SC dose (375 mg/m2), 34 patients on 2 SC dose (625 mg/m2) and 40 patients on dose 3 SC (800 mg/m2). Prior to randomization, eligible patients were treated with at least one IV dose of Rituximab at 375 mg/m2 in the maintenance setting. For patients randomized to one of the SC cohorts, a single IV dose was replaced by an SC dose. Patients received Rituximab on either a 2-monthly (q2m) or 3-monthly (q3m) regimen, as per local practice. Safety data are available from a total of 119 patients. Rituximab SC was generally well tolerated. No clinically significant observations or serious treatment-related adverse events were reported. A total of 95 adverse events (AEs) were reported in 46 patients (39%). The most commonly documented AE was "administration-associated reaction" (AAR, which includes irritation, erythema, and mild discomfort). These AARs were reversible, predominantly mild in intensity and only 1 event required any treatment (metoclopramide for nausea). In general, the profile of AE is not significantly different from that expected in patients treated with IV Rituximab (after AAR, the most frequent events were gastrointestinal disorders and mild infections). Four serious adverse events (SAEs) were reported in 4 separate patients, all reported as unrelated to study medication. There were no AEs that led to death, withdrawal or discontinuation of treatment.
[0212] The total volume administered SC to each patient in the range from 4.4 to 15.0 mL. The mean injection duration was 2 mL/min. Peak Rituximab serum concentrations in the SC cohorts occurred between Day 2 and Day 8 (48 h and 168 h). Pharmacokinetic parameters were linear with respect to doses beyond the SC administered dose range (375, 625 and 800 mg/m2). Rituximab concentrations on Day 28 (C28) and the extent of serum exposure (AUCo-57) in patients given 625 mg/m2 of Rituximab SC were comparable to those in patients given the standard IV Rituximab dose of 375 mg/m2 SC.
[0213] In conclusion, subcutaneous Rituximab can be released quickly, comfortably and safely while achieving comparable serum exposures to the approved intravenous formulation in FL patients during maintenance treatment. The patient's experience was favorable. These results support additional subcutaneous testing of Rituximab and a fixed dose of 1400 mg Rituximab SC was selected for the formal Ctrough non-inferiority test at stage 2 of the examination. EXAMPLE 4: RITUXIMABESQ VS. RITUXIMABEIV IN PATIENTS WITH FOLLICULAR NON-HODGKIN LYMPHOMA
[0214] Patients with previously untreated follicular lymphoma (lower grade) are treated with maintenance treatment with either: (a) Rituximab SC Formulation (prepared according to Example 1, Formulation A) in combination with CHOP or CVP, or (b ) Rituximab IV in combination with CHOP or CVP.
[0215] Patients will be randomized to receive 375 mg/m2 of Rituximab as an intravenous infusion or 1400 mg of Rituximab given subcutaneously. In addition, patients will receive standard chemotherapy (CVP or CHOP). Patients who achieve a complete or partial response after 8 treatment cycles will receive maintenance treatment for an additional maximum number of 12 cycles. Maintenance treatment cycles will be repeated every 8 weeks. The anticipated time in study treatment is 96 weeks.
[0216] Treatment with 1400mg anti-CD20 antibody Rituximab SQ as a maintenance treatment every 8 weeks for up to 12 cycles is expected to be safe and effective in the treatment of follicular lymphoma, optionally in combination with chemotherapy (which includes CHOP or CVP).

