HK1208176B - Highly concentrated pharmaceutical formulations comprising anti - cd20 antibody - Google Patents

Description

Highly concentrated pharmaceutical formulations comprising anti-CD20 antibodies

The present application is a divisional application of an invention patent application filed on September 10, 2010, with application number 201080050468.1 (international application number PCT/EP2010/063271), entitled “Highly concentrated pharmaceutical formulation comprising anti-CD20 antibodies”.

The present invention relates to highly concentrated, stable pharmaceutical formulations of pharmaceutically active anti-CD20 antibodies or mixtures of such antibody molecules for subcutaneous injection. Such formulations contain, in addition to a relatively high amount of the anti-CD20 antibody or mixture thereof, a buffer, a stabilizer or a mixture of two or more stabilizers, a nonionic surfactant, and an effective amount of at least one hyaluronidase enzyme. The present invention also relates to methods for preparing such formulations and uses of such formulations.

Background of the Invention

The pharmaceutical use of antibodies has increased over the past few years. In many cases, such antibodies are injected or infused via an intravenous (IV) route. Unfortunately, the amount of antibody that can be administered via the intravenous route is limited by the physicochemical properties of the antibody, particularly by its solubility and stability in a suitable liquid formulation and by the volume of the infusion liquid. Alternative routes of administration are subcutaneous or intramuscular injection. These injection routes require a 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): 1390-1402; Roskos, L.K., Davis C.G. et al., "The clinical pharmacology of therapeutic antibodies", Drug Development Research 2004; 61(3): 108-120]. In order to increase the volume, and thereby the therapeutic dose, which can be safely and comfortably administered subcutaneously, the use of glycosaminoglycanase enzymes to increase the interstitial space into which antibody formulations can be injected has been proposed [WO 2006/091871].

Examples of stable formulations of pharmaceutically active antibodies currently marketed for therapeutic use are as follows:

Rituximab is a chimeric antibody that binds to the CD20 antigen on B cells. The commercial formulation is a sterile, clear, colorless, preservative-free liquid concentrate for intravenous (IV) administration. Rituximab is supplied at a concentration of 10 mg/mL in 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated in 9 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dehydrate, 0.7 mg/mL polysorbate 80, and water for injection. The pH is adjusted to 6.5. An alternative liquid formulation of rituximab suitable for IV administration is disclosed in U.S. Patent No. 6,991,790.

HERCEPTIN ^™ (Trastuzumab) is a monoclonal antibody against the HER2 receptor (anti-HER2) that is currently sold in Europe as a 150 mg lyophilized powder (containing the antibody, α,α-trehalose dihydrate, L-histidine and L-histidine hydrochloride, and polysorbate 20) that should be reconstituted with water for injection for infusion to yield an injection dose of approximately 21 mg/ml. In the United States and many other countries, multidose vials containing 440 mg of trastuzumab are sold.

AVASTIN ^™ (Bevacizumab) is a monoclonal antibody against vascular endothelial growth factor (VEGF) that is currently sold in Europe as a liquid formulation in two vials: a) 100 mg bevacizumab in 4 ml and b) 400 mg 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.

Although it has been found that the above-mentioned antibody formulations are suitable for intravenous administration, it is desirable to provide a highly concentrated stable pharmaceutical formulation of the therapeutically active antibody for subcutaneous injection. The advantage of subcutaneous injection is that it allows medical practitioners to implement it on patients in a relatively short intervention. In addition, patients can be trained to implement subcutaneous injections themselves. Typically, injections via the subcutaneous route are limited to about 2 ml. For patients who need multiple doses, multiple unit dose formulations can be injected at multiple locations on the body surface.

The following two antibody products are commercially available for subcutaneous administration.

HUMIRA ^™ (Adalimumab) is a monoclonal antibody against tumor necrosis factor alpha (TNFα) that is currently marketed in Europe in a 40 mg dose in a 0.8 ml injection volume for subcutaneous use (concentration: 50 mg antibody/ml injection volume).

XOLAIR ^™ (Omalizumab) is a monoclonal antibody against immunoglobulin E (anti-IgE) that is currently sold as a 150 mg lyophilized powder (containing the antibody, sucrose, histidine and histidine hydrochloride monohydrate, and polysorbate 20) that should be reconstituted with water for subcutaneous injection to produce a 125 mg/ml injectable dose.

Highly concentrated, stable pharmaceutical anti-CD20 antibody formulations suitable for subcutaneous administration are currently not commercially available. It would therefore be desirable to provide such highly concentrated, stable pharmaceutical formulations of therapeutically active antibodies for subcutaneous injection.

Subcutaneous injection of parenteral drugs is generally limited to volumes of less than 2 ml due to the viscoelastic resistance to hydraulic conduction in the subcutaneous (SC) tissue and the back pressure generated after injection [Aukland K. and Reed R., "Interstitial-Lymphatic Mechanisms in the control of Extracellular Fluid Volume", Physiology Reviews, 1993; 73: 1-78], and due to the perception of pain.

The preparation of high concentration protein formulations is very challenging and requires that each formulation be tailored to the specific protein being used, as each protein has different aggregation behaviors. Aggregates are suspected to cause immunogenicity of therapeutic proteins in at least some cases. Immunogenic responses to protein or antibody aggregates can result in neutralizing antibodies that can render the therapeutic protein or antibody ineffective. It appears that the immunogenicity of protein aggregates is most problematic in association with subcutaneous injection, whereby repeated administration increases the risk of an immune response.

Although antibodies have very similar overall structures, they differ in amino acid composition (particularly in the CDR regions responsible for antigen binding) and glycosylation patterns. In addition, there may be post-translational modifications, such as charge and glycosylation variants.

SUMMARY OF THE INVENTION

The present invention provides highly concentrated stable pharmaceutical formulations of pharmaceutically active anti-CD20 antibodies or mixtures of such antibody molecules, preferably for subcutaneous injection.

More specifically, the highly concentrated, stable pharmaceutical formulation of the pharmaceutically active anti-CD20 antibody formulation of the present invention comprises:

- about 20 to 350 mg/ml anti-CD20 antibody;

- about 1 to 100 mM buffer providing a pH of 5.5 ± 2.0;

- about 1 to 500 mM stabilizer or a mixture of two or more stabilizers, wherein optionally methionine is used as a second stabilizer, preferably in a concentration of 5 to 25 mM;

- about 0.01 to 0.1% nonionic surfactant; and

-Preferably, an effective amount of at least one hyaluronidase enzyme.

Detailed Description of the Invention

The term "antibody" herein is used in the broadest sense and specifically covers full-length antibodies, genetically engineered antibodies such as monoclonal antibodies, or recombinant antibodies, polyclonal antibodies, multispecific antibodies formed from at least two full-length antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, fully human antibodies, and fragments of such antibodies, so long as they exhibit the desired biological activity.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for possible variants (such variants generally exist in small amounts) that can be generated during the generation of monoclonal antibodies, the antibody individuals constituting the population are identical and/or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Beyond its specificity, the advantage of monoclonal antibodies is that they are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that an antibody is a feature obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibody generation by any specific method. For example, it can be generated by the hybridoma method described by et al., Nature, 256:495 (1975) for the first time, or it can be generated by recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567) to generate the monoclonal antibody to be used according to the present invention. "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clarkson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991). As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules composed of a single amino acid. Thus, the term "human monoclonal antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences, exhibiting a single binding specificity. In one embodiment, human monoclonal antibodies are generated by hybridomas comprising B cells obtained from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene, fused to an immortalized cell. The term "monoclonal antibody" herein specifically includes so-called chimeric antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World Monkeys, apes, etc.) and human constant region sequences.

"Antibody fragments" comprise a portion of a full-length antibody, generally comprising 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. Additionally, antibody fragments include single-chain polypeptides that possess the characteristics of a VH chain, i.e., are capable of assembling with a VL chain to bind to the CD20 antigen. "Antibody fragments" also include fragments that, by themselves, are incapable of providing effector function (ADCC/CDC), but that provide this function in a manner consistent with the present invention when combined with appropriate antibody constant domains.

A "full-length antibody" is an antibody comprising an antigen-binding variable region and a light chain constant domain (CL) and heavy chain constant domains CHI, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, a full-length antibody has one or more effector functions.

"Amino acid sequence variant" antibodies herein are antibodies having an amino acid sequence that is different from the main species antibody. Typically, amino acid sequence variants will have at least about 70% homology with the main species antibody, and preferably, they will have at least about 80%, more preferably at least about 90% homology with the main species antibody. Amino acid sequence variants have substitutions, deletions, and/or additions at certain positions within or near the amino acid sequence of the main species antibody. Examples of amino acid sequence variants herein include acidic variants (e.g., deamidated antibody variants), basic variants, antibodies with an amino-terminal leader extension (e.g., VHS-) on one or both of its light chains, antibodies with a C-terminal lysine residue on one or both of its heavy chains, and the like, and include combinations of changes to the amino acid sequence of the heavy and/or light chains. Antibody variants of particular interest herein are antibodies that comprise an amino-terminal leader extension on one or both of its light chains, optionally further comprising other amino acid sequence and/or glycosylation differences, relative to the main species antibody.

As used herein, a "glycosylation variant" antibody is an antibody that has one or more carbohydrate moieties attached that are different from the one or more carbohydrate moieties attached to the main species antibody. Examples of glycosylation variants herein include antibodies having a G1 or G2 oligosaccharide structure attached to its Fc region instead of a G0 oligosaccharide structure, antibodies having one or two carbohydrate moieties attached to one or both of its light chains, antibodies without carbohydrates attached to one or both of its heavy chains, and the like, as well as combinations of glycosylation changes. In addition, the term "glycosylation variant" also includes glycoengineered antibodies such as those described in WO 1'331'266 and USP 7'517'670.

Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), and the like.

Full-length antibodies can be assigned to different "classes" based on the amino acid sequence of the heavy chain constant domain. There are five major classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are called α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. The subunit structures and three-dimensional structures of the different classes of immunoglobulins are well known.

As used herein, the "biological activity" of a monoclonal antibody refers to the ability of the antibody to bind to an antigen and produce a measurable biological response that can be measured in vitro or in vivo. Such activity can be antagonistic (e.g., in the case where the antibody is a CD20 antibody) or agonistic.

The term "humanized antibody" refers to an antibody in which the framework or "complementary determining region" (CDR) has been modified to include the CDRs of an immunoglobulin of a different specificity than that of the parent immunoglobulin. In a preferred embodiment, mouse CDRs are grafted into the framework region of a human antibody to prepare a "humanized antibody." Particularly preferred CDRs correspond to the sequences of the recognition antigens described below for chimeric and bifunctional antibodies. Generally, humanized antibodies refer to human immunoglobulins (acceptor antibodies) in which residues from the hypervariable region of an acceptor are replaced with residues from the hypervariable region of a non-human species (donor antibody) such as a mouse, rat, rabbit, or non-human primate with desired specificity, affinity, and performance. In some cases, the framework region (FR) residues of a human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may be included in residues not found in the acceptor antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will comprise at least one, typically two, substantially all variable domains, in which all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all FRs are those of a human immunoglobulin sequence. Optionally, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (see, e.g., Riechmann, L. et al., Nature 332 (1988) 323-327; and Neuberger, M.S. et al., Nature 314 (1985) 268-270).

The term "chimeric antibody" refers to a monoclonal antibody, typically prepared by recombinant DNA technology, comprising a variable region, i.e., a binding region, from one source or species and at least a portion of a constant region derived from a different source or species. Chimeric antibodies comprising a murine variable region and a human constant region are particularly preferred. Such murine/human chimeric antibodies are the product of expressed immunoglobulin genes comprising 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 changed from that of the original antibody. Such "chimeric" antibodies are also referred to as "class-switched antibodies." Methods for generating chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art (see, for example, Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US 5,202,238 and US 5,204,244).

As used herein, the term "human antibody" is intended to include antibodies having variable and 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) 368-374). Based on such technology, human antibodies can be generated against a wide variety of targets. Examples of human antibodies are described in, for example, Kellermann, S.A. et al., Curr. Opin. Biotechnol. 13 (2002) 593-597.

As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from host cells such as NS0 or CHO cells or from transgenic animals (e.g., mice) expressing human immunoglobulin genes or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences in a rearranged form. The recombinant human antibodies according to the present invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that may not naturally occur in the human antibody germline repertoire in vivo, although derived and associated from human germline VH and VL sequences.

As used herein, "specific binding" refers to an antibody that specifically binds to the CD20 antigen. Preferably, the binding affinity is a Kd value of ^10-9 mol/l or less (e.g., ^10-10 mol/l), preferably a Kd value of ^10-10 mol/l or less (e.g., ^10-12 mol/l). Binding affinity is determined using standard binding assays, such as surface plasmon resonance technology.

As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

The "constant domains" are not involved directly in binding the antibody to antigen, but are involved in effector functions (ADCC, complement fixation, and CDC).

As used herein, "variable region" (light chain variable region (VL), heavy chain variable region (VH)) means each of the light and heavy chain pairs that are directly involved in antibody binding to antigen. Human light and heavy chain variable domains have the same general structure, and each domain comprises four framework (FR) regions whose sequences are extensively conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β-sheet conformation, while the CDRs can form loops connecting the β-sheet structure. The CDRs in each chain are maintained in their three-dimensional structure by the framework regions and, together with the CDRs from the other chain, form an antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the present invention, and therefore provide a further object of the present invention.

The terms "antigen-binding portion of an antibody" or "hypervariable region" as used herein refer to the amino acid residues in an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs". The "framework" or "FR" regions are those variable domain regions that differ from the hypervariable region residues as defined herein. Thus, the light and heavy chains of an antibody comprise, from N- to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding. The CDR and FR regions are determined according to the standard definitions of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) and/or those residues from the "hypervariable loops".

The terms "CD20" and "CD20 antigen" are used interchangeably herein and include any variants, isoforms, and species homologs of human CD20 that are naturally expressed by cells or expressed on cells transfected with the CD20 gene. Binding of the antibodies of the present invention to the CD20 antigen mediates killing of cells expressing CD20 (e.g., tumor cells) by inactivating CD20. Killing of cells expressing CD20 can occur through one or more of the following mechanisms: cell-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), induction of cell death and/or apoptosis, homotypic aggregation, and the like.

As recognized in the art, synonyms for CD20 include B lymphocyte antigen CD20, B lymphocyte surface antigen Bl, Leu-16, and Bp35.

The term "anti-CD20 antibody" according to the present invention is an antibody that specifically binds to the CD20 antigen. Based on the binding properties and biological activities of anti-CD20 antibodies directed against the CD20 antigen, two types of anti-CD20 antibodies (type I and type II anti-CD20 antibodies) can be distinguished according to Cragg, M.S. et al., Blood 103 (2004) 2738-2743; and Cragg, M.S. et al., Blood 101 (2003) 1045-1051, see Table 1.

