HK1147688A

HK1147688A - Antibody formulation

Info

Publication number: HK1147688A
Application number: HK11101829.2A
Authority: HK
Inventors: Michael Adler; Hanns-Christian Mahler; Christine Wurth
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2007-12-21
Filing date: 2008-12-11
Publication date: 2011-08-19

Description

Antibody formulations

The present invention relates to an anti-CD 20 monoclonal antibody preparation, a method of preparing said preparation and the use of said preparation.

Background

The CD20 molecule (also known as B-lymphocyte-restricted differentiation antigen or Bp35) is a hydrophobic transmembrane protein of approximately 35kD molecular weight on pre-B and mature B lymphocytes (Valentine et al (1989) J.biol. chem.264 (19): 11282-11287 and Einfield et al (1988) EMBO J.7 (3): 711-717). CD20, which is expressed during early pre-B cell development and persists until plasma cell differentiation, is found on the surface of more than 90% of B cells from peripheral blood or lymphoid organs. CD20 is present on normal B cells as well as malignant B cells. In particular, CD20 was expressed on more than 90% of B cell non-Hodgkin's lymphomas (NHL) (Anderson et al (1984) Blood 63 (6): 1424-.

The 85 amino acid carboxy-terminal region of the CD20 protein is located within the cytoplasm. This region is of a length comparable to other B cell-specific surface structures (e.g., IgM, IgD and IgG heavy chains) or histocompatibility class I1 antigen alpha or beta chains, and has relatively short cytoplasmic domains of 3, 28, 15 and 16 amino acids, respectively (Komaromy et al (1983) NAR 11: 6775-6785). Of the last 61 carboxy-terminal amino acids, 21 are acidic residues and only 2 are basic, indicating that this region has a strong net negative charge. GenBank accession number is NP-690605. It is thought that CD20 may be involved in regulating early stages in the process of B cell activation and differentiation (Tedder et al (1986) Eur. J. Immunol.2516: 881-887) and may function as a calcium channel (Tedder et al (1990) J. cell. biochem. 14D: 195).

There are two different types of anti-CD 20 antibodies that differ significantly in their CD20 binding pattern and biological activity (Cragg, m.s., et al, Blood, 103(2004) 2738-. Type I antibodies, such as Rituximab (Rituximab), are effective in complement-mediated cytotoxicity, while type II antibodies, such as Tositumomab (Tositumomab) (Toximab)B1) 11B8 and AT80, promoted target cell death by caspase-independent apoptosis with concomitant exposure of phosphatidylserine.

Shared common features of type I and type II anti-CD 20 antibodies are summarized in table 1.

Type I anti-CD 20 antibodies	Type II anti-CD 20 antibodies
		Type I CD20 epitope	Type II CD20 epitope
Positioning CD20 in lipid membrane rafts	Not localizing CD20 in lipid membrane rafts
		Increased CDC (if IgG1 isotype)	Reduced CDC (if IgG1 isotype)
ADCC Activity (if IgG1 isotype)	ADCC Activity (if IgG1 isotype)
		Full binding capacity	Reduced binding capacity

Homotypic aggregation	Stronger homotypic aggregation
		Induction of apoptosis upon crosslinking	Induce strong cell death without cross-linking

Table 1: characterization of type I and type II anti-CD 20 antibodies

Summary of The Invention

In one aspect, the present invention relates to a pharmaceutical formulation comprising:

1 to 150mg/mL of an anti-CD 20 antibody;

1 to 100mM buffer;

optionally 0.001 to 1% of a surfactant; and

optionally 1 to 800mM of a tonicity agent;

the pH is in the range of 4.5 to 7.0.

Preferably, the anti-CD 20 antibody is a type II antibody. More preferably, the anti-CD 20 antibody is a humanized B-Ly1 antibody.

Detailed Description

The term "antibody" includes antibodies in a variety of different forms, including, but not limited to, whole antibodies, human antibodies, humanized antibodies, and genetically engineered antibodies (e.g., monoclonal antibodies, chimeric antibodies, or recombinant antibodies) and fragments of such antibodies, so long as the characteristic features of the invention are retained.