权利要求:
Claims (23)
[0001]
1. STABLE PHARMACEUTICAL FORMULATION, highly concentrated, for subcutaneous administration of a pharmaceutically active anti-CD20 antibody, characterized by the fact that it comprises: (a) 50 to 350 mg/ml of anti-CD20 antibody; (b) 1 to 100 mM of a buffering agent that provides a pH of 5.5 ± 2.0; (c) 1 to 500 mM of a stabilizer or a mixture of two or more stabilizers; (d) 0.01 to 0.1% of a nonionic surfactant; and (e) 1,000 to 16,000 U/ml of a hyaluronidase enzyme, preferably 2,000 U/ml or 12,000 U/ml.
[0002]
2. FORMULATION according to claim 1, characterized in that the concentration of anti-CD20 antibody is 100 to 150 mg/ml, preferably 120 ± 20 mg/ml.
[0003]
3. FORMULATION according to any one of claims 1 to 2, characterized in that the buffering agent is in a concentration of 1 to 50 mM.
[0004]
4. FORMULATION according to any one of claims 1 to 3, characterized in that the buffering agent provides a pH of 5.5 to 6.5, preferably selected from the group consisting of 5.5, 6, 0, 6.1 and 6.5.
[0005]
5. FORMULATION according to any one of claims 1 to 4, characterized in that the buffering agent is a histidine buffer.
[0006]
6. FORMULATION according to any one of claims 1 to 5, characterized in that the stabilizer is a saccharide, such as, for example, α,α-trehalose dihydrate or sucrose.
[0007]
7. FORMULATION, according to any one of claims 1 to 6, characterized in that the stabilizer is in a concentration of 15 to 250 mM.
[0008]
8. FORMULATION according to any one of claims 6 to 7, characterized in that methionine is used as a second stabilizer.
[0009]
9. FORMULATION according to claim 8, characterized in that methionine is present in a concentration of 5 to 25 mM.
[0010]
10. FORMULATION according to any one of claims 1 to 9, characterized in that the nonionic surfactant is a polysorbate, preferably selected from the group consisting of polysorbate 20, polysorbate 80, and polyethylene-polypropylene copolymer.
[0011]
11. FORMULATION according to claim 10, characterized in that the concentration of polysorbate is 0.02% (w/v) to 0.08% (w/v).
[0012]
12. FORMULATION according to any one of claims 1 to 11, characterized in that the enzyme hyaluronidase is rHuPH20.
[0013]
13. FORMULATION according to any one of claims 1 to 12, characterized in that the anti-CD20 antibody is Rituximab.
[0014]
14. FORMULATION according to any one of claims 1 to 12, characterized in that the anti-CD20 antibody is Ocrelizumab.
[0015]
15. FORMULATION according to any one of claims 1 to 12, characterized in that the anti-CD20 antibody is HuMabe<CD20>.
[0016]
16. FORMULATION according to any one of claims 1 to 15, characterized in that it is stable upon freezing and thawing.
[0017]
17. FORMULATION according to any one of claims 1 to 16, characterized in that it is in liquid form.
[0018]
18. FORMULATION according to any one of claims 1 to 16, characterized in that it is in lyophilized form.
[0019]
19. FORMULATION, according to any one of claims 1 to 18, characterized in that it comprises 120 mg/ml of Rituximab, 20 mM of L-histidine, 210 mM of trehalose dihydrate, 10 mM of methionine, 0.06% of polysorbate 80, 2000 U/ml rHuPH20 at pH 5.5.
[0020]
20. USE OF A FORMULATION as defined in any one of claims 1 to 18, characterized in that it is for the preparation of a drug useful for the treatment of a disease or disorder sensitive to treatment with an anti-CD20 antibody, preferably cancer or a non-malignant disease.
[0021]
21. USE, according to claim 20, characterized in that said drug comprises a fixed dose of 1200 mg to 2200 mg of anti-CD20 antibody.
[0022]
22. USE, according to claim 20, characterized in that said drug comprises a fixed dose of 1200 mg to 1800 mg of anti-CD20 antibody.
[0023]
23. USE, according to claim 20, characterized in that said drug comprises a fixed dose of 1600 mg to 2200 mg of anti-CD20 antibody.

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法律状态:
2020-11-03| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-11-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-12-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-01-19| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-08-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-09-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 10/09/2010, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

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2021-09-21| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: REF. RPI 2644 DE 08/09/2021 QUANTO AO ENDERECO. |

优先权:

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申请号 | 申请日 | 专利标题

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EP09170110.2|2009-09-11|

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EP09170110|2009-09-11|

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PCT/EP2010/063271|WO2011029892A2|2009-09-11|2010-09-10|Highly concentrated pharmaceutical formulations|

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