Table 1: Characteristics of Type I and Type II anti-CD20 antibodies

A crucial property of type I and type II anti-CD20 antibodies is their binding mode. Thus, type I and type II anti-CD20 antibodies can be classified by the ratio of their binding properties to CD20 on Raji cells (ATCC-No. CCL-86) compared to rituximab.

As used herein, an "anti-CD20 antibody" may be a type I or type II antibody. Preferably, it is a type I antibody, and most preferably, it is rituximab.

Type I anti-CD20 antibodies have a ratio of the binding properties of said anti-CD20 antibodies to CD20 on Raji cells (ATCC-No. CCL-86) compared to rituximab of 0.8 to 1.2, preferably 0.9 to 1.1. Examples of such type I anti-CD20 antibodies include, for example, rituximab as described in EP2000149B1 (Anderson et al., see, e.g., Figures 4 and 5), 1F5 IgG2a (ECACC, hybridoma; Press et al., Blood 69/2:584-591 (1987)), HI47 IgG3 (ECACC, hybridoma), 2C6 IgG1 (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 (e.g., variants disclosed in Tables 1 and 2). Preferably, the type I anti-CD20 antibody is a monoclonal antibody that binds to the same epitope as rituximab.

Type II anti-CD20 antibodies have a ratio of their binding to CD20 on Raji cells (ATCC No. CCL-86) compared to rituximab of 0.3 to 0.6, preferably 0.35 to 0.55, and more preferably 0.4 to 0.5. Examples of such Type II anti-CD20 antibodies include, for example, tositumomab (B1 IgG2a), humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody, as disclosed in WO 2005/044859), 11B8 IgG1 (as disclosed in WO 2004/035607), and AT80 IgG1. Preferably, the Type II anti-CD20 antibody is a monoclonal antibody that binds to the same epitope as the humanized B-Ly1 antibody (as disclosed in WO 2005/044859).

The "ratio of the binding capacity of anti-CD20 antibodies to CD20 on Raji cells (ATCC-No. CCL-86) compared to rituximab" was determined by direct immunofluorescence measurement (measuring mean fluorescence intensity (MFI)) using the anti-CD20 antibodies conjugated to Cy5 and rituximab conjugated to Cy5 in a FACSArray (Becton Dickinson) using Raji cells (ATCC-No. CCL-86) and calculated as follows:

The ratio of binding performance to CD20 on Raji cells (ATCC-No. CCL-86) =

MFI refers to mean fluorescence intensity. As used herein, "Cy5 labeling ratio" means the number of Cy5 label molecules per molecule of antibody.

Typically, said type I anti-CD20 antibody has a ratio of the binding properties of said first anti-CD20 antibody to CD20 on Raji cells (ATCC-No. CCL-86) compared to rituximab of 0.8 to 1.2, preferably 0.9 to 1.1.

Typically, the type II anti-CD20 antibody has a ratio of the binding properties of the second anti-CD20 antibody to CD20 on Raji cells (ATCC-No. CCL-86) compared to rituximab of 0.3 to 0.6, preferably 0.35 to 0.55, more preferably 0.4 to 0.5.

In a preferred embodiment, the type II anti-CD20 antibody, preferably a humanized B-Ly1 antibody, has increased antibody-dependent cellular cytotoxicity (ADCC).

"An antibody with increased antibody-dependent cellular cytotoxicity (ADCC)" means an antibody with increased ADCC as determined by any suitable method known to one of ordinary skill in the art, as that term is defined herein. One recognized in vitro ADCC assay is as follows:

1) The assay uses target cells known to express the target antigen recognized by the antigen-binding region of the antibody;

2) The assay uses human peripheral blood mononuclear cells (PBMCs) isolated from the blood of randomly selected healthy donors as effector cells;

3) Perform the assay according to the following protocol:

i) PBMCs were isolated using a standard density centrifugation protocol and suspended in RPMI cell culture medium at 5 x 10 ⁶ cells/ml;

ii) target cells were cultured by standard tissue culture methods, harvested from the exponential growth phase with a viability greater than 90%, washed in RPMI cell culture medium, labeled with 100 microcuries ⁵¹ CI, washed twice with cell culture medium, and resuspended in cell culture medium at a density of 10 ⁵ cells/ml;

iii) transferring 100 μl of the final target cell suspension to each well of a 96-well microtiter plate;

iv) serially diluting the antibody from 4000 ng/ml to 0.04 ng/ml in cell culture medium and adding 50 μl of the resulting antibody solution to target cells in a 96-well microtiter plate, testing multiple antibody concentrations covering the entire concentration range in triplicate;

v) For the maximum release (MR) control, three additional wells of the plate containing labeled target cells received 50 μl of a 2% (VN) aqueous solution of nonionic detergent (Nonidet, Sigma, St. Louis) instead of the antibody solution (point iv above);

vi) For spontaneous release (SR) controls, three additional wells of the plate containing labeled target cells received 50 μl of RPMI cell culture medium instead of the antibody solution (point iv above);

vii) The 96-well microtiter plate was then centrifuged at 50 x g for 1 minute and incubated at 4°C for 1 hour;

viii) 50 μl of the PBMC suspension (point i above) was added to each well to produce an effector to target cell ratio of 25:1, and the plate was placed in an incubator at 37°C in a 5% _CO2 atmosphere for 4 hours;

ix) harvesting the cell-free supernatant from each well and quantifying the experimentally released radioactivity (ER) using a gamma counter;

x) calculating the percentage of specific lysis for each antibody concentration according to the formula (ER-MR)/(MR-SR) x 100, where ER is the mean radioactivity quantified (see point ix above) for the antibody concentration, MR is the mean radioactivity quantified (see point ix above) for the MR control (see point v above), and SR is the mean radioactivity quantified (see point ix above) for the SR control (see point vi above);

4) "Increased ADCC" is defined as an increase in the maximum percentage of specific lysis observed within the antibody concentration range tested above and/or a decrease in the antibody concentration required to achieve half the maximum percentage of specific lysis observed within the antibody concentration range tested above. The increase in ADCC is relative to ADCC mediated by the same antibody as measured by the above assay, produced by the same type of host cells using the same standard production, purification, formulation, and storage methods known to those skilled in the art, but not produced by host cells engineered to overexpress GnTIII.

Such "increased ADCC" can be obtained by glycoengineering of the antibody, which means enhancing the natural, cell-mediated effector function of a monoclonal antibody by engineering its oligosaccharide components, as described in Umana, P. et al., Nature Biotechnol. 17:176-180 (1999) and U.S. Patent No. 6,602,684.

The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells by an antibody according to the present invention in the presence of complement. Preferably, CDC is measured by treating a preparation of CD20-expressing cells with an anti-CD20 antibody according to the present invention in the presence of complement. CDC is detected if the antibody induces 20% or more tumor cell lysis (cell death) after 4 hours at a concentration of 100 nM. Preferably, the assay is performed using tumor cells labeled with ⁵¹ Cr or Eu and measurement of released ⁵¹ Cr or Eu. A control comprises incubating the tumor target cells with complement but not with the antibody.

Typically, type I and type II anti-CD20 antibodies of the IgG1 isotype exhibit characteristic CDC properties. Compared to each other, type I anti-CD20 antibodies have increased CDC (if IgG1 isotype), while type II anti-CD20 antibodies have decreased CDC (if IgG1 isotype). Preferably, both type I and type II anti-CD20 antibodies are IgG1 isotype antibodies.

The "rituximab" antibody is a genetically engineered chimeric monoclonal antibody directed against the human CD20 antigen containing a human γ1 murine constant domain. This chimeric antibody contains a human γ1 constant domain and is identified by the name "C2B8" in EP2000149B1 (Anderson et al., see, e.g., Figures 4 and 5). Rituximab is approved for the treatment of patients with relapsed or refractory low-grade or follicular, CD20-positive, B-cell non-Hodgkin's lymphoma. In vitro mechanism of action studies have shown that rituximab exhibits human complement-dependent cytotoxicity (CDC) (Reff et al., Blood 83(2):435-445 (1994)). In addition, it exhibits significant activity in an assay measuring antibody-dependent cellular cytotoxicity (ADCC).

The term "humanized B-Ly1 antibody" refers to a humanized B-Ly1 antibody as disclosed in WO2005/044859, which is obtained from the 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: 131-139 (1987)) by chimerization with human constant domains from IgG1 followed by humanization (see WO2005/044859). These "humanized B-Ly1 antibodies" are disclosed in detail in WO2005/044859.

Preferably, the "humanized B-Ly1 antibody" has a heavy chain variable region (VH) selected from the group consisting of SEQ ID No: 3 to SEQ ID No: 20 (B-HH2 to B-HH9 and B-HL8 to B-HL17 of WO2005/044859). Particularly preferred are Seq. ID Nos: 3, 4, 7, 9, 11, 13, and 15 (B-HH2, B-HH3, B-HH6, B-HH8, B-HL8, B-HL11, and B-HL13 of WO2005/044859). Most preferably, the VH is BHH6. Preferably, the "humanized B-Ly1 antibody" has a light chain variable region (VL) of SEQ ID No: 20 (B-KV1) of WO2005/044859. Furthermore, preferably, the humanized B-Ly1 antibody is an IgG1 antibody. Preferably, such humanized B-Ly1 antibodies are glycoengineered (GE) in the Fc region according to the procedures described in WO 2005/044859, WO 2004/065540, Umana, P. et al., Nature Biotechnol. 17:176-180 (1999), and WO 99/154342. Most glycoengineered humanized B-Ly1 antibodies have an altered glycosylation pattern in the Fc region, preferably with reduced levels of fucose residues. Preferably, at least 40% or more (in one embodiment, between 40% and 60%, in another embodiment, at least 50%, and in yet another embodiment, at least 70% or more) of the Fc region oligosaccharides are non-fucosylated. Furthermore, preferably, the Fc region oligosaccharides are bisected. Most preferably, the "humanized B-Ly1 antibody" comprises VH B-HH6 and VL B-KV1 of WO2005/044859. As used herein, the antibody is also referred to as "HuMab <CD20>". The antibody is named INN Afutuzumab. In another most preferred embodiment, the antibody has reduced levels of fucose residues, as defined above, and/or most preferably, the oligosaccharides in the Fc region are bisected. In another most preferred embodiment, the antibody exhibits increased ADCC, as defined herein.

Oligosaccharide components can significantly affect the properties related to the efficacy of therapeutic glycoproteins, including physical stability, resistance to protease attack, interaction with the immune system, pharmacokinetics, and specific biological activity. Such properties may depend not only on the presence or absence of oligosaccharides, but also on the specific structure of the oligosaccharides. Some generalizations between oligosaccharide structure and glycoprotein function can be made. For example, certain oligosaccharide structures mediate the rapid removal of glycoproteins from the bloodstream via interactions with specific carbohydrate binding proteins, while other oligosaccharide structures can be bound by antibodies and trigger unwanted immune responses. (Jenkins et al., Nature Biotechnol. 14: 975-81 (1996)).

Mammalian cells are the preferred host for producing therapeutic glycoproteins due to their ability to glycosylate proteins in a form most compatible with human applications (Cumming et al., Glycobiology 1:115-30 (1991); Jenkins et al., Nature Biotechnol. 14:975-81 (1996)). Bacterial glycosylation of proteins is very rare, and like other common host types, such as yeast, filamentous fungi, insects, and plant cells, they produce glycosylation patterns that are associated with rapid clearance from the bloodstream, unwanted immune interactions, and (in some specific cases) reduced biological activity. Among mammalian cells, Chinese hamster ovary (CHO) cells have been the most commonly used over the past two decades. In addition to providing an appropriate glycosylation pattern, these cells allow the consistent generation of genetically stable, highly productive clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media, allowing the development of safe and reproducible bioprocesses. Other commonly used animal cells include baby hamster kidney (BHK) cells, NSO- and SP2/0 mouse melanoma cells. More recently, the generation of self-transgenic animals has also been tested (Jenkins et al., Nature Biotechnol. 14:975-981 (1996)).

All antibodies contain carbohydrate structures at conserved positions in the heavy chain constant region, with each isotype possessing a unique array of N-linked carbohydrate structures that variably affect protein assembly, secretion, or functional activity (Wright, A. and Morrison, S.L., Trends Biotech. 15:26-32 (1997)). Depending on the degree of processing, the attached N-linked carbohydrate structures vary considerably and can include high-mannose, multibranched, and biantennary complex oligosaccharides. In general, there is heterogeneous processing of the core oligosaccharide structure attached at specific glycosylation sites, such that even monoclonal antibodies exist in multiple glycoforms. Similarly, significant differences in antibody glycosylation have been shown between cell lines, and even slight differences are seen for a given cell line cultured under different culture conditions (Lifely, M.R. et al., Glycobiology 5(8):813-22 (1995)).

One way to achieve a substantial increase in potency while maintaining a simple production process and potentially avoiding significant, unwanted side effects is to enhance the natural, cell-mediated effector functions of monoclonal antibodies by engineering their oligosaccharide components, as described in Umana, P. et al., Nature Biotechnol. 17:176-180 (1999) and U.S. Patent No. 6,602,684. IgG1 antibodies (i.e., the most commonly used antibodies in cancer immunotherapy) are glycoproteins with a conserved N-linked glycosylation site at Asn297 in each CH2 domain. Two complex biantennary oligosaccharides attached to Asn297 are buried between each CH2 domain, forming extensive contacts with the polypeptide backbone, and their presence is crucial for antibody-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, M.R. et al., Glycobiology 5:813-822 (1995); Jefferis, R. et al., Immunol. Rev. 163:59-76 (1998); Wright, A. and Morrison, S.L., Trends Biotechnol. 15:26-32 (1997)).

Overexpression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisected oligosaccharides, in Chinese hamster ovary (CHO) cells was previously shown to significantly enhance the in vitro ADCC activity of an anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced by engineered CHO cells (see Umana, P. et al., Nature Biotechnol. 17:176-180 (1999); and WO 99/154342, incorporated herein by reference in their entirety). Antibody chCE7 belongs to a large class of unconjugated monoclonal antibodies that have high tumor affinity and specificity, but have too little potency to be clinically useful when produced in standard industrial cell lines lacking the GnTIII enzyme (P. et al., Nature Biotechnol. 17:176-180 (1999)). The studies show for the first time that a substantial increase in ADCC activity can be obtained by engineering antibody-producing cells to express GnTIII, which also results in an increase in the proportion of constant region (Fc)-bound bisected oligosaccharides, including bisected non-fucosylated oligosaccharides, above the levels found in naturally occurring antibodies.

The term "expression of the CD20 antigen" is intended to indicate the expression of a significant level of the CD20 antigen on the cell surface of a cell, preferably a B cell, more preferably a B cell from a tumor or cancer, preferably a non-solid tumor. Patients with a "CD20-expressing cancer" can be determined by standard assays known in the art. Preferably, "expression of the CD20 antigen" is also intended to indicate the expression of a significant level of the CD20 antigen on the cell surface of a cell, preferably a B cell, more preferably a B cell of an autoimmune disease. CD20 antigen expression is measured using, for example, immunohistochemistry (IHC) assays, FACS, or PCR-based detection of the corresponding mRNA.