An "antibody fragment" includes a portion of a full-length antibody, typically including at least an antigen-binding portion or variable region thereof. Examples of antibody fragments include diabodies, single chain antibody molecules, immunotoxins, and multispecific antibodies formed from antibody fragments. In addition, antibody fragments include single chain polypeptides having the characteristics of a VH chain, i.e., capable of assembling together with a VL chain or having the characteristics of a VL chain that binds to CD20 antigen, i.e., capable of assembling with a VH chain into a functional antigen-binding pocket.

"antibody fragments" also include fragments which do not themselves provide effector function (ADCC/CDC), but which, upon binding to the appropriate antibody constant regions, provide such function in a manner according to the invention.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules consisting of a single amino acid. Thus, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity, having variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is prepared from a hybridoma comprising a B cell obtained from a transgenic non-human animal (e.g., 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 "chimeric antibody" refers to a monoclonal antibody comprising a variable region (i.e., a binding region) derived from one source or species and at least a portion of a constant region derived from a different source or species, typically prepared by recombinant DNA techniques. Chimeric antibodies comprising murine variable regions and human constant regions are particularly preferred. Such murine/human chimeric antibodies are the expression products of 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 invention are those in which the class or subclass of the original antibody is modified or altered. Such "chimeric" antibodies are also referred to as "switch-like antibodies". Methods for the preparation of chimeric antibodies involve conventional recombinant DNA and gene transfection techniques known in the art. See, e.g., Morrison, S.L. et al, Proc. Natl. Acad Sci. USA 81(1984) 6851-6855; US 5,202,238 and US 5,204,244.

The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDRs) are modified to include immunoglobulin CDRs that are specifically different from the parent immunoglobulin. In a preferred embodiment, murine CDRs are grafted into the framework regions of a human antibody to make a "humanized antibody". See, for example, Riechmann, L.et al, Nature 332(1988) 323-. For chimeric and bifunctional antibodies, particularly preferred CDRs correspond to those representative sequences that recognize the above-described antigens.

The term "human antibody" as used herein 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 this technology, human antibodies can be made against a wide variety of targets. Examples of human antibodies are described, for example, in Kellermann, S.A. et al, Curr Opin Biotechnol.13(2002) 593-.

The term "recombinant human antibody" as used herein is intended to include all human antibodies that are prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from a host cell (e.g., NSO or CHO cells) or from a transgenic animal (e.g., mouse) for human immunoglobulin genes, or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences in rearranged forms. The recombinant human antibodies of the invention have been hypervariable in vivo in somatic cells. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

As used herein, "specifically binds" refers to an antibody that specifically binds to the CD20 antigen. Preferably, the binding affinity is 10^-9mol/l or 10^-9mol/l or less (e.g., 10)^-10mol/l), preferably 10^-10mol/l or 10^-10mol/l or less (e.g., 10)^-12mol/l) KD value. Determination of binding affinity using standard binding assays, e.g. surface plasmon resonance (Biacore))。

As used herein, "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.

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

As used herein, "variable region" (light chain variable region (VL), heavy chain variable region (VH)) refers to each pair of light and heavy chains that are directly involved in antibody antigen binding. The variable regions of human light and heavy chains have the same general structure, each region comprising four Framework (FR) regions, widely conserved in sequence, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a b-sheet conformation and the CDRs can form loops connecting the b-sheet structure. The CDRs in each chain retain their three-dimensional structure through the framework regions and form together with the CDRs of the other chain an antigen binding site. The antibody heavy and light chain CDR3 regions play an important role in the binding specificity/affinity of antibodies according to the invention and thus provide another object of the invention.

The term "hypervariable region" or "antigen-binding portion of an antibody" when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region comprises the amino acid residues of a "complementarity determining region" or "CDR". The "framework" or "FR" regions are those variable region regions other than the hypervariable region residues defined herein. Thus, the heavy and light chains of an antibody comprise, from N-terminus to C-terminus, the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. In particular, the CDR3 of the heavy chain is the region that contributes most to antigen binding. The CDR regions and FR regions are determined according to the standard definition of Kabat et al, Sequences of Proteins of immunological interest, fifth edition, department of public health, national ministry of health, Besserda, Maryland. (1991)) and/or those residues from "hyper-variable loops".

The terms "CD 20" and "CD 20 antigen" are used interchangeably herein and include any variant, isoform and species homolog of human CD20 that is naturally expressed by a cell or expressed on a cell transfected with the CD20 gene. Binding of the antibodies of the invention to the CD20 antigen mediates killing of cells (e.g., tumor cells) expressing CD20 by inactivating CD 20. Killing of cells expressing CD20 may occur by one or more of the following mechanisms:

synonyms for CD20 that have been identified in the prior art include the B-lymphocyte antigen CD20, the B-lymphocyte surface antigen B1, Leu-16, Bp35, BM5 and LF 5.

The term "anti-CD 20 antibody" according to the present invention is an antibody that specifically binds to the CD20 antigen. Depending on the binding properties and biological activity of anti-CD 20 antibodies to the CD20 antigen, anti-CD 20 antibodies can be distinguished into two types according to Cragg, M.S. et al, Blood 103(2004) 2738-.

Type I anti-CD 20 antibodies	Type II anti-CD 20 antibodies
		Type I CD20 epitope	Type II CD20 epitope
Positioning CD20 in lipid membrane rafts	Not localizing CD20 in lipid membrane rafts
		Increased CDC (if IgG1 isotype)	Reduced CDC (if IgG1 isotype)
ADCC Activity (if IgG1 isotype)	ADCC Activity (if IgG1 isotype)
		Full binding capacity	Reduced binding capacity
Homotypic aggregation	Stronger homotypic aggregation
		Induction of apoptosis upon crosslinking	Induce strong cell death without cross-linking

Table 2: characterization of type I and type II anti-CD 20 antibodies

An important property of type I and type II anti-CD 20 antibodies is their mode of binding. Type I and type II anti-CD 20 antibodies can therefore be classified by the ratio of the binding capacity of the anti-CD 20 antibody to rituximab for CD20 on Raji cells (ATCC-No. ccl-86).

As used herein, an "anti-CD 20 antibody" can be a type I or type II antibody. Preferably, it is a type II antibody.

The ratio of the type I anti-CD 20 antibody relative to the ability of rituximab to bind to CD20 on Raji cells (ATCC-No. ccl-86) is 0.8 to 1.2, preferably 0.9 to 1.1. Examples of such type I anti-CD 20 antibodies include, for example, rituximab, (WO94/11026), 1F5IgG2a (ECACC, hybridoma; Press et al, Blood 69/2: 584-591(1987)), H147IgG3(ECACC, hybridoma), 2C6IgG1 (as disclosed in WO2005/103081), 2F2IgG1 (as disclosed in WO2004/035607 and WO2005/103081) and 2H 7IgG 1 (as disclosed in WO 2004/056312). Preferably the type I anti-CD 20 antibody is a monoclonal antibody that binds to the same epitope as rituximab. The ratio of the type II anti-CD 20 antibody relative to the ability of rituximab to bind to CD20 on Raji cells (ATCC-No. ccl-86) is 0.3 to 0.6, preferably 0.35 to 0.55, more preferably 0.4 to 0.5. Examples of such type II anti-CD 20 antibodies include, for example, tositumomab (B1 IgG2a), humanized B-Ly1 antibody IgG1 (e.g., chimeric humanized IgG1 antibody disclosed in WO2005/044859), 11B8 IgG1 (e.g., disclosed in WO 2004/035607), and AT80IgG 1. Preferably the type II anti-CD 20 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-CD 20 antibody to rituximab to CD20 on Raji cells (ATCC-No. ccl-86)" was determined by direct immunofluorescence assay (determination of Mean Fluorescence Intensity (MFI)) using the Cy 5-conjugated anti-CD 20 antibody and Cy 5-conjugated rituximab in facsarray (becton dickinson) as described in example 2, and calculated as follows:

MFI is the mean fluorescence intensity. As used herein, "Cy 5 labeling ratio" means the number of Cy5 labeled molecules per molecule of antibody.

The ratio of the binding capacity of the first anti-CD 20 antibody to rituximab for CD20 on Raji cells (ATCC-No. ccl-86), typically the type I anti-CD 20 antibody is 0.8 to 1.2, preferably 0.9 to 1.1.

The ratio of the binding capacity of the second anti-CD 20 antibody to rituximab for CD20 on Raji cells (ATCC-No. ccl-86), typically the type II anti-CD 20 antibody is 0.3 to 0.6, preferably 0.35 to 0.55, more preferably 0.4 to 0.5.

In preferred embodiments, the type II anti-CD 20 antibody (preferably a humanized B-Ly1 antibody) has increased Antibody Dependent Cellular Cytotoxicity (ADCC).