"Patient" or "subject" refers to any mammal, and preferably a human, suffering from a condition or disease in accordance with the present invention.

As used herein, preferably, the term "CD20-expressing cancer" refers to lymphoma (preferably B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemia. Such lymphomas and lymphocytic leukemias include, for example: (a) follicular lymphoma, (b) small non-cleaved cell lymphoma/Burkitt's lymphoma (including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma and non-Burkitt's lymphoma), (c) marginal zone lymphoma (including extranodal marginal zone B cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT), nodal marginal zone B cell lymphoma and splenic marginal zone lymphoma), (d) mantle cell lymphoma (MCL), (e) large cell lymphoma (including B cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic leukemia), (f) leukemia (including B cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic leukemia), (g) leukemia (including B cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic leukemia), (h ...i) leukemia (including B cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic leukemia), (ii) leukemia (including B cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic leukemia), (iii) leukemia (including B cell diffuse large cell lymphoma (DLCL), diffuse mixed cell lymphoma, immunoblastic leukemia), ( (i) plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasma cell neoplasms, and (j) Hodgkin's disease.

Preferably, the cancer expressing CD20 is a B-cell non-Hodgkin's lymphoma (NHL). Other examples of cancers expressing CD20 include: mantle cell lymphoma (MCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell diffuse large cell lymphoma (DLCL), Burkitt's lymphoma, hairy cell leukemia, follicular lymphoma, multiple myeloma, marginal zone lymphoma, post-transplant lymphoproliferative disorder (PTLD), HIV-associated lymphoma, Waldenstrom's macroglobulinemia, or primary CNS lymphoma.

As used herein, "autoimmune disease" refers to a disease or condition caused by and directed against an individual's own tissues. Examples of autoimmune diseases or conditions include, but are not limited to, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic cleroderma and sclerosis, responses associated with inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome, and inflammatory bowel disease. syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving T-cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), juvenile onset diabetes, multiple sclerosis, allergic encephalomyelitis encephalomyelitis), immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis, agranulocytosis, 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 leukocytosis, inflammatory disorders of the central nervous system (CNS), multiple organ injury syndrome, myasthenia gravis gravis), antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bechet disease, Castleman syndrome, Goodpasture syndrome, Lambert-Eaton myasthenic syndrome, Reynaud syndrome, Sjögren syndrome, Stevens-Johnson syndrome, pemphigoid bullous, pemphigus, autoimmune polyendocrinopathies, nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura purpura (ITP), thrombotic throbocytopenic purpura (TTP), autoimmune thrombocytopenia, autoimmune testicular and ovarian disorders, including autoimmune orchitis and oophoritis, and primary hypothyroidism; autoimmune endocrine disorders, including autoimmune thyroiditis, chronic thyroiditis (Hashimoto’s Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison’s disease, Graves’ disease, and autoimmune polyglandular syndrome. syndromes) (or polyglandular endocrinopathy syndrome), type 1 diabetes mellitus, also known as insulin-dependent diabetes mellitus (IDDM) and Sheehan's syndrome; autoimmune hepatitis, lymphoid interstitial pneumonia (HIV), bronchiolitis obliterans (non-graft) versus NSIP, Guillain-Barre's syndrome, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), medium vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis ( progressive glomerulonephritis), primary biliary cirrhosis, Celiac sprue (gluten enteropathy), cryoglobulinemia, amyotrophic lateral sclerosis (ALS), coronary artery disease, etc.

As used herein, "growth inhibitory agent" refers to a compound or composition that inhibits the growth of cells, particularly cancer cells expressing CD20, in vitro or in vivo. Thus, a growth inhibitory agent can be one that significantly reduces the percentage of cells expressing CD20 that are in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a position other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. For more information, see "The Molecular Basis of Cancer," Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs," Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13.

"Treatment" refers to both therapeutic treatment and preventative or prophylactic measures. Subjects in need of treatment include those already suffering from a disease as well as those in whom the disease is to be prevented. Thus, a patient to be treated herein may already be diagnosed as suffering from a disease or may have a predisposition to or be susceptible to the disease.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell destruction. The term is intended to include radioisotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and radioisotopes 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.

"Chemotherapeutic agent" refers to a chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide (CYTOXAN ^™ ); alkyl sulfonates; sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodepa, carboquone, meturedepa, and uredepa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL ^™ ); β-lapachone; lapachol; colchicines; betulinic acid; camptothecins (including the synthetic analogs topotecan (HYCAMTIN ^™ ), CPT-11 (irinotecan, CAMPTOSAR ^™ ), acetylcamptothecin, scopoletin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); podophyllotoxin; podophyllinic acid acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, and chlorambucil. hydrochloride), melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as the enediynes (e.g., calicheamicin, especially calicheamicin gamma and calicheamicin omega (see, e.g., Angewandte, Chemie Intl. Ed. Engl., 33:183-186 (1994); anthracyclines (dynemicins), including anthracycline A; esperamicin; and neocarzinostatin chromophores and related chromophores (enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN ^™ , morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolidodoxorubicin and liposomal doxorubicin hydrochloride injection (DOXIL ^™ ), liposomal doxorubicin TLC D-99 (MYOCET ^™ ), PEGylated liposomal doxorubicin (CAELYX ^™ ), ), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR ^™ ), tegafur (UFTORAL ^™ ), capecitabine (XELODA ^™ ), epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azathioprine, dapoxetine, thiabendazole, dapoxetine, thioguanine, pyrimidine analogs such as ancitabine, azathioprine, dapoxetine, dapoxetine, thioguan ... azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK ^™ polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verrucarin A, roridin A, and anguidin); urethan; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");thiotepa; taxoids, such as paclitaxel (TAXOL ^™ ), albumin-engineered nanoparticle formulations of paclitaxel (ABRAXANE ^™) , ) and doxetaxel (TAXOTERE ^™ ); chloranbucil; thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin, and carboplatin; and the vincas (which prevent tubulin from polymerizing to form microtubules), including vinblastine (VELBAN ^™ ), vincristine (ONCOVIN ^™ ), vindesine (ELDISINE ^™ ), FILDESIN ^™ , and vinorelbine (NAVELBINE™ ⁾ . )); etoposide (VP-16); ifosfamide; mitoxantrone; leucovovin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid, including bexarotene (TARGRETIN ^™ ); bisphosphonates, such as clodronate (e.g., BONEFOS ^™ or OSTAC ^™ ), etidronate (DIDROCAL™), and daunomycin (Daunomycin ^™ ). ), NE-58095, zoledronic acid/zoledronate (ZOMETA™), alendronate (FOSAMAJX ^™ ), pamidronate (AREDIA ^™ ), tiludronate (SKELID ^™ ), or risedronate (ACTONEL ^™ ) ^; troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly antisense oligonucleotides that inhibit the expression of genes in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE ^™ vaccine and gene therapy vaccines, such as ALLOVECTIN ^™ vaccine, LEUVECTIN ^™ vaccine, and VAXID ^™ vaccines; topoisomerase 1 inhibitors (e.g., LURTOTECAN ^™ ); rmRH (e.g., ABARELIX ^™ ); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE ^™ ); CCI-779; tipifarnib (R1 1577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersensodium (GENASENSE ^™ ); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing substances; and combinations of two or more of the foregoing substances, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy (optionally further comprising interferon (CHVP/interferon))), FOLFOX (abbreviation for the treatment regimen of oxaliplatin (ELOXATIN ^™ ) in combination with 5-FU and folinic acid), CVP (cyclophosphamide, vincristine, and prednisolone), MCP (mitoxantrone, 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.

"Anti-angiogenic agents" refer to compounds that block or interfere with the formation of blood vessels to some extent. Anti-angiogenic factors can, for example, be small molecules or antibodies that bind to growth factors or growth factor receptors involved in promoting angiogenesis. Preferred anti-angiogenic agents herein are antibodies that bind to vascular endothelial growth factor (VEGF), such as bevacizumab (AVASTIN ^™ ).

The term "cytokine" refers to a general term for proteins released by one cell population that act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cytokines include growth hormones 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 follicle-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-related peptide, inhibin; activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), nerve growth factors such as NGF-β, platelet growth factor; transforming growth factor (TGF) such as TGF-α and TGF-β, insulin-like growth factor-I and -II, erythropoietin (EPO), osteoinductive factors; interferons such as interferon-α, -β and -γ, colony stimulating factors (CSF) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (IL) 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, tumor necrosis factors such as TNF-α or TNF-β, and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes biologically active equivalents of proteins and native sequence cytokines from natural sources or from recombinant cell culture.

The term "effective amount" refers to an amount that provides the desired effect. In the case of formulation ingredients such as hyaluronidase enzymes according to the present invention, an effective amount is the amount necessary to improve the dispersion and absorption of the co-administered anti-CD20 antibody in the following manner so that the anti-CD20 antibody can act in a therapeutically effective manner, as outlined above. In the case of a pharmaceutical drug substance, it is the amount of the active ingredient that effectively treats the disease in the patient. In the case where the disease is cancer, an effective amount of the drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down and preferably stop to some extent) the infiltration of cancer cells into surrounding organs; inhibit (i.e., slow down and preferably stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate one or more symptoms associated with cancer to some extent. To the extent that the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. An effective amount can prolong progression-free survival, produce an objective response (including partial response PR or complete response CR), increase overall survival time, and/or improve one or more cancer symptoms.

The term "pharmaceutical formulation" refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile.

A "sterile" formulation is sterile or free from all viable microorganisms and their spores.

A "stable" formulation is one in which all proteins substantially retain their physical stability and/or chemical stability and/or biological activity after storage at the intended storage temperature, e.g., 2-8°C. Preferably, the formulation substantially retains its physical and chemical stability, and its biological activity after storage. Generally, the storage period is selected based on the intended shelf life of the formulation. In addition, preferably, the formulation is stable after freezing (to, e.g., -20°C) and thawing the formulation, e.g., after one or more cycles of freezing and thawing. Various analytical techniques for measuring protein stability are available in the art and are reviewed in, e.g., Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, New York, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). 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 assessing aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity, and/or by visual inspection); assessing charge heterogeneity using cation exchange chromatography or capillary zone electrophoresis; SDS-PAGE analysis to compare reduced and intact antibodies; assessing the biological activity or antigen binding function of the antibody; etc. Instability can involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation), isomerization (e.g., Asp isomerization), clipping/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, etc.

Therapeutic formulations of antibodies for use according to the present invention are prepared by mixing an antibody having the desired degree of purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of a lyophilized formulation or an aqueous solution for storage. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.

As used herein, the term "surfactant" means a pharmaceutically acceptable surfactant. In the formulation of the present invention, the amount of surfactant is described in terms of a percentage expressed in weight/volume. The most commonly used weight/volume unit is mg/mL. Suitable examples of pharmaceutically acceptable surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene olefin ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium lauryl sulfate (SDS). The most suitable polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20 ^™ ) and polysorbate 80 (sold under the trademark Tween 80 ^™ ). The most suitable polyethylene-polypropylene copolymers are those sold under the names F68 or Poloxamer 188 ^™ . Preferred polyoxyethylene-stearate are those sold under the trademark Myrj ^™ . The most suitable polyoxyethylene alkyl ethers are those sold under the trademark Brij ^™ . The most suitable hydrocarbylphenol polyoxyethylene ethers are sold under the trademark Triton-X.

As used herein, the term "buffer" means a pharmaceutically acceptable buffer. As used herein, the term "buffer providing a pH of 5.5 ± 2.0" refers to an agent that provides a solution comprising the buffer to resist pH changes through the action of its acid/base conjugate components. Suitable pharmaceutically acceptable buffers according to the present invention include, but are not limited to, histidine buffer, citrate buffer, gluconate buffer, succinate buffer, acetate buffer, glycylglycine and other organic acid buffers, and phosphate buffer. Preferred buffers include L-histidine or a mixture of L-histidine and L-histidine hydrochloride, with an isotonic agent and potential pH adjustment with an acid or base as known in the art. Most preferably, L-histidine.

"Histidine buffer" is a buffer comprising the amino acid histidine. Examples of histidine buffers include histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate. It was found that the preferred histidine buffer identified in the examples herein is histidine hydrochloride. In a preferred embodiment, the histidine hydrochloride buffer is prepared by titrating L-histidine (free base, solid) with dilute hydrochloric acid or by dissolving L-histidine and L-histidine hydrochloride (e.g., in the form of a monohydrate) in defined amounts and ratios.

"Isotonic" means that the formulation of interest has substantially the same osmotic pressure as human blood. An isotonic formulation will generally have an osmotic pressure of about 300 mOsm/kg. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

As used herein, the term "isotonic agent" means a pharmaceutically acceptable isotonic agent. Isotonic agents are used to provide isotonic formulations. Isotonic formulations are liquids or solid forms, such as the liquid reconstructed in a lyophilized form, and mean some other solutions compared therewith, such as physiological saline solutions and serum with the same tension force. Suitable isotonic agents include but are not limited to salts, include but are not limited to sodium chloride (NaCl) or potassium chloride, saccharides and sugar alcohols, include but are not limited to glucose, sucrose, trehalose or glycerol and any component from the group consisting of amino acids, saccharides, salts and combinations thereof. Isotonic agents are generally used in a total amount of about 5mM to about 350mM.

The term "liquid" as used herein in connection with a formulation according to the present invention means a formulation that is liquid at a temperature of at least about 2 to about 8°C.

The term "lyophilized" as used herein in connection with the formulation according to the present invention means a formulation that has been dried, ie the formulation has been frozen and then ice has been sublimed from the frozen contents by any freeze-drying method known in the art, such as commercial freeze-drying apparatus.

As used herein, "salt" means a salt in an amount of about 1 mM to about 500 mM. Non-limiting examples of salts include salts of any combination of the cations sodium, potassium, calcium or magnesium and the anions chloride, phosphate, citrate, succinate, sulfate or mixtures thereof.

As used herein, the term "amino acid" means an amino acid in an amount of about 1 to about 100 mg/mL, including, but not limited to, arginine, glycine, ornithine, glutamine, asparagine, lysine, histidine, glutamic acid, asparagine, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, and proline.

As used herein, "saccharides" encompass the general composition ( _CH2O ) _n and its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. Examples of sugars herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, and the like. Also included within the definition according to the present invention are glucosamine, N-methylglucosamine (so-called "methylglucamine"), galactosamine, and neuraminic acid, as well as combinations of sugars according to the present invention. Preferred sugars herein are non-reducing disaccharides, such as trehalose or sucrose. The most preferred carbohydrate according to the present invention is trehalose.

The term "stabilizer" refers to pharmaceutically acceptable stabilizers such as, for example but not limited to, amino acids and sugars as described in the above section and commercial dextran of any kind and molecular weight as known in the art.