By "antibody with increased antibody-dependent cellular cytotoxicity (ADCC)" is meant the term antibody as defined herein, which has increased ADCC as determined by any suitable method known to the person of ordinary skill in the art. One method accepted in the in vitro ADCC assay is as follows:

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

2) the assay uses human Peripheral Blood Mononuclear Cells (PBMCs) isolated from blood of randomly selected healthy donors as effector cells;

3) the assay was performed according to the following protocol:

i) PBMC were isolated using standard density centrifugation procedure and were treated at 5X 10⁶Individual cells/ml were suspended in RPMI cell culture medium;

ii) growing the target cells by standard tissue culture methods, collected from an exponential growth phase with viability greater than 90%, washed in RPMI cell culture medium, labeled with 100 microcurie "CI-, washed twice with cell culture medium, and then resuspended in cell culture medium at a density of 10' cells/ml;

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

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

v) for the Maximum Release (MR) control, 50 μ l of 2% (VN) aqueous non-ionic detergent (Nonidet, Sigma, st louis) solution was added to 3 additional wells of the plate containing labeled target cells instead of antibody solution (point iv above);

vi) for the Spontaneous Release (SR) control, 50 microliters of RPMI cell culture medium was added to 3 additional wells containing labeled target cells in the plate instead of the antibody solution (point iv above);

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

viii) 50 microliters of PBMC suspension (point i above) was added to each well to give an effector to target cell ratio of 25: 1, and then the plates were placed in an incubator at 37 ℃ under an atmosphere of 5% CO2 for 4 hours;

ix) cell-free supernatants were collected from each well and radioactivity released from the Experiment (ER) was quantified using a gamma counter;

x) calculating the specific lysis percentage for each antibody concentration according to the formula (ER-MR)/(MR-SR) x 100, wherein ER is the average radioactivity quantified for that antibody concentration (see above points ix), MR is the average radioactivity quantified for the MR control (see above points V) (see above points ix), and SR is the average radioactivity quantified for the SR control (see above points vi) (see above points ix);

4) "increased ADCC" is defined as an increase in the maximum percent specific lysis observed over the range of antibody concentrations tested above, and/or a decrease in antibody concentration required to achieve half the maximum percent specific lysis observed over the range of antibody concentrations tested above. The ADCC increase is relative to ADCC, determined by the above assay, mediated by the same antibody, produced by the same type of host cell, using the same standard preparation, purification, formulation and storage methods, which are known to those skilled in the art, but which have not been produced by host cells engineered to overexpress GnTIII.

The "increased ADCC" can be obtained by glycoengineering the antibody, which means that the antibody is produced as described by Umana, p. et al, Nature biotechnol.17: 176-180(1999) and U.S. Pat. No. 6,602,684, enhance the natural, cell-mediated effector functions of the monoclonal antibodies by engineering their oligosaccharide components.

The term "complement-dependent cytotoxicity (CDC)" refers to the lysis of human tumor target cells by an antibody according to the invention in the presence of complement. CDC is preferably determined by a preparation of CD20 expressing cells treated with an anti-CD 20 antibody according to the invention in the presence of complement. CDC was found if antibody at 100nM concentration induced lysis (cell death) of 20% or more of the tumor cells after 4 hours. Preferably by⁵¹Cr or Eu labelling of tumour cells and measuring release⁵¹Cr or Eu. Controls included incubating tumor target cells with complement but without antibodies.

Typically, type I and type II anti-CD 20 antibodies of the IgG1 isotype exhibit characteristic CDC properties. Type I anti-CD 20 antibodies have increased CDC (if of the IgG1 isotype) and type II anti-CD 20 antibodies have decreased CDC (if of the IgG1 isotype) relative to each other. Preferably, both the type I and type II anti-CD 20 antibodies are of the IgG1 isotype.

The "rituximab" antibody is a monoclonal antibody directed against the human CD20 antigen containing a genetically engineered chimeric human gamma 1 murine constant region. Such chimeric antibodies contain a human gamma 1 constant region and are identified by the designation "C2B 8" in WO94/11026(Anderson et al). Rituximab is approved for the treatment of patients with relapsed or refractive low-grade malignancy or follicular, CD 20-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, rituximab showed significant activity in assays to determine 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-CD 20 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 the human constant regions of IgG1 and subsequent humanization (see WO 2005/044859). These "humanized B-Ly1 antibodies" are disclosed in detail in WO 2005/044859.

Preferably, the "humanized B-Ly1 antibody" has a heavy chain variable region (VH) selected from SEQ ID No.3 to SEQ ID No.20 (B-HH 2 to B-HH9 and B-HL8 to B-HL17 in WO 2005/044859). Particularly preferred are seq.ID Nos. 3, 4, 7, 9, 11, 13 and 15 (B-HH 2, BHH-3, B-HH6, B-HH8, B-HL8, B-HL11 and B-HL13 in WO 2005/044859). Most preferably, the VH is BHH 6. Preferably, the "humanized B-Ly1 antibody" has the light chain variable region (VL) of SEQ ID No.20(B-KV1) in WO 2005/044859. Furthermore, the humanized B-Ly1 antibody is preferably an IgG1 antibody. Preferably, the Fc region of such a humanized B-Ly1 antibody is encoded by a light chain according to WO2005/044859, WO 2004/065540, Umana, p. et al, Nature biotechnol.17: 176-180(1999) and WO 99/154342 describe methods for Glycoengineering (GE). The Fc region of the somewhat glycoengineered humanized B-Ly1 antibody has an altered glycosylation pattern, preferably with reduced levels of fucose residues. Preferably, at least 40% or more than 40% (in one embodiment between 40% and 60%, in another embodiment at least 50%, in yet another embodiment at least 70% or more than 70%) of the oligosaccharides in the Fc region are nonfucosylated. Furthermore, the oligosaccharides in the Fc region are preferably bisected. Most preferably, the "humanized B-Ly1 antibody" comprises VH B-HH6 and VL B-KV1 of WO 2005/044859. As used herein, the antibody is also referred to as "HuMab < CD20 >. In another most preferred embodiment, the antibody has a reduced level of fucose residues as defined above and/or the oligosaccharides in the Fc region are most preferably bisected. In yet another most preferred embodiment, said antibody exhibits increased ADCC as defined herein.

The oligosaccharide component can significantly affect properties related to the efficacy of the therapeutic glycoprotein, 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 clearance of glycoproteins from the bloodstream by interacting with specific glycobinding proteins, while other oligosaccharides are bound by antibodies and initiate undesirable immune responses (Jenkins et al, Nature Biotechnol. 14: 975-81 (1996)).

Mammalian cells are preferred hosts for the production of therapeutic glycoproteins due to their ability to glycosylate proteins in the most compatible form for human use. (Cumming et al, Glycobiology 1: 115-30 (1991); Jenkins et al, Nature Biotechnol.14: 975-81 (1996)). Bacteria rarely glycosylate proteins and, like other types of common hosts, such as yeast, filamentous fungi, insect and plant cells, produce glycosylation patterns that are accompanied by rapid clearance from the blood stream, undesirable immune interactions, and, in some particular cases, reduced biological activity. Among mammalian cells, Chinese Hamster Ovary (CHO) cells have been most commonly used in the last two decades. In addition to providing a suitable glycosylation pattern, these cells allow for the consistent production of genetically stable, highly productive clonal cell lines. They can be cultured to high densities in simple bioreactors using serum-free media and allow the development of safe and reproducible bioprocesses. Other commonly used animal cells include Baby Hamster Kidney (BHK) cells, NSO-and SP 2/0-mouse myeloma cells. Recently, production from transgenic animals has also been tested. (Jenkins et al, Nature Biotechnol.14: 975-981 (1996)).

All antibodies contain sugar structures at conserved positions in the heavy chain constant region, with each isotype having a different array of N-linked sugar structures that variably affect protein assembly, secretion, or functional activity. (Wright, A. and Monison, S.L., Trends Biotech.15: 26-32 (1997)). The structure of the attached N-linked sugar varies considerably depending on the degree of processing and may include high mannose, multi-branched and biantennary complex oligosaccharides. (Wright, A. and Morrison, S.L., Trends Biotech.15: 26-32 (1997)). Typically, heterogeneous processing of the core oligosaccharide structures attached at specific glycosylation sites exists such that monoclonal antibodies exist even in the polysaccharide form. Also, it has been shown that major differences in antibody glycosylation occur between cell lines, with even minor differences seen for a given cell line grown under different culture conditions. (Life, M.R. et al, Glycobiology 5 (8): 813-22 (1995)).