The term "antioxidant" means a pharmaceutically acceptable antioxidant. This may include excipients such as methionine, benzyl alcohol or any other excipient used to minimize oxidation.

The term "treatment method" or its equivalent, when applied to, for example, cancer, refers to a procedure or course of action 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 another proliferative disorder" does not necessarily mean that cancer cells or other disorders will actually be eliminated, that the number of cells or disorders will actually be reduced, or that the symptoms of the cancer or other disorder will actually be alleviated. Often, a method of treating cancer will be implemented even if the probability of success is low, but it is still believed to induce an overall beneficial course of action in view of the patient's medical history and estimated survival expectancy.

Therefore, the problem to be solved by the present invention is to provide novel, highly concentrated, stable pharmaceutical formulations for subcutaneous injection of pharmaceutically active anti-CD20 antibodies or mixtures of such antibody molecules. In addition to a relatively high amount of anti-CD20 antibodies or mixtures thereof, such formulations contain a buffer, a stabilizer or a mixture of two or more stabilizers, a non-ionic surfactant, and preferably an effective amount of at least one hyaluronidase enzyme. The preparation of highly concentrated antibody formulations is challenging due to the potential increase in viscosity at higher protein concentrations and the potential increase in protein aggregation, a phenomenon that is essentially concentration-dependent. High viscosity negatively affects the processing capabilities (e.g., aspiration and filtration steps) and administration (e.g., syringe capacity) of the antibody formulation. High viscosity can be reduced in some cases by adding excipients. The control and analysis of protein aggregation are increasingly challenging. Aggregation is potentially encountered during various steps of the manufacturing process (including fermentation, purification, formulation) and during storage. Various factors, such as temperature, protein concentration, agitation stress, freezing and thawing, solvent and surfactant effects, and chemical modifications can affect the aggregation behavior of therapeutic proteins. During the development of highly concentrated antibody formulations, the aggregation tendency of the protein must be monitored and controlled by the addition of various excipients and surfactants [Kiese S. et al., J. Pharm. Sci., 2008; 97(10); 4347-4366].

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 such antibody molecules for parenteral administration. Preferably, the route of administration is intravenous administration, either as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, or intrathecal routes. Intravenous or subcutaneous administration of the antibody is preferred; subcutaneous injection is most preferred. As outlined above, it is non-trivial to generate a highly concentrated, stable pharmaceutical formulation of a CD20 antibody that is substantially free of particles. If the formulation is intended for subcutaneous application, in a preferred embodiment, the formulation is combined with a hyaluronidase enzyme.

More particularly, the highly concentrated, stable pharmaceutical formulation of the pharmaceutically active anti-CD20 antibody formulation of the present invention comprises:

- about 20 to 350 mg/ml anti-CD20 antibody;

- about 1 to 100 mM buffer providing a pH of 5.5 ± 2.0;

- about 1 to 500 mM stabilizer or a mixture of two or more stabilizers, wherein optionally methionine is used as a second stabilizer, preferably in a concentration of 5 to 25 mM;

- 0.01 to 0.1% nonionic surfactant; and

-Preferably, an effective amount of at least one hyaluronidase enzyme.

The highly concentrated, stable pharmaceutical formulations of the pharmaceutically active anti-CD20 antibody formulations of the present invention can be provided in liquid form or can be provided in lyophilized form. The antibody concentration in the reconstituted formulation can be increased by reconstituting the lyophilized formulation to provide a protein concentration in the reconstituted formulation that is about 2-40 times greater than the protein concentration in the mixture before the lyophilization step.

The preferred anti-CD20 antibody concentration is 50 to 150 mg/ml, more preferably 75 to 150 mg/ml, even more preferably 120 ± 20 mg/ml, and most preferably about 120 mg/ml.

The preferred concentration of the buffer is 1 to 50 mM, more preferably 10 to 30 mM; the most preferred concentration is about 20 mM. Various buffers are known to those skilled in the art, as outlined above. Preferred buffers are selected from the group consisting of histidine buffer, acetate buffer, and citrate buffer, with L-histidine/HCl buffer being the most preferred. The histidine buffer according to the present invention is used in an amount of about 1 mM to about 50 mM, preferably about 10 mM to about 30 mM, and even more preferably about 20 mM. The acetate buffer according to the present invention is preferably about 10 mM to about 30 mM, and most preferably about 20 mM. The citrate buffer according to the present invention is preferably about 10 mM to about 30 mM, and most preferably about 20 mM.

Regardless of the buffer used, the pH is adjusted to a value comprised between about 4.5 and about 7.0, and preferably, between about 5.5 and about 6.5, further preferably selected from the group consisting of 5.5, 6.0, 6.1, and 6.5. This pH can be achieved by adjusting with acids or bases known in the art, or by using a mixture of appropriate buffer components, or both.

Preferably, the stabilizer (used synonymously with the term "stabilizer" in this patent specification) is selected from the group consisting of salts, carbohydrates, sugars, and amino acids, more preferably carbohydrates or sugars, more preferably sugars approved by the authorities as suitable additives or excipients in pharmaceutical formulations, and most preferably selected from the group consisting of α,α-trehalose dihydrate, NaCl, and methionine. The preferred concentration of the stabilizer is 15 to 250 mM, or more preferably 150 to 250 mM. A concentration of approximately 210 mM is most preferred. The formulation may contain a second stabilizer, preferably methionine, preferably at a concentration of 5 to 25 mM, more preferably 5 to 15 mM. The most preferred methionine concentration is approximately 10 mM.

Preferably, the nonionic surfactant is a polysorbate, more preferably, selected from the group consisting of polysorbate 20, polysorbate 80, and polyethylene-polypropylene copolymer. The concentration of the nonionic surfactant is 0.01 to 0.1% (w/v), or 0.02 to 0.08% (w/v), and preferably 0.02 to 0.06% (w/v), most preferably about 0.06% (w/v).

As used herein, the term "sugar" means a pharmaceutically acceptable sugar used in an amount of about 25 mM to about 500 mM. Preferably, it is 100 to 300 mM. More preferably, it is 180 to 240 mM. Most preferably, it is 210 mM.

The concentration of the hyaluronidase enzyme depends on the actual hyaluronidase enzyme used in the preparation of the formulation of the present invention. Based on the further disclosure below, those skilled in the art can easily determine the effective amount of the hyaluronidase enzyme. It should be provided in sufficient amounts so that the dispersion and absorption of the anti-CD20 antibodies administered together are possible. Preferably, the effective amount of the hyaluronidase enzyme is about 1'000 to 16'000 U/ml, wherein based on the assumed specific activity of 100'000 U/mg, the amount corresponds to about 0.01mg to 0.15mg protein. The preferred concentration of the hyaluronidase enzyme is about 1'500 to 12'000 U/ml. Most preferably, a concentration of about 2'000 U/ml or about 12'000 U/ml. The amount specified above corresponds to the amount of the hyaluronidase enzyme initially added to the formulation. The hyaluronidase enzyme is present in the final formulation of the combination or is used for co-application, for example, as a co-formulation as further described below. An important issue for the claimed formulation is that when it is ready for use and/or injection it has the claimed composition.

The hyaluronidase enzyme can be derived from animal, human samples or produced based on recombinant DNA technology, as further described below.

More particularly, the highly concentrated, stable pharmaceutical formulation according to the present invention has one of the following preferred compositions:

a) 100 to 150 mg/ml anti-CD20 antibody, preferably rituximab, ocrelizumab or HuMab<CD20>; 1 to 50 mM histidine buffer, preferably L-histidine/HCl (pH about 5.5); 15 to 250 mM stabilizer, preferably α,α-trehalose dihydrate and optionally methionine at a concentration of 5 to 25 mM as a second stabilizer; a non-ionic surfactant selected from the group consisting of polysorbate 20 and polysorbate 80, preferably 0.02 to 0.06% (w/v), and optionally 1'000 to 16'000 U/ml hyaluronidase, preferably rHuPH20, most preferably at a concentration of 2'000 U/ml or 12'000 U/ml.

b) 120±20 mg/ml anti-CD20 antibody, wherein preferably, the antibody is rituximab, ocrelizumab or HuMab<CD20>; 10 to 30 mM, preferably 20 mM histidine buffer, preferably L-histidine/HCl (pH about 5.5); 150 to 250 mM, preferably 210 mM stabilizer, which is preferably α,α-trehalose dihydrate and optionally a concentration of 5 to 25 mM, preferably 5 to 1 5 mM, most preferably 10 mM methionine as a second stabilizer; a non-ionic surfactant selected from the group consisting of polysorbate 20 and polysorbate 80, preferably 0.02 to 0.06% (w/v), and optionally 1'000 to 16'000 U/ml, preferably 1'500 to 12'000 U/ml, most preferably 2'000 U/ml or 12'000 U/ml hyaluronidase enzyme, preferably rHuPH20.

c) 120 mg/ml anti-CD20 antibody, wherein preferably, the antibody is rituximab, ocrelizumab or HuMab<CD20>; 10 to 30 mM, preferably 20 mM histidine buffer, preferably L-histidine/HCl (pH about 5.5); 150 to 250 mM, preferably 210 mM stabilizer, which is preferably α,α-trehalose dihydrate and optionally a concentration of 5 to 25 mM, preferably 5 to 15 mM. mM, most preferably 10 mM methionine as a second stabilizer; a non-ionic surfactant selected from the group consisting of polysorbate 20 and polysorbate 80, preferably 0.02 to 0.06% (w/v), and optionally 1'000 to 16'000 U/ml, preferably 1'500 to 12'000 U/ml, most preferably 2'000 U/ml or 12'000 U/ml hyaluronidase enzyme, preferably rHuPH20.

d) 120 mg/ml anti-CD20 antibody, preferably rituximab; 20 mM histidine buffer, preferably L-histidine/HCl (pH 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 consisting of polysorbate 20 and polysorbate 80, preferably 0.02 to 0.06% (w/v), and optionally 2'000 U/ml or 12'000 U/ml hyaluronidase enzyme, preferably rHuPH20.

e) A lyophilized formulation comprising 120 mg/ml anti-CD20 antibody, preferably rituximab; 20 mM histidine buffer, preferably L-histidine/HCl (pH 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 consisting of polysorbate 20 and polysorbate 80, preferably 0.02 to 0.06% (w/v), and optionally 2'000 U/ml or 12'000 U/ml hyaluronidase enzyme, preferably rHuPH20.

Provided are stable pharmaceutical formulations of a pharmaceutically active anti-CD20 antibody comprising about 30 mg/ml to 350 mg/ml, e.g., about 30 mg/ml to 100 mg/ml (including about 30 mg/ml, about 50 mg/ml, or about 100 mg/ml) of ocrelizumab (e.g., humanized 2H7.v16); about 1 to 100 mM buffer (e.g., sodium acetate) providing a pH of 5.5±2.0 (e.g., pH 5.3); about 15 to 250 mM stabilizer or a mixture of two or more stabilizers (including trehalose, e.g., about 8% trehalose dihydrate); about 0.01 to 0.1% (w/v) nonionic surfactant; and optionally, an effective amount of at least one hyaluronidase enzyme (e.g., rhHUPH20), preferably in an amount of about 1,500 U/ml to about 12,000 U/ml.

Alternative compositions of preferred formulations are given in the examples.

It has been proposed to promote the subcutaneous injection of therapeutic proteins and antibodies by using a small amount of soluble hyaluronidase glycoprotein (sHASEGP); See WO2006/091871. It has been shown that the addition of such soluble hyaluronidase glycoproteins (as a combined formulation or by co-administration) promotes the administration of therapeutic drugs to the subcutaneous tissue. By rapidly depolymerizing the hyaluronan HA in the extracellular space, sHASEGP reduces the viscosity of the interstitial space, thereby improving hydraulic conduction, and allowing larger volumes to be safely and comfortably administered into the subcutaneous tissue. The elevated hydraulic conduction induced by sHASEGP via the reduced interstitial viscosity allows for greater dispersion, which potentially improves the systemic bioavailability of the therapeutic drugs administered by SC.

Therefore, the highly concentrated stable pharmaceutical formulation of the present invention comprising soluble hyaluronidase glycoprotein is particularly suitable for subcutaneous injection. It is well understood by those skilled in the art that such formulations comprising anti-CD20 antibodies and soluble hyaluronidase glycoproteins can be provided to be used in a single combined formulation form or alternatively in two separate formulation forms that can be mixed before subcutaneous injection. Alternatively, anti-CD20 antibodies and soluble hyaluronidase glycoproteins can be used in different parts of the body by separate injection, preferably in closely adjacent positions. It is also possible to inject the therapeutic agent present in the formulation of the present invention in a continuous injection form, for example, first soluble hyaluronidase glycoprotein, followed by injection of anti-CD20 antibody formulations. These injections can also be implemented in the opposite order, i.e., by first injecting the anti-CD20 antibody formulation, followed by injection of the soluble hyaluronidase glycoprotein. In the case where the anti-CD20 antibody and the soluble hyaluronidase glycoprotein are administered as separate injections, one or both of the proteins must be provided with a buffer, stabilizer, and nonionic surfactant at the concentrations specified in the appended claims, but excluding the hyaluronidase enzyme. The hyaluronidase enzyme can then be provided in, for example, L-histidine/HCl buffer (pH about 6.5), 100 to 150 mM NaCl, and 0.01 to 0.1% (w/v) polysorbate 20 or polysorbate 80. In a preferred embodiment, the anti-CD20 antibody is provided with a buffer, stabilizer, and nonionic surfactant at the concentrations specified in the appended claims.

As noted above, it can be considered that soluble hyaluronidase glycoprotein is another excipient in the anti-CD20 preparaton.Soluble hyaluronidase glycoprotein can be added to the anti-CD20 preparaton when manufacturing the anti-CD20 preparaton or can be added shortly before injection.Alternatively, soluble hyaluronidase glycoprotein can be provided with separate injections.In the latter case, soluble hyaluronidase glycoprotein can be provided in a lyophilized form in a separate vial and must be reconstituted with a suitable diluent before subcutaneous injection occurs, or can be provided by the manufacturer with a liquid preparaton.Anti-CD20 preparaton and soluble hyaluronidase glycoprotein can be obtained as separate entities or can also be provided with a kit comprising both injection components and instructions for subcutaneous administration thereof.Suitable instructions suitable for reconstructing and/or administering one or both of the preparatons can also be provided.

Thus, the present invention also provides a pharmaceutical composition consisting of a highly concentrated, stable pharmaceutical formulation of a pharmaceutically active anti-CD20 antibody or a mixture of such antibodies and a suitable amount of at least one hyaluronidase enzyme in the form of a kit containing both the injection components and suitable instructions for its subcutaneous administration.

Yet another aspect of the present invention relates to an injection device comprising a highly concentrated, stable pharmaceutical formulation according to the present invention. Such a 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 additionally comprise a soluble hyaluronidase glycoprotein as a combined formulation or as a separate formulation for co-administration.