One way to achieve a large increase in potency while maintaining a simple manufacturing process and potentially avoiding significant, undesirable side effects is to use a solution such as Umana, p, et al, Nature biotechnol.17: 176-180(1999) and U.S. Pat. No. 6,602,684 describe enhancing the natural, cell-mediated effector functions of monoclonal antibodies by engineering their oligosaccharide components. IgG 1-type antibodies, the most commonly used antibodies in cancer immunotherapy, are glycoproteins with conserved N-linked glycosylation sites at Asn297 in the respective CH2 domain. Two double antenna complex oligosaccharides attached to Asn297 are buried between the CH2 domains, making extensive contact with the polypeptide backbone, and their presence is essential for antibody-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) (Life, 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)).

It was previously demonstrated that overexpression of β (1, 4) -N-acetylglucosaminyltransferase I11 ("GnTII 17y), a glycosyltransferase that catalyzes the formation of truncated oligosaccharides, by Chinese Hamster Ovary (CHO) cells significantly increased 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, the entire contents of which are hereby incorporated by reference). The antibody chCE7 belongs to a large class of unconjugated monoclonal antibodies that have high tumor affinity and specificity but have too little potential to be clinically useful when prepared in standard industrial cell lines lacking the GnTIII enzyme (Umana, P., et al, Nature Biotechnol.17: 176-180 (1999)). This study was the first to demonstrate that a large increase in ADCC activity could be obtained by engineering antibody producing cells to overexpress GnTIII, which also resulted in an increase in the proportion of constant region (Fc) -bound, truncated oligosaccharides (including truncated non-fucosylated oligosaccharides) over that found in naturally occurring antibodies.

The term "expression of CD20 antigen" is used to indicate a significant level of expression of CD20 antigen in cells, preferably on the cell surface of T-or B-cells, more preferably on the cell surface of B-cells, derived from a tumor or cancer (preferably a non-solid tumor), respectively. Patients with "CD 20 expressing cancer" can be determined by standard assays known in the art. "expression of CD20 antigen" is also preferably used to indicate a significant level of expression of CD20 antigen in cells of an autoimmune disease, preferably on the cell surface of T-or B-cells, more preferably on the cell surface of B-cells. CD20 antigen expression is determined, for example, using Immunohistochemistry (IHC) detection, FACS or PCR-based detection of the corresponding mRNA.

The term "CD 20 expressing cancer" as used herein preferably refers to lymphomas (preferably B-cell non-Hodgkin's lymphoma (NHL) and lymphocytic leukemias. such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, B) small non-nucleated/Burkitt's lymphoma (including endemic, sporadic 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 lymphoma, primary mediastinal B-cell lymphoma, primary noncytotic B-cell lymphoma, secondary lymphoma, and primary noncytotic lymphoma, Angiocentric lymphoma-pulmonary B cell lymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma, waldenstrom's macroglobulinemia, h) Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Leukemia (SLL), B cell prolymphocytic leukemia, i) plasma cell neoplasm, plasma cell myeloma, multiple myeloma, plasmacytoma, j) hodgkin's disease.

Preferably, the CD 20-expressing cancer is a B-cell non-hodgkin's lymphoma (NHL). CD20 expressing cancers are in particular 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 central nervous system lymphoma.