A variety of anti-CD20 antibodies are known in the art. Preferably, such antibodies are monoclonal antibodies. They may be so-called chimeric antibodies, humanized antibodies, or fully human antibodies. They may be full-length anti-CD20 antibodies; anti-CD20 antibody fragments having the same biological activity; and include 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, ocrelizumab, and afutuzumab (HuMab<CD20>). The most successful therapeutic anti-CD20 antibody is rituximab, sold by Genentech Inc. and F. Hoffmann-La Roche Ltd. under the trade names MABTHERA ^™ or RITUXAN ^™ .

Preferably, the anti-CD20 antibody as defined herein is selected from the group consisting of rituximab (see, e.g., U.S. Pat. No. 7,381,560 and EP2000149B1 to Anderson et al., see, e.g., Figures 4 and 5), ocrelizumab (as disclosed in WO 2004/056312), and WO 2006/084264 (e.g., variants disclosed in Tables 1 and 2), preferably variants v.16, v.114, or v.511, and HuMab <CD20> (see WO 2005/044859). The most preferred anti-CD20 antibody is rituximab. The terms "rituximab," "ocrelizumab," and "HuMab <CD20>" encompass all corresponding anti-CD20 antibodies that meet the requirements necessary to obtain marketing authorization as an identical or biosimilar product in a country or region selected from the United States, Europe, and Japan. Rituximab has the CDR regions defined in US Patent No. 7,381,560 and EP2000149B1.

Many 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.

The SC (subcutaneous) interstitial matrix is composed of a network of fibrillar proteins embedded within a viscoelastic gel of glycosaminoglycans. Hyaluronan (HA), a non-sulfated, repeating linear disaccharide, is the major glycosaminoglycan of SC tissue. HA is secreted into the interstitium by fibroblasts as a high-molecular-weight, megadalton, viscous polymer. It is subsequently degraded locally, in the lymph, and in the liver by the action of lysosomal hyaluronidases and exoglycosidases. Approximately 50% of the hyaluronan in the body is produced by SC tissue, where it is found at approximately 0.8 mg/gm wet weight of tissue [Aukland K. and Reed R., "Interstitial-Lymphatic Mechanisms in the control of Extracellular Fluid Volume", Physiology Reviews", 1993; 73: 1-78]. An average 70 kg adult is estimated to contain 15 grams of HA, of which 30% is turned over (synthesized and degraded) daily [Laurent L.B. et al., "Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver", Exp. Physiol. 1991; 76: 695-703]. As the main component of the gel-like component of the subcutaneous matrix, HA contributes significantly to its viscosity.

Glycosaminoglycans (GAGs) are complex linear polysaccharides of the extracellular matrix (ECM). GAGs are characterized by a repeating disaccharide structure of N-substituted hexosamine and uronic acid (in the case of hyaluronan (HA), chondroitin sulfate (CS), chondroitin (C), dermatan sulfate (DS), heparan sulfate (HS), and heparin (H)), or galactose (in the case of keratan sulfate (KS)). Except for HA, all exist in a covalently bound manner to a core protein. GAGs and their core proteins are structurally referred to as proteoglycans (PGs).

In mammals, hyaluronan (HA) is found primarily in connective tissue, skin, cartilage, and in synovial fluid. Hyaluronan is also a major component of the vitreous humor of the eye. In connective tissue, the water of hydration associated with hyaluronan creates a hydrated matrix between tissues. Hyaluronan plays a key role in biological phenomena related to cell motility, including rapid development, regeneration, repair, embryogenesis, embryological development, wound healing, angiogenesis, and tumorigenesis (Toole, Cell Biol. Extracell. Matrix, Hay (ed.), Plenum Press, New York, 1991; pp. 1384-1386; Bertrand et al., Int. J. Cancer 1992; 52: 1-6; Knudson et al., FASEB J. 1993; 7: 1233-1241). In addition, hyaluronan levels are correlated with tumor aggressiveness [Ozello et al., Cancer Res. 1960; 20: 600-604; Takeuchi et al., Cancer Res. 1976; 36: 2133-2139; Kimata et al., Cancer Res. 1983; 43: 1347-1354].

HA is found in the extracellular matrix of many cells, particularly in soft connective tissue. Various physiological functions have been assigned to HA, such as in water and plasma protein homeostasis [Laurent T.C. et al., FASEB J., 1992; 6: 2397-2404]. HA production increases in proliferating cells and may play a role in mitosis. It has also been implicated in motility and cell migration. HA appears to play an important role in cell regulation, development, and differentiation [Laurent et al., supra].

HA has been widely used in clinical medicine. Its tissue protection and rheological properties have been shown to be useful in ophthalmic surgery (e.g., 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 of various organs [Laurent et al., supra].

Hyaluronan-protein interactions are also involved in the structure of the extracellular matrix or "ground matter."

Hyaluronidases are a group of generally neutral or acidic active enzymes found throughout the animal kingdom. Hyaluronidases vary in terms of substrate specificity and mechanism of action (WO 2004/078140). There are three major classes of hyaluronidases:

1. Mammalian hyaluronidases (EC 3.2.1.35) are endo-β-N-acetylhexosaminidase enzymes with tetrasaccharides and hexasaccharides as the main end products. They have both hydrolytic and transglycosidase activities and can degrade hyaluronan and chondroitin sulfate (CS), generally C4-S and C6-S.

2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan and, to varying degrees, CS and DS. They are endo-β-N-acetylhexosaminidase enzymes that operate via β-elimination reactions that primarily generate disaccharide end products.

3. Hyaluronidases from leeches, other parasites, and crustaceans (EC 3.2.1.36) are endo-β-glucuronidases that hydrolyze β1-3 linkages to generate tetrasaccharide and hexasaccharide end products.

Mammalian hyaluronidase can be further divided into two groups: neutral activity and acid activity enzyme. There are 6 kinds of hyaluronidase-like genes in the human genome, i.e. HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 and PH20/SPAM1. HYALP1 is a pseudogene, and it has not yet been shown that HYAL3 has enzymatic activity for any known substrate. HYAL4 is a chondroitinase, and shows little activity for hyaluronan. HYAL1 is a prototype acid activity enzyme, and PH20 is a prototype neutral activity enzyme. Generally, acid activity hyaluronidase, such as HYAL1 and HYAL2, lacks catalytic activity at neutral pH (i.e. pH 7). For example, HYAL1 has little catalytic activity in vitro above pH 4.5 [Frost I.G. and Stern, R., "A microtiter-based assay for hyaluronidase activity not requiring specialized reagents", Anal. Biochemistry, 1997; 251: 263-269]. HYAL2 is an acid-active enzyme with very low specific activity in vitro.

Hyaluronidase-like enzymes can also be characterized as those that are generally locked to the plasma membrane via a glycosylphosphatidylinositol anchor, such as human HYAL2 and human PH20 [Danilkovitch-Miagkova et al., Proc. Natl. Acad. Sci. USA, 2003; 100(8):4580-4585; Phelps et al., Science 1988; 240(4860):1780-1782], and those that are generally soluble, such as human HYAL1 [Frost, IG et al., "Purification, cloning, and expression of human plasma hyaluronidase", Biochem. Biophys. Res. Commun. 1997; 236(1):10-15]. However, there are variations between species: for example, bovine PH20 is very loosely attached to the plasma membrane and is not anchored via a phospholipase-sensitive anchor [Lalancette et al., Biol. Reprod., 2001; 65(2): 628-36]. This unique feature of bovine hyaluronidase has allowed the use of soluble bovine testicular hyaluronidase enzymes as extracts for clinical use (Wydase ^™ , Hyalase ^™ ). Other PH20 species are lipid-anchored enzymes that are generally insoluble without the use of detergents or lipases. For example, human PH20 is anchored to the plasma membrane via a GPI anchor. Attempts to generate human PH20 without introducing a lipid anchor into the polypeptide resulted in a catalytically inactive enzyme or an insoluble enzyme [Arming et al., Eur. J. Biochem., 1997; 1; 247(3): 810-4]. Naturally occurring macaque sperm hyaluronidase is found in both soluble and membrane-bound forms. Although the 64 kDa membrane-bound form possesses enzymatic 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): 142-53]. Thus, soluble forms of PH20 often lack enzymatic activity under neutral conditions.

As noted above and in accordance with the teachings in WO2006/091871 and U.S. Patent No. 7,767,429, a small amount of soluble hyaluronidase glycoprotein (sHASEGP) can be introduced into a formulation so that therapeutic drugs are administered into the hypodermis. By rapidly depolymerizing the HA in the extracellular space, sHASEGP reduces interstitial viscosity, thereby improving hydraulic conduction, and allowing larger volumes to be safely and comfortably administered into SC tissues. The elevated hydraulic conduction induced by sHASEGP via reduced interstitial viscosity allows for greater dispersion, which potentially improves the systemic bioavailability of the therapeutic drugs administered by SC.

When injected into the hypodermis, sHASEGP localizes the depolymerization of HA to the injection site in the SC tissue. Experimental evidence shows that sHASEGP is locally inactivated in the interstitial space with a half-life of 13 to 20 minutes in mice, and there is no detectable systemic absorption in the blood after a single intravenous dose in CD-1 mice. In the vascular compartment, sHASEGP shows a half-life of 2.3 and 5 minutes in mice and macaques, respectively, at doses up to 0.5 mg/kg. Combined with the continuous synthesis of HA substrate in SC tissue, sHASEGP's rapid clearance leads to a transient and locally active permeation enhancement of other co-injected molecules, an effect that is fully reversible within 24 to 48 hours after administration [Bywaters G.L. et al., "Reconstitution of the dermal barrier to dye spread after Hyaluronidase injection", Br. Med. J., 1951; 2(4741): 1178-1183].

In addition to its influence on local liquid dispersion, sHASEGP also acts as an absorption enhancer. Macromolecules greater than 16 kilodaltons (kDa) are mostly excluded from absorption via diffusion through capillaries, and are mostly absorbed via drainage lymph nodes. Therefore, macromolecules administered subcutaneously, such as therapeutic antibodies (molecular weight of about 150kDa) must pass through the interstitial matrix, then reach the drainage lymphatic system, and are subsequently absorbed into the vascular compartment. By improving local dispersion, sHASEGP increases the absorption rate (Ka) of many macromolecules. Relative to SC administration in the absence of sHASEGP, this results in elevated peak blood levels ( _Cmax ), and potentially leads to elevated bioavailability [Bookbinder LH, et al., "Arecombinant humanenzyme for enhanced interstitial transport of therapeutics", J.Control.Release2006; 114: 230-241].

Hyaluronidase products of animal origin have been used clinically for over 60 years, primarily to enhance the dispersion and absorption of other co-administered drugs and for subcutaneous infusion (SC injection/infusion of large volumes of fluids) [Frost GI, "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007; 4: 427-440]. Details on the mechanism of action of hyaluronidase are 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; 69-81; Chain E., “A mucolytic enzyme in testes extracts”, Nature 1939; 977-978; Weissmann B., “The transglycosylative action of testicular hyaluronidase”, J. Biol. Chem., 1955; 216: 783-94; Tammi, R., Saamanen, AM, Maibach, HI, Tammi 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: 126-130; Laurent, UBG, Dahl, LB, Reed, RK, "Catabolism of hyaluronan in rabbitskin takes place locally, in lymph nodes and liver", Exp. Physiol. 1991; 76: 695-703; Laurent, TC and Fraser, JRE, "Degradation of Bioactive Substances: Physiology and Pathophysiology", Henriksen, JH (Ed) CRC Press, Boca Raton, FL; 1991. Pages 249-265; Harris, EN et al., "Endocytic function, glycosaminoglycanspecificity, and antibody sensitivity of the recombinant human 190-kDahyaluronan receptor for receptor endocytosis (HARE)”, J. Biol. Chem. 2004; 279: 36201-36209; Frost, GI, “Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration”, Expert Opinion on Drug Delivery, 2007; 4: 427-440. Hyaluronidase products approved in EU countries include “Dessau” and animal-derived hyaluronidase products approved in the United States include Vitrase ^™ , Hydase ^™ , and Amphadase ^™ .

The safety and efficacy of hyaluronidase products have been widely established. The most significant safety risk identified is hypersensitivity and/or allergenicity, which is believed to be related to the lack of purity of animal-derived preparations [Frost, G.I., "Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration", Expert Opinion on Drug Delivery, 2007; 4: 427-440]. It should be noted that there are differences in the approved dosages of animal-derived hyaluronidases between the UK, Germany, and the US. In the UK, a common dose as an adjuvant for subcutaneous or intramuscular injection is 1500 units added directly to the injection. In the US, a common dose used for this purpose is 150 units. In subcutaneous infusions, hyaluronidase is used to facilitate the subcutaneous administration of relatively large volumes of fluid. In the UK, 1500 units of hyaluronidase are generally given for every 500 to 1000 ml of fluid used subcutaneously. In the United States, 150 units are considered sufficient for each liter of subcutaneous infusion solution. In Germany, 150 to 300 units are considered sufficient for this purpose. In the United Kingdom, the diffusion of the local anesthetic is accelerated by adding 1500 units. In Germany and the United States, 150 units are considered sufficient for this purpose. Although there are dosage differences (the dosage in the United Kingdom is 10 times higher than in the United States), there are no reports of significant differences in the safety profiles of animal-derived hyaluronidase products sold in the United States and the United Kingdom, respectively.

On December 2, 2005, Halozyme Therapeutics Inc. received approval from the FDA for an injectable formulation of recombinant human hyaluronidase rHuPH20 (HYLENEX ^™ ). The FDA approved a 150-unit dose of HYLENEX ^™ for SC administration for the following indications:

- Acts as an adjuvant to enhance the absorption and dispersion of other injected drugs

-For subcutaneous infusion

-As an adjunct in SC urography to improve absorption of radiopaque agents.

As part of the regulatory review, it was established that rHuPH20 possesses the same properties of previously approved animal-derived hyaluronidase preparations for enhancing the dispersion and absorption of other injectable drugs, but with an improved safety profile. Specifically, the use of recombinant human hyaluronidase (rHuPH20) minimizes the potential risk of contamination with animal pathogens and transmissible spongiform encephalopathies compared to animal-derived hyaluronidase.

Soluble hyaluronidase glycoprotein (sHASEGP), its preparation method and its use in pharmaceutical compositions have been described in WO 2004/078140.

Detailed experimental work as outlined further below has shown that, surprisingly, the claimed formulations have an advantageous storage stability and meet all necessary requirements for approval by health authorities.

It is believed that the hyaluronidase enzyme in the formulation according to the present invention enhances the delivery of anti-CD20 antibodies to the systemic circulation, for example by increasing the absorption of the active substance (it acts as a penetration enhancer). It is believed that the hyaluronidase enzyme also improves the delivery of therapeutic anti-CD20 antibodies to the systemic circulation via the subcutaneous application route by reversibly hydrolyzing hyaluronan, an extracellular component of the SC interstitial tissue. The hydrolysis of hyaluronan in the subcutaneous tissue temporarily opens channels in the interstitial space of the SC tissue and thereby improves the delivery of therapeutic anti-CD20 antibodies to the systemic circulation. In addition, administration has shown that humans experience reduced pain and less swelling derived from the volume of the SC tissue.