As used herein, "autoimmune disease" refers to a disease or disorder that is produced by and is directed against the patient's own tissues. Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, reactions associated with 15 inflammatory bowel disease, crohn's disease, ulcerative colitis, respiratory distress syndrome, Adult Respiratory Distress Syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving T-cell exudation and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, Systemic Lupus Erythematosus (SLE), juvenile diabetes, multiple sclerosis, allergic encephalomyelitis, immune responses related to acute and delayed-type reactions mediated by cytokines and T-lymphocyte hypersensitivity reactions, inflammatory disorders, inflammatory bowel disease, inflammatory bowel, Tuberculosis, sarcoidosis, granulomatosis (including wegener's granulomatosis), agranulocytosis, vasculitis (including ANCA), aplastic anemia, bunyasu's anemia, immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), pernicious anemia, mono-Pure Red Cell Aplasia (PRCA), factor VIII deficiency, hemophilia a, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, Central Nervous System (CNS) inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, behcet's disease, macrolymph node hyperplasia syndrome (Castleman's syndrome), goodpasture's syndrome, Lambert-Eaton myasthenia syndrome, Raynaud's syndrome, Sjorgen's syndrome, Stevens Johnson syndrome, bullous pemphigoid, pemphigus, autoimmune polyendocrinopathy, nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, autoimmune diseases of the testis and ovary (including orchitis and oophoritis), primary hypothyroidism, autoimmune endocrine disorders (including autoimmune thyroiditis), chronic thyroiditis (hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, edison's disease, Grave's disease, autoimmune polycystic gland syndrome (or polyadenylic I endocrinopathy syndrome), Type I diabetes is also known as Insulin Dependent Diabetes Mellitus (IDDM) and hahn's syndrome, autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), obstructive bronchiolitis (non-transplant) versus NSIP, guillain-barre syndrome, macrovasculitis (including polymyalgia rheumatica and giant cell (homo-ampere) arteritis), medium vascular vasculitis (including Sichuan and polyarteritis nodosa), ankylosing spondylitis, buerger's disease (IgA nephropathy), acute glomerulonephritis, primary biliary cirrhosis, sprue (non-tropical sprue), cryoglobulinemia, Amyotrophic Lateral Sclerosis (ALS), coronary heart disease, and the like.

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

The term "surfactant" as used herein refers to a pharmaceutically acceptable surfactant. In the formulations of the present invention, the amount of surfactant is expressed as a weight/volume percentage. The most commonly used weight/volume unit is mg/ml. Suitable pharmaceutically acceptable surfactants include, but are not limited to, nonionic surfactants, such as TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG). Also, the surfactant includes, but is not limited to, polyoxyethyleneSorbitan ether fatty acid esters, polyoxyethylenated polypropylene glycols, polyoxyethylene stearates and sodium lauryl sulfate. A preferred polyoxyethylene sorbitan is polyoxyethylene (20) sorbitan ester (also known as polysorbate 20 under the trademark Tween (Tween) 20)^TMSold) and polyoxyethylene (20) sorbitan monooleate (also known as polysorbate 80, under the trademark tween 80^TMSales). The preferred polyoxyethylated polypropylene glycol is PluronicF68 or Poloxamer 188^TMThose sold. Most preferred is poloxamer 188^TM. Preferred polyoxyethylene stearates are under the trade mark Myrj^TMThose sold. Preferred polyoxyethylene monolaurate is under the trademark Brij^TMThose sold. When polyoxyethylene sorbitan-polyoxyethylene (20) sorbitan ester (Tween 20) is used^TM) And polyoxyethylene (20) sorbitan monooleate (Tween 80)^TM) When used, they are generally used in amounts of about 0.001 to about 1%, preferably about 0.005 to about 0.1% and more preferably about 0.01% to about 0.04% w/v.

The term "buffer" as used herein refers to a pharmaceutically acceptable buffer. Suitable pharmaceutically acceptable buffers include, but are not limited to, histidine buffers, citrate buffers, succinate buffers, acetate buffers, and phosphate buffers. Preferred buffers include L-histidine or L-histidine with L-histidine hydrochloride and isotonic agents, and pH adjustment is possible with acids or bases known in the art. Most preferred is L-histidine. The histidine-buffer described above is generally used in an amount of about 1mM to about 100mM, preferably about 5mM to about 50mM and still more preferably about 20 mM. The pH is adjusted to a value comprising from about 4.5 to about 7.0, preferably from about 5.5 to about 6.5 and more preferably about 6.0, independently of the buffer used, by adjustment with an acid or base known in the art or by using a mixture of suitable buffering ingredients or both.

The term "isotonic agent" as used herein refers to pharmaceutically acceptable isotonic agents. Isotonic agents are used to provide isotonic formulations. Isotonic preparations are liquids or liquids reconstituted from solid forms (e.g., lyophilized forms) and refer to solutions having the same tonicity as some other solution to which it is compared (e.g., physiological saline solution and serum). Suitable isotonic agents include, but are not limited to, salts including, but not limited to, sodium chloride (NaCl) or potassium chloride; sugars, including but not limited to glucose, sucrose, trehalose or glycerol and any ingredient from the group of amino acids, sugars, salts and combinations thereof. Isotonic agents are generally used in total amounts of about 5mM to about 350 mM.