Hyaluronidase has its full effect locally when applied topically. In other words, hyaluronidase is locally inactivated and metabolized within minutes, and no systemic or long-term effects have been noted. Its rapid inactivation within minutes when hyaluronidase enters the bloodstream eliminates the practical ability to conduct comparable biodistribution studies between different hyaluronidase products. This characteristic also minimizes any potential systemic safety concerns, as hyaluronidase products cannot function at distant sites.

The unifying feature of all hyaluronidase enzymes according to the present invention is their ability to depolymerize hyaluronan, regardless of differences in chemical structure, species origin, tissue origin, or batches of pharmaceutical products derived from the same species and tissue. They are distinguished by the fact that, despite having different structures, their activities are identical (except for potency).

The hyaluronidase enzyme of the formulation according to 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. In addition, the hyaluronidase enzyme merely modifies the delivery of the anti-CD20 antibody to the systemic circulation, but does not possess any properties that can provide or contribute to the therapeutic effect of the anti-CD20 antibody absorbed systemically. The hyaluronidase enzyme is not systemically bioavailable and does not adversely affect the molecular integrity of the anti-CD20 antibody under the recommended storage conditions of the stable pharmaceutical formulation according to the present invention. Therefore, it is considered an excipient in the anti-CD20 antibody formulation according to the present invention. Because it does not exert a therapeutic effect, it represents a pharmaceutical form component other than the therapeutically active anti-CD20 antibody.

Many suitable hyaluronidase enzymes according to the present invention are known from the prior art. The preferred enzyme is human hyaluronidase, most preferably the enzyme known as rHuPH20. rHuPH20 is a member of the neutral and acidic active β-1,4 glycosyl hydrolase family that depolymerizes hyaluronan by hydrolyzing the β-1,4 linkage between the _C1 position of N-acetylglucosamine and the _C4 position of glucuronic acid. Hyaluronan is a polysaccharide found in the intracellular matrix of connective tissues, such as subcutaneous interstitial tissue, and certain specialized tissues, such as the umbilical cord and vitreous humor. The hydrolysis of hyaluronan temporarily reduces the viscosity of the interstitial tissue and promotes the dispersion of injected fluids or local transudates or exudates, thus facilitating their absorption. The effects of hyaluronidase are local and reversible with complete reconstitution of tissue hyaluronan 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: 427-440]. Increased connective tissue permeability through hydrolysis of hyaluronan is associated with the efficacy of hyaluronidase in improving the dispersion and absorption of co-administered molecules.

The human genome contains several hyaluronidase genes. Only the PH20 gene product possesses efficient hyaluronidase activity under physiological extracellular conditions and acts as a spreading agent, while acid-active hyaluronidases do not have this property.

rHuPH20 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 possesses effective hyaluronidase activity under physiological extracellular conditions and acts as a diffusing agent. The naturally occurring human PH20 protein has a lipid anchor attached to the carboxyl-terminal amino acid that anchors it to the plasma membrane. The rHuPH20 enzyme developed by Halozyme is a truncated deletion variant that lacks the amino acids at the carboxyl terminus responsible for lipid attachment. This produces a soluble, neutral pH-active enzyme 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 authentic N-terminal amino acid sequence that is orthologous to the N-terminal amino acid sequence found in some bovine hyaluronidase preparations.

PH20 hyaluronidases (including animal-derived PH20 and recombinant human rHuPH20) depolymerize hyaluronan by hydrolyzing the β-1,4 linkage between the C ₁ position of N-acetylglucosamine and the C ₄ position of glucuronic acid. Tetrasaccharides are the smallest digestion products [Weissmann, B., "The transglycosylative action of testicular hyaluronidase", J.Biol.Chem., 1955; 216: 783-94]. This N-acetylglucosamine/glucuronic acid structure is not found in the N-linked glycans of recombinant biological products, and therefore, rHuPH20 does not affect the glycosylation of antibodies formulated with it, such as, for example, rituximab. The rHuPH20 enzyme itself has 6 N-linked glycans per molecule, with a core structure similar to that found in monoclonal antibodies. As expected, these N-linked structures do not change over time, confirming the lack of enzymatic activity of rHuPH20 on these N-linked glycan structures. The short half-life of rHuPH20 and the continuous synthesis of hyaluronan result in a short and localized effect of the enzyme on tissues.

Preferably, the hyaluronidase enzyme used as an excipient in the subcutaneous formulation according to the present invention is prepared by using recombinant DNA technology. Thus, it is ensured that the same protein (same amino acid sequence) is always obtained and allergic reactions caused by contaminating proteins co-purified during tissue extraction are avoided. Preferably, the hyaluronidase enzyme used in the formulation according to the present invention is a human enzyme, most preferably rHuPH20.

The amino acid sequence of rHuPH20 (HYLENEX ^™ ) is known and is available as CAS Registry No. 75971-58-7. The approximate molecular weight is 61 kDa (see also US Patent No. 7,767,429).

Multiple structural and functional comparisons have been performed between mammalian hyaluronidases of natural origin and PH-20 cDNA clones from humans and other mammals. The PH-20 gene is the gene used in the recombinant product rHuPH20; however, the recombinant drug product is a 447-amino acid truncated version of the complete protein encoded by the PH-20 gene. Structural similarity in terms of amino acid sequence rarely exceeds 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, showing that the clinical safety and unit efficacy of hyaluronidase are equivalent regardless of its source.

The use of rHuPH20 in the anti-CD20 antibody SC formulation according to the present invention allows for the administration of higher volumes of drug product and potentially enhances the absorption of subcutaneously administered CD20 antibodies, preferably rituximab, into the systemic circulation.

The osmolarity of the stable pharmaceutical formulation according to the present invention is 350±50 mOsm/kg.

Preferably, the stable pharmaceutical formulation according to the present invention is substantially free of visible (to human eye) particles. Sub-visible particles (as measured by light obscuration) should meet the following criteria:

- Maximum number of particles ≥10 μm per vial -> 6000

- Maximum number of particles ≥25 μm per vial -> 600

In another aspect, the present invention provides a use of a formulation for the preparation of a medicament for treating a disease or condition amenable to treatment with an anti-CD20 antibody, such as, preferably, cancer or a non-malignant disease, in a subject, comprising administering to the subject a formulation described herein in an amount effective to treat the disease or condition. Preferably, the anti-CD20 antibody is co-administered concomitantly or sequentially with a chemotherapeutic agent.

In yet another aspect, the present invention provides a method of treating a disease or condition (e.g., cancer (preferred) or a non-malignant disease) in a subject that is amenable to treatment with an anti-CD20 antibody, comprising administering to the subject a formulation described herein in an amount effective to treat the disease or condition. The cancer or non-malignant disease will typically involve CD20-expressing cells, such that the CD20 antibody in the SC therapeutic formulation according to the invention is able to bind to the affected cells. Preferably, the cancer is a CD20-expressing cancer. Preferably, the non-malignant disease treatable with the composition according to the invention is an autoimmune disease, as defined herein. Preferably, the anti-CD20 antibody is co-administered concomitantly or sequentially with a chemotherapeutic agent.

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 according to the present invention improves the dispersion, absorption, and bioavailability of therapeutic antibodies. Macromolecules (i.e., greater than 16 kDa) administered via the SC route are preferentially absorbed into the vascular compartment via the draining lymph [Supersaxo, A. et al., "Effect of Molecular Weight on the Lymphatic Absorption of Water-Soluble Compounds Following Subcutaneous Administration", 1990; 2: 167-169; Swartz, M.A., "Advanced Drug Delivery Review, The physiology of the lymphatic system", 2001; 50: 3-20]. Thus, the rate at which these macromolecules are introduced into the systemic circulation is slowed relative to intravenous infusion, thereby potentially leading to a reduction in the frequency/intensity of infusion-related reactions.

The production of subcutaneous CD20 antibody (preferably rituximab) formulations according to the present invention requires a high antibody concentration (about 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 of CD20 antibodies, preferably rituximab. The highly concentrated, stable pharmaceutical anti-CD20 antibody formulations according to the present invention can also be provided as stabilized protein formulations that can be reconstituted with a suitable diluent to produce a reconstituted formulation with a high anti-CD20 antibody concentration.

Preferably, the CD20 antibody SC formulation according to the invention is used to treat cancer, preferably a CD20 expressing cancer.

As used in this patent specification, the term "about" is intended to indicate that the particular numerical value provided may vary to some extent, such as, for example, to indicate that a range of ±10%, preferably ±5%, and most preferably ±2% is included in a given numerical value.

In addition to the assays described above, a variety of in vivo assays are available to the skilled practitioner. For example, cells within a patient's body can be exposed to an antibody, optionally labeled with a detectable marker, such as a radioisotope, and binding of the antibody to cells in the patient can be assessed, for example, by external scanning for radioactivity or by analyzing a biopsy taken from a patient previously exposed to the antibody.

It is contemplated that the CD20 antibody SC formulations according to the invention may also be used to treat various non-malignant diseases or disorders, including autoimmune diseases as defined herein; endometriosis; scleroderma; restenosis; polyps, such as colon polyps, nasal polyps or gastrointestinal polyps; fibroadenomas; respiratory diseases; cholecystitis; neurofibromatosis; polycystic kidney disease; inflammatory diseases; skin disorders, including psoriasis and dermatitis; vascular diseases; conditions involving abnormal proliferation of vascular epithelial cells; gastrointestinal ulcers; Menetrier's disease, secretory adenoma or protein loss syndrome; renal disorders; angiogenic disorders (angiogenic ocular diseases such as age-related macular degeneration, presumed ocular histoplasmosis syndrome, retinal neovascularization from proliferative diabetic retinopathy, retinal vascularization, diabetic retinopathy, or age-related macular degeneration; bone-related pathologies such as osteoarthritis, rickets, and osteoporosis; injury following a cerebral ischemic event; fibrotic or edematous diseases such as cirrhosis of the liver, pulmonary fibrosis, carcoidosis, throiditis, hyperviscosity syndrome systemic, Osier Weber-Rendu disease, chronic obstructive pulmonary disease, or edema following burns, trauma, radiation, stroke, hypoxia, or ischemia; hypersensitivity reactions of the skin; diabetic retinopathy and diabetic nephropathy; Guillain-Barre syndrome syndrome; graft-versus-host disease or transplant rejection; Paget's disease; bone or joint inflammation; photoaging (e.g., caused by UV radiation to human skin); benign prostatic hypertrophy; certain microbial infections, including microbial pathogens selected from the group consisting of adenovirus, hantavirus, Borrelia burgdorferi, Yersinia spp., and Bordetella pertussis. pertussis); thrombosis caused by platelet aggregation; reproductive conditions such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding, or menorrhagia; synovitis; atheroma; acute and chronic kidney disease (including proliferative glomerulonephritis and diabetes-induced nephropathy); eczema; hypertrophic scarring; endotoxic shock and fungal infections; familial adenomatous polyposis; neurodegenerative diseases (e.g., Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy, and cerebellar degeneration); myelodysplastic syndrome; aplastic anemia; ischemic injury; fibrosis of the lung, kidney, or liver; T-cell-mediated hypersensitivity disorders; infantile hypertrophic pyloric stenosis; urinary obstructive syndrome (UOS); syndrome); psoriatic arthritis; and Hashimoto's thyroiditis. Preferred non-malignant indications for therapy are as defined herein.

In the case where the indication is cancer, the combination treatment patient of antibody formulation and chemotherapeutics can be used.Joint administration includes co-administration or simultaneous administration (it uses separate formulation or single drug formulation to carry out) and continuous administration in any order, wherein preferably, there is the time period in which all activating agents apply their biological activity simultaneously.So, chemotherapeutics can be used before or after administration according to the antibody formulation of the present invention.In this embodiment, the opportunity between at least one administration of chemotherapeutics and at least one administration according to the antibody formulation of the present invention is preferably about 1 month or less, and most preferably about 2 weeks or less.Or, chemotherapeutics and according to the antibody formulation of the present invention can be used simultaneously to the patient in a single formulation or separate formulation.

Treatment with the antibody formulation can result in an improvement in the signs or symptoms of a cancer or disease. For example, where the disease being treated is cancer, such therapy can result in an improvement in survival (overall survival and/or progression-free survival) and/or can result in an objective clinical response (partial or complete). In addition, combined treatment with a chemotherapeutic agent and an antibody formulation can produce a synergistic or greater than superimposed therapeutic benefit to the patient.

Preferably, the antibody in the administered formulation is a naked antibody. However, the administered antibody can be conjugated to a cytotoxic agent. Preferably, the immunoconjugate and/or the antigen of its combination is internalized by the cell, causing the therapeutic efficacy of the immunoconjugate in killing the cancer cells it combines to increase. In a preferred embodiment, the cytotoxic agent targets or interferes with the nucleic acid in the cancer cell. The example of such cytotoxic agent includes maytansinoids, calicheamicin (calioheamicin), ribonuclease and DNA endonuclease. Preferred immunoconjugates are rituximab-maytansinoid immunoconjugates similar to trastuzumab-DM1 (T-DM1), as they are described in WO2003/037992, more preferably the immunoconjugate T-MCC-DM1.

For subcutaneous delivery, the formulation can be administered via a suitable device such as, but not limited to, a syringe; an injection device (e.g., INJECT-EASE ^™ and GENJECT ^™ devices); an infusion pump (such as, for example, Accu-Chek ^™ ); an injection pen (such as GENPEN ^™ ); a needle-free device (e.g., MEDDECTOR ^™ and BIOJECTOR ^™ ); or via a subcutaneous patch delivery system.

The amount and timing of administration of the anti-CD20 antibody formulation for the prevention or treatment of a disease will depend on the type (species, sex, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. Also important for determining the appropriate dose is the course of the disease, whether the antibody is being administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history, and his or her response to the antibody. Final dosage determination is at the discretion of the attending physician. The antibody is appropriately administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 50 mg/kg (e.g., 0.1-20 mg/kg) of the anti-CD20 antibody is a candidate initial dose for administration to the patient.

The preferred dosage range of the anti-CD20 antibody will be about 0.05 mg/kg to about 30 mg/kg body weight. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, 10 mg/kg, or 30 mg/kg (or any combination thereof) can be administered to the patient. Depending on the type (species, sex, age, weight, etc.) and condition of the patient and the type of anti-CD20 antibody, the first dose may be different from the second dose of the anti-CD20 antibody. Such doses may be administered daily or intermittently, for example, every three to six days or even every one to three weeks. An initial higher loading dose may be administered, followed by one or more lower doses. Based on clinical studies (see also non-limiting illustrative Examples 3 and 4 for rituximab), the preferred dosage range is 300 mg/m ² to 900 mg/m ^2. More preferably, the preferred dosage range of the anti-CD20 antibody is about 375 mg/m ² to about 800 mg/m ² . Preferred specific doses of the anti-CD20 antibody are doses of about 375 mg/m ² , about 625 mg/m ² and about 800 mg/m ^2. Also preferred are fixed doses of the anti-CD20 antibody.

In one embodiment, the fixed dose for B-cell lymphoma, preferably non-Hodgkin's lymphoma, is as follows. Preferred is a dose of about 1200 mg to about 1800 mg of the anti-CD20 antibody per dose. More preferred is a dose selected from the group consisting of about 1300 mg, about 1500 mg, about 1600 mg, and about 1700 mg of the anti-CD20 antibody per dose. Most preferably, the fixed dose for patients with B-cell lymphoma, preferably non-Hodgkin's lymphoma, is about 1400 mg of the anti-CD20 antibody (e.g., rituximab) per dose, which can be administered according to a variety of schedules, including about every 2 months (including about every 8 weeks), about every 3 months (including about every 12 weeks), for about 2 years (or more), etc. (see also non-limiting examples 3 and 4 for rituximab).

In another embodiment, the fixed dose for leukemia patients, preferably chronic lymphocytic leukemia (CLL) patients, is as follows. Preferably, the dose is about 1600 mg to about 2200 mg of the anti-CD20 antibody per dose. More preferably, the dose is selected from the group consisting of about 1700 mg, about 1800 mg, about 1900 mg, and about 2100 mg of the anti-CD20 antibody per dose. In one embodiment, the fixed dose for leukemia patients, preferably CLL patients, is about 1870 mg of the anti-CD20 antibody (e.g., rituximab) per dose.

In yet another embodiment, the fixed dose for patients with autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, lupus nephritis, diabetes, ITP, and vasculitis is as follows: preferably, about 1200 mg to about 2200 mg of the anti-CD20 antibody per dose, for example, about 1500 mg of the anti-CD20 antibody (e.g., rituximab) per dose.

If a chemotherapeutic agent is administered, it is typically administered at its known dose, or optionally reduced due to the combined effects of the drugs or negative side effects attributable to the administration of the chemotherapeutic agent. The preparation and dosing schedules for such chemotherapeutic agents can be used according to the manufacturer's instructions or as determined empirically by a skilled practitioner. The preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M.C. Perry, Williams & Wilkins, Baltimore, MD (1992).

Preferably, the stable pharmaceutical formulation of the pharmaceutically active anti-CD20 antibody according to the present invention is administered by subcutaneous injection, wherein the administration is preferably repeated several times at intervals of 3 weeks (q3w). Most preferably, the entire volume of the injection fluid is administered over a period of 1 to 10 minutes, preferably 2 to 6 minutes, and most preferably 3 ± 1 minutes. Most preferably, 2 ml/minute, i.e., for example, about 240 mg/minute, is administered. For many patients who are not given other intravenous (IV) chemotherapeutic agents, such subcutaneous administration results in increased patient convenience and the potential for self-administration at home. This results in improved compliance and can reduce/eliminate costs associated with IV administration (i.e., nursing costs for IV administration, couch rental, patient travel, etc.). Subcutaneous administration according to the present invention is most likely to be associated with a reduction in the frequency and/or intensity of infusion-related reactions.

In a preferred embodiment, the drug can be used to prevent or reduce metastasis or further spread in such patients with cancers that express CD20. The drug can be used to prolong the duration of survival of such patients, prolong the progression-free survival of such patients, prolong the duration of response, and result in a statistically significant and clinically meaningful improvement in treated patients, as measured by duration of survival, progression-free survival, response rate, or duration of response. In a preferred embodiment, the drug can be used to increase the response rate in a patient group.

In the context of the present invention, one or more additional growth inhibitory agents, cytotoxic agents, chemotherapeutic agents, anti-angiogenic agents, anticancer agents or cytokines, or compounds that enhance the effects of such agents, may be used in anti-CD20 antibody treatment of cancers expressing CD20. Preferably, anti-CD20 antibody treatment is not used with such additional cytotoxic agents, chemotherapeutic agents or anticancer agents, or compounds that enhance the effects of such agents.

Such agents include, for example, alkylating agents or agents having an alkylating effect, such as cyclophosphamide (CTX; for example, chlorambucil (CHL); for example, cisplatin (CisP); for example, busulfan (e.g., melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, etc.; anti- Metabolites, such as methotrexate (MTX), etoposide (VP16; for example, 6-mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g., xeloda, dacarbazine (DTIC), etc.; antibiotics, such as actinomycin D ( D), doxorubicin (DXR; for example, doxorubicin, daunorubicin (daunomycin), bleomycin, mithramycin, etc.; alkaloids, such as vinca alkaloids such as vincristine (VCR), vinblastine, etc.; and other antitumor agents, such as paclitaxel (e.g., taxol and paclitaxel derivatives, cytostatics, glucocorticoids such as dexamethasone (DEX; for example, decatetron and corticosteroids such as prednisone, nucleosidase inhibitors such as hydroxyurea, amino acid depleting enzymes ( Enzymes such as asparaginase, leucovorin and other folic acid derivatives, and similar various anti-tumor agents. The following agents may also be used as additional agents: arnifostine (e.g., dactinomycin), mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, lomustine (CCNU), doxorubicin liposome (e.g., gemcitabine (e.g., daunorubicin liposome) lipo) (e.g., procarbazine), mitomycin, docetaxel (e.g., taxotere, aldesleukin, 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, tane), pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil. Preferably, anti-CD20 antibody therapy is not used with such additional agents.

The use of the cytotoxic and anticancer agents described above and antiproliferative target-specific anticancer drugs, such as protein kinase inhibitors, in chemotherapy regimens is generally well characterized in the field of cancer therapy, and their use herein is subject to the same considerations regarding monitoring tolerance and efficacy and regarding controlling the route of administration and dosage, with some adjustments. For example, the actual dose of the cytotoxic agent may vary with the response of the patient's cultured cells as determined using tissue culture methods. Generally, the dose will be reduced compared to the amount used in the absence of additional other agents.

Typical dosages of effective cytotoxic agents may be within the range 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 magnitude in concentration or amount. Thus, the actual dosage will depend on the physician's judgment, the patient's condition, and the efficacy of the treatment method, which is based on the in vitro responsiveness of primary cultured malignant cells or tissue culture tissue samples, or the responses observed in appropriate animal models.

In the context of the present invention, in addition to anti-CD20 antibody treatment of CD20-expressing cancers, an effective amount of ionizing radiation and/or a radiopharmaceutical may be administered. The radiation source may be external or internal to the patient being treated. When the source is external to the patient, the treatment is known as external beam radiation therapy (EBRT). When the radiation source is internal to the patient, the treatment is known as brachytherapy (BT). The radioactive atoms used in the context of the present 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 iodine-111. It is also possible to label the antibody with such radioisotopes. Preferably, anti-CD20 antibody treatment is not used in conjunction with such ionizing radiation.

Radiation therapy is a standard treatment for controlling unresectable or inoperable tumors and/or tumor metastases. Improved results have been seen when combining radiation therapy with chemotherapy. Radiation therapy is based on the principle that high doses of radiation delivered to a target area will cause the death of reproductive cells in both tumors and normal tissues. Radiation dosage regimens are generally defined in terms of radiation absorbed dose (Gy), time, and fractionation, and must be carefully defined by an oncologist. The amount of radiation a patient receives will depend on various considerations, but the two most important are the location of the tumor relative to other vital structures or organs of the body and the extent to which the tumor has spread. A typical course of treatment for a patient undergoing radiation therapy would be a treatment schedule over a period of 1 to 6 weeks, with a total dose of 10-80 Gy administered to the patient five days a week in a single daily fraction of approximately 1.8 to 2.0 Gy. In a preferred embodiment of the present invention, synergy is present when treating tumors in human patients using the combination therapy of the present invention and radiation. In other words, the inhibition of tumor growth by a pharmaceutical agent comprising a CD20 antibody formulation of the present invention is enhanced when combined with radiation, optionally with an additional chemotherapeutic or anticancer agent. For example, parameters for adjuvant radiotherapy are contained in WO 99/60023.

Other therapeutic regimens can be combined with the antibody, including but not limited to a second (third, fourth, etc.) chemotherapeutic agent (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 a cytokine, etc.; or any suitable combination thereof.

In addition to the above treatment options, patients may undergo surgery to remove cancer cells and/or radiation therapy.

In another embodiment of the present invention, there is provided a product containing the pharmaceutical formulation of the present invention and providing its use instructions. This product comprises a container. Suitable containers include, for example, bottles, vials (e.g., multiple or dual-chamber vials), syringes (such as multiple or dual-chamber syringes) and test tubes. The container can be formed from a variety of materials such as glass or plastic. The container holds the formulation, and a label on the container or in combination with the container can indicate use instructions. The container holding the formulation can be a multiple-use vial, which allows for repeated use (e.g., 2 to 6 uses) of the reconstituted formulation. The product can further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts printed with use instructions.

Antibodies formulated according to the invention are preferably substantially pure, and desirably substantially homogeneous (i.e., free of contaminating proteins, etc., wherein the hyaluronidase enzyme in the formulation according to the invention is not considered a contaminating protein of the anti-CD20 monoclonal antibodies according to the invention).

The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention. All literature and patent citations are incorporated herein by reference.

Example

Anti-CD20 formulations for subcutaneous administration according to the present invention were developed based on the experimental results provided below using the general preparative and analytical methods and assays as outlined below.

Example 1: Preparation of highly concentrated liquid formulations

Rituximab is manufactured by techniques generally known from recombinant protein production. A genetically engineered Chinese hamster ovary cell line (CHO), prepared as described in EP-B-2000149, is expanded in cell culture from a mother cell bank. Rituximab monoclonal antibody is harvested from the cell culture fluid and purified using immobilized protein A affinity chromatography, cation exchange chromatography, a filtration step to remove viral contamination, followed by anion exchange chromatography and an ultrafiltration/diafiltration step.

rHuPH20 is manufactured by generally generating known techniques from recombinant proteins. The process begins by melting cells from a working cell bank (WCB) or a mother cell bank (MCB) and is expanded via a series of cell cultures in roller bottles. Up to 6 liters of cell culture are used to provide a continuous source of cells maintained under the selective pressure of methotrexate. When expanded to approximately 36 liters, the culture is transferred to a 400-liter bioreactor to obtain a final batch volume of approximately 300 liters. The production bioreactor is operated in a fed-batch mode in the absence of selective pressure, and the duration of the production phase is approximately 2 weeks. rHuPH20 is secreted into the culture fluid. A 1000-liter bioreactor can also be used to obtain a final batch volume of 500 liters. After completing the production phase, the harvest is clarified by filtration and then treated with a solvent/detergent to inactivate the virus. Then, the protein is purified by a series of four column chromatography processes to remove process and product-related impurities. A virus filtration step was performed, and the filtered bulk was then concentrated and formulated into the final buffer: 10 mg/mL rHuPH20 in 20 mM L-histidine/HCl buffer, pH 6.5, 130 mM NaCl, 0.05% (w/v) polysorbate 80. The rHuPH20 bulk was stored at -70°C.

The other excipients of the formulation according to the present invention are widely used in practice and are known to those skilled in the art. Therefore, there is no need to explain them in detail here.

A liquid drug product formulation for subcutaneous administration according to the present invention was developed as follows.

To prepare a liquid formulation, rituximab is buffer exchanged against a diafiltration buffer containing the desired buffer composition and, if necessary, concentrated by diafiltration to an antibody concentration of approximately 200 mg/ml. After completion of the diafiltration operation, excipients (e.g., trehalose, rHuPH20, surfactants) are added to the antibody solution as stock solutions. Finally, the protein concentration is adjusted with buffer to a final rituximab concentration of approximately 120 mg/ml.

All formulations were sterile filtered through a 0.22 μm low protein binding filter and aseptically filled into sterile 6 ml glass vials, sealed with ETFE (copolymer of ethylene and tetrafluoroethylene) coated rubber stoppers and Alucrimp caps. The filling volume was approximately 3.0 ml. These formulations were stored for different time intervals under different climatic conditions (5° C., 25° C., and 40° C.) and were stressed by shaking (at 5° C. and 25° C. for 1 week at a shaking frequency of 200 rpm) and freeze-thaw stress. Samples were analyzed by the following analytical methods before and after the stress test:

1) UV spectrophotometry;

2) size exclusion chromatography (SEC);

3) by ion exchange chromatography (IEC);

4) by the turbidity of the solution;

5) in terms of visible particles; and

6) In terms of rHuPH20 activity.

UV spectrophotometry for protein content determination was performed on a Perkin Elmer λ35 UV spectrophotometer in the wavelength range of 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:

The UV light absorption at 280 nm was corrected for light scattering at 320 nm and multiplied by the dilution factor (determined from the weighted mass and density of the pure sample and dilution buffer). The molecule was divided by the product of the path length d of the cuvette and the extinction coefficient ε.

Use size exclusion chromatography (SEC) to detect soluble high molecular weight species (aggregates) and low molecular weight hydrolysates (LMW) in the formulation. This method uses a suitable HPLC instrument equipped with a UV detector (detection wavelength 280nm) and a TosoHaas TSKG3000SWXL post (7.8x300mm). Use 0.2M dipotassium hydrogen phosphate, 0.25M potassium chloride, pH 7.0 to separate complete monomer, aggregates and hydrolysates by an isocratic elution profile with a flow rate of 0.5ml/minute.

Ion exchange chromatography (IEC) was performed to detect chemical degradation products that change the net charge of rituximab in the formulation. For this purpose, rituximab was digested with papain. The method used a suitable HPLC instrument equipped with a UV detector (detection wavelength 280nm) and a Polymer Labs PL-SCX 1000A analytical cation exchange column. 10mM MES, pH 6.0 and 10mM MES, 0.2M sodium chloride, pH 6.0 were used as mobile phases A and B, respectively, with a flow rate of 1ml/min.

To determine turbidity, opalescence was measured in FTU (turbidity units) using a HACH 2100AN turbidimeter at room temperature.

The samples were analyzed for visible particles using a Seidenader V90-T visual inspector.

Use rHuPH20 as the in vitro enzyme assay of hyaluronidase as activity determination method.This assay method is based on forming insoluble precipitate when hyaluronan (sodium hyaluronate) is in conjunction with cationic precipitation agent.By incubating rHuPH20 with hyaluronan substrate, then measure enzyme activity with the serum albumin (horse serum) precipitation of acidification undigested hyaluronan.Measure turbidity with the wavelength of 640nm, and the turbidity reduction that is derived from the enzyme activity to hyaluronan substrate is the measurement of enzyme activity.Use the standard curve operation this procedure that measures the dilution that produces with reference to standard product with rHuPH20, and read sample activity from curve.