The term "liquid" as used herein in connection with the formulation according to the present invention refers to a formulation which is liquid at a temperature of at least about 2 to about 8 ℃.

The term "lyophilized" as used herein in connection with the formulation according to the present invention refers to a formulation that is dried by freezing the formulation and then subliming ice from the frozen contents using any freeze-drying method known in the art (e.g., commercially available freeze-drying equipment).

The term "salt" as used herein refers to an amount of salt of from about 1mM to about 500 mM. Non-limiting examples of such salts include salts of any combination of the cations sodium, potassium, calcium or magnesium with the anions chloride, phosphate, citrate, succinate, sulfate, or mixtures thereof.

The term "amino acid" as used herein refers to an amount of amino acid from about 1 to about 100mg/mL, which includes, but is not limited to, arginine, glycine, ornithine, glutamine, asparagine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline.

The term "sugar" as used herein refers to a pharmaceutically acceptable sugar in an amount of about 25mM to about 500 mM. Preferably 100 to 300 mM. More preferably 220 to 260 mM. Most preferably 240 mM. Suitable sugars include, but are not limited to, monosaccharides and disaccharides. Non-limiting examples of sugars according to the present invention include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucamine (commonly known as "meglumine"), galactosamine, and neuraminic acid and combinations thereof. Most preferred 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 any type and molecular weight of commercially available dextran known in the art.

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

The term "method of treatment" or its equivalent when used in reference to, for example, cancer, refers to a procedure or process for reducing or eliminating the number of cancer cells in a patient or alleviating the symptoms of cancer. "method of treatment" of cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will actually be eliminated, that the number of cells or disorder will actually be reduced or that the symptoms of the cancer or other disorder will actually be alleviated. Generally, methods for treating cancer are performed even with a low likelihood of success, but are still considered to induce an overall beneficial course of action, given the patient's medical history and estimated survival expectations.

In one aspect, the invention relates to an anti-CD 20 antibody formulation comprising:

about 1 to about 150mg/mL of an anti-CD 20 antibody,

-from about 0.001 to about 1% of at least one surfactant, and

-from about 1 to about 100mM of a buffer,

-a pH of about 4.5 to about 7.0.

In a more preferred embodiment, the formulation according to the invention comprises:

about 1 to about 150mg/mL of an anti-CD 20 antibody,

-from about 0.005 to about 0.05% of at least one surfactant, and

-from about 1 to about 100mM of a buffer,

-a pH of about 4.5 to about 7.0.

In a preferred embodiment, the formulation according to the invention comprises:

about 10 to about 30mg/mL of a type II anti-CD 20 antibody,

-20mM of L-histidine,

240mM of trehalose, and

-0.02% w/v polysorbate 20,

-a pH of about 6.

In a further preferred embodiment, the formulation according to the invention comprises:

about 10 to about 30mg/mL of a type II anti-CD 20 antibody,

-0.02% w/v poloxamer 188^TM，

-20mM L-histidine, and

-240mM of trehalose,

-a pH of about 6.

Preferably, the anti-CD 20 antibody is a type I antibody. More preferably, the anti-CD 20 antibody is a type II antibody. Even more preferably, the anti-CD 20 antibody is a "humanized B-Ly1 antibody" as disclosed in detail in WO 2005/044859. More preferably, the antibody is HuMab < CD20 >. The formulation comprises from 1 to about 150mg/ml of the antibody, more preferably from about 5 to about 100mg/ml of the antibody, even more preferably from about 10 to about 30mg/ml of the antibody, or selected from about 5, 10, 15, 20, 25 or 30mg/ml of the antibody, and most preferably 25mg/ml of the antibody.

The formulation according to the invention may be in liquid form, lyophilized form or reconstituted liquid form from lyophilized form.

In one embodiment, the formulation according to the invention is a lyophilized formulation. The lyophilized formulation according to the invention has the advantage of improved stability towards the formation of higher molecular weight particles and aggregates, which is often difficult to achieve with liquid formulations of the same concentration of the anti-CD 20 antibody.

The formulations according to the invention may be administered by intravenous (i.v.), subcutaneous (s.c.) or any other parenteral administration such as those known in the pharmaceutical art.

Furthermore, the preparations according to the invention may contain preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; chelating agents, such as EDTA; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes).

The