The results of the stability testing of Formulations A to J are provided in the table appended below.

Example 2: Preparation of humanized 2H7 anti-CD20 liquid formulation

To prepare the liquid formulation, recombinant humanized 2H7 anti-CD20 antibody (2H7.v16, as disclosed in WO2006/084264) was buffer exchanged against a diafiltration buffer containing the desired buffer composition and, if necessary, concentrated to antibody concentrations of approximately 60 and 120 mg/ml. After reaching the target concentration, excipients (e.g., trehalose, rHuPH20, polysorbate 20) were then added to the antibody solution as stock solutions. Finally, the protein concentration was adjusted to approximately 30, 50, and 100 mg/ml of humanized 2H7 using the final formulation buffer.

All formulations were sterile filtered through a 0.22 μm low protein binding filter and aseptically filled into sterile 3 ml glass vials, stoppered with fluororesin-laminated butyl rubber stoppers and capped with aluminum/plastic flip-off seals. The fill volume was approximately 1.2 ml. These formulations were stored at different temperatures (5° C., 25° C., and 40° C.) for various time intervals. Samples were analyzed at each stability time point using the following analytical methods:

1) UV spectrophotometry;

2) size exclusion chromatography (SEC);

3) ion exchange chromatography (IEC);

4) Complement-dependent cytotoxicity (CDC) assay for humanized 2H7 activity

5) Turbidimetric Assay of rHuPH20 Activity.

1) Determine protein concentration by UV absorption spectroscopy using an Agilent 8453 spectrophotometer in the wavelength range of 240 nm to 400 nm. Dilute the sample to approximately 0.5 mg/ml by weight with the corresponding formulation buffer. Calculate protein concentration using Equation 1:

Protein concentration = ((A _max - A ₃₂₀ ) x DF) / (ε (cm ² /mg) x d (cm)) (Equation 1) where DF is the dilution factor, d is the cuvette path length, and ε is the extinction coefficient, which is 1.75 (cm ² /mg ^-1 ) for 2H7 at A _max . UV light absorption at A max (typically 278 to 280 nm) is corrected for light scattering at 320 nm and multiplied by the dilution factor (determined from the weighted mass and density of the pure sample and dilution buffer). Divide the numerator by the product of the cuvette path length d and the extinction coefficient ε.

2) Size exclusion chromatography (SEC) was used to detect soluble high molecular weight species (aggregates) and low molecular weight hydrolysis products (fragments) in the formulation. SEC was performed on an Agilent Technologies, Inc. 1100 series HPLC equipped with a UV detector (detection wavelength 280 nm) and a TSK G3000SWXL column (7.8 x 300 mm). Intact monomers, aggregates, and hydrolysis products were separated by an isocratic elution profile using 0.20 M potassium phosphate and 0.25 M potassium chloride (pH 6.2) at a flow rate of 0.3 ml/min.

3). Ion exchange chromatography (IEC) was performed to detect chemical degradation products in the formulation that altered the net charge of the anti-CD20 antibody. For this purpose, the anti-CD20 antibody was 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 280 nm) and a Dionex ProPac WCX-10 (4 x 250 mm) column. A linear gradient of 25 mM potassium phosphate (pH 6.9) (mobile phase A) and 120 mM potassium chloride dissolved in 25 mM potassium phosphate (mobile phase B) was used to separate the acidic and basic variants at a flow rate of 0.5 mL/min.

4). A complement-dependent cytotoxicity (CDC) assay was performed to determine the in vitro activity of the anti-CD20 antibodies. The complement-dependent cytotoxicity (CDC) potency assay was used to measure the ability of the antibodies to lyse human B lymphoblastoid (WIL2-S) cells in the presence of human complement. The assay was performed in 96-well tissue culture microtiter plates. In this assay, different concentrations of anti-CD20 antibody reference substances, controls, or samples diluted in assay diluent were incubated with WIL2-S cells (50,000 cells/well) in the presence of a fixed amount of human complement. The plates were incubated at 37°C/5% CO2 in a humidified incubator for 1 to 2 hours. At the end of the incubation period, 50 μL of the redox dye ALAMARBLUE ^™ was added to each well, and the plates were incubated for 15 to 26 hours. ALAMARBLUE ^™ is a redox dye that fluoresces with an excitation wavelength of 530 nm and an emission wavelength of 590 nm when reduced by living cells. Therefore, the change in color and fluorescence is proportional to the number of viable cells.The results expressed as relative fluorescence units (RFU) were plotted against the anti-CD20 antibody concentration and the parallel lines procedure was used to assess the activity of the anti-CD20 antibody samples relative to the reference material.

5). Hyaluronidase activity and enzyme concentration were determined using a turbidimetric assay. This method is based on the formation of an insoluble precipitate when hyaluronic acid binds to acidified serum albumin. Briefly, serial dilutions of rhuPH20 hyaluronidase (Halozyme, Inc.) working reference standard were prepared in enzyme diluent (70 mM NaCl, 25 mM PIPES, pH 5.5, 0.66 mg/ml gelatin hydrolysate, 0.1% human serum albumin) ranging from 2.5 U/ml to 0.25 U/ml. The test sample was diluted in enzyme diluent to a final concentration of 1.5 U/ml. 30 μl of standard and sample dilutions were transferred into a "black clean bottom" 96-well plate (Nunc). The plate was then covered and preheated at 37°C for 5 minutes. Then, the reaction was started by adding 30 μl of preheated 0.25 mg/ml hyaluronic acid substrate solution (70 mM NaCl, 25 mM PIPES, pH 5.5, 0.25 mg/ml dry sodium hyaluronate, Lifecore Biomedical). The plate was briefly shaken and incubated at 37 ° C for 10 minutes. After this incubation step, the reaction was stopped by adding 240 μl of serum working solution (2.5% horse serum, 500 mM potassium acetate, pH 4.25). After a 30-minute development period at room temperature, the turbidity of the reaction was measured at a wavelength of 640 nm on a microplate reader. The reduction in turbidity derived from the enzyme activity on the hyaluronic acid substrate is a measurement of hyaluronidase activity. Sample activity was measured relative to a calibration curve generated with dilutions of the rhuPH20 working reference standard.

The results obtained with various humanized 2H7 antibody formulations are shown in the table below:

Example 3: Treatment of patients with formulations

The regimen containing rituximab has become the standard of care for patients with various CD20-positive B-cell malignancies. Currently, rituximab is administered as an intravenous (IV) infusion over several hours. These long infusion times and the side effects associated with the infusion are cited by some patients as the uncomfortable consequences of current therapeutic treatments. In addition, it is believed that the procedures required to establish intravenous access are invasive and can be painful, particularly in patients with malignant diseases who are being repeatedly treated. Subcutaneous (SC) administration can significantly simplify the process, shortening administration to less than 10 minutes and improving the patient experience. Recombinant human hyaluronidase (rHuPH20) has been developed and approved to improve the dispersion and absorption of co-administered drugs. It has been combined with rituximab to allow for safe and comfortable SC administration with an injection volume greater than 10 mL. The objectives of this treatment were to select a dose of a SC rituximab formulation with rHuPH20 (Formulation A), prepared as described in Example 1, that gives comparable exposure to IV rituximab and to assess its safety and tolerability in male and female follicular lymphoma (FL) patients during maintenance therapy.

This example provides Phase 1 data from a randomized, open-label, multicenter adaptive Phase Ib study. 124 patients were randomized into one of four rituximab maintenance treatment groups: 16 patients IV control, 34 patients SC dose 1 (375 mg/m ² ), 34 patients SC dose 2 (625 mg/m ² ), and 40 patients SC dose 3 (800 mg/m ² ). Before randomization, eligible patients were treated with at least one dose of IV rituximab at 375 mg/m ² in the maintenance setting. For patients randomized to one of the SC groups, a single IV dose was replaced with the SC dose. Patients received rituximab every 2 months (q2m) or every 3 months (q3m) according to local practice. Safety data were available for a total of 119 patients. In general, rituximab SC was well tolerated. No clinically significant observations or treatment-related serious adverse events have been reported. In 46 patients (39%), a total of 95 adverse events (AE) were reported. The most frequently recorded AE was "administration-related reaction" (AAR, including rash, erythema and mild discomfort). These AARs are reversible, mainly mild in intensity, and only 1 event requires any treatment (metoclopramide (metoclopramide) for nausea). Overall, the AE profiles are not significantly different from the AE profiles expected in the patient treated with rituximab IV (after AAR, the most common event is gastrointestinal disorders and mild infection). 4 serious adverse events (SAEs) were reported in 4 different patients, all reported as being unrelated to study drug. There was no AE resulting in death, withdrawal or treatment interruption.

The total volume of SC administration per patient ranged from 4.4–15.0 mL. The mean infusion duration was 2 mL/minute. Maximum serum concentrations of rituximab in the SC group occurred between days 2 and 8 (48 and 168 hours). Pharmacokinetic parameters were linear across the administered SC dose range (375, 625, and 800 mg/m ² ). Rituximab concentrations (C ₂₈ ) and extent of serum exposure (AUC _0-57 ) on day 28 in patients who received 625 mg/m ² of rituximab SC were comparable to those in patients who received a standard IV dose of 375 mg/m ² of rituximab SC.

In summary, subcutaneous rituximab can be delivered quickly, comfortably, and safely, while maintaining serum exposure comparable to the approved intravenous formulation in FL patients during treatment. The patient experience was favorable. These results support further testing of subcutaneous rituximab, and a fixed dose of 1400 mg rituximab SC has been selected for the formal C- _trough non-inferiority test in the Phase 2 trial.

Example 4: Rituximab SQ versus Rituximab IV in Patients with Follicular Non-Hodgkin's Lymphoma

Patients with previously untreated follicular (low-grade) lymphoma were treated with maintenance therapy with (a) a SC formulation of rituximab in combination with CHOP or CVP (prepared according to Example 1, Formulation A), or (b) rituximab IV in combination with CHOP or CVP.

Patients will be randomized to receive either 375 mg/m ^rituximab as an intravenous infusion or 1400 mg rituximab administered subcutaneously. Additionally, patients will receive standard chemotherapy (CVP or CHOP). Patients who achieve a complete or partial response after eight treatment cycles will receive maintenance therapy for a maximum of 12 cycles. Maintenance therapy cycles will be repeated every eight weeks. The expected duration of study treatment is 96 weeks.

Treatment with 1400 mg SQ rituximab anti-CD20 antibody every 8 weeks as maintenance treatment for up to 12 cycles is safe and effective in treating follicular lymphoma, optionally in combination with chemotherapy including CHOP or CVP.

Claims (24)

1. A highly concentrated and stable pharmaceutical formulation of a pharmaceutically active anti-CD20 antibody for subcutaneous administration, comprising: a. 100-120 mg/ml anti-CD20 antibody, wherein the anti-CD20 antibody is rituximab; b. A buffer of 10 to 30 mM selected from acetic acid, citric acid and histidine, which provides a pH selected from the group consisting of 5.5, 6.0, 6.1 and 6.5; c. A stabilizer and 5 to 25 mM of methionine as a second stabilizer; wherein the stabilizer is 210 to 250 mM α,α-trehalose dihydrate or 120 to 150 mM sodium chloride; d. 0.02% (w/v) to 0.06% (w/v) of a nonionic surfactant, wherein the nonionic surfactant is polysorbate; and e. An effective amount of 2000 U/ml or 12000 U/ml of a hyaluronidase, wherein the hyaluronidase is rHuPH20. 2. The highly concentrated and stable anti-CD20 antibody formulation according to claim 1, wherein the buffer is a histidine buffer. 3. A highly concentrated and stable anti-CD20 antibody formulation according to claim 1 or 2, wherein the stabilizer is α,α-trehalose dihydrate. 4. A highly concentrated and stable anti-CD20 antibody formulation according to claim 1 or 2, wherein the stabilizer is sodium chloride. 5. A highly concentrated, stable anti-CD20 antibody formulation according to any one of claims 1-4, wherein the nonionic surfactant is selected from the group consisting of polysorbate 20 and polysorbate 80. 6. A highly concentrated and stable anti-CD20 antibody formulation according to any one of claims 1 to 5, which is stable after freezing and thawing. 7. A highly concentrated and stable anti-CD20 antibody formulation according to any one of claims 1 to 6, wherein the formulation is in liquid form. 8. A highly concentrated and stable anti-CD20 antibody formulation according to any one of claims 1 to 6, wherein the formulation is in lyophilized form. 9. A highly concentrated, stable anti-CD20 antibody formulation according to any one of claims 1 to 8, wherein the antibody is conjugated to a cytotoxic agent. 10. A highly concentrated, stable anti-CD20 antibody formulation according to claim 9, wherein the cytotoxic agent is a maytansine alkaloid, galicariin, ribonuclease, or DNA endonuclease. 11. A highly concentrated, stable anti-CD20 antibody formulation according to any one of claims 1 to 10, which is administered by subcutaneous injection, wherein the administration is repeated several times at 3-week intervals (q3w). 12. A highly concentrated, stable anti-CD20 antibody formulation according to claim 11, administered by subcutaneous injection, wherein the entire volume of the injection fluid is administered over a period of 1 to 10 minutes. 13. A highly concentrated, stable anti-CD20 antibody formulation according to claim 12, administered by subcutaneous injection, wherein the entire volume of the injection fluid is administered over a period of 2 to 6 minutes. 14. A highly concentrated and stable anti-CD20 antibody formulation according to claim 13, administered by subcutaneous injection, wherein the entire volume of the injection fluid is administered over a period of 3 ± 1 minutes. 15. A highly concentrated and stable anti-CD20 antibody formulation according to claim 14, administered by subcutaneous injection at a rate of 2 ml/min. 16. A highly concentrated and stable anti-CD20 antibody formulation according to claim 15, administered subcutaneously at a rate of 240 mg/min. 17. A device for subcutaneous delivery comprising a highly concentrated, stable anti-CD20 antibody formulation according to any one of claims 1 to 16. 18. The device according to claim 17, which is a syringe, injection device, infusion pump, injection pen, needle-free device, or subcutaneous patch delivery system. 19. An article comprising a highly concentrated, stable anti-CD20 antibody formulation according to any one of claims 1 to 16 and instructions for use. 20. Use of a formulation according to any one of claims 1 to 16 for preparing a medicament suitable for treating a cancer or non-malignant disease in a subject, comprising administering the formulation described herein to the subject in an amount effective for treating said cancer or non-malignant disease. 21. The use according to claim 20, wherein the formulation is administered concurrently with or sequentially with chemotherapy. 22. The use according to claim 20 or 21, wherein a fixed dose of 1200 mg to 2200 mg of anti-CD20 antibody is administered to the subject in need. 23. The use according to claim 20 or 21, wherein a fixed dose of 1200 mg to 1800 mg of anti-CD20 antibody is administered to the subject in need. 24. The use according to claim 20 or 21, wherein a fixed dose of 1600 mg to 2200 mg of anti-CD20 antibody is administered to the subject in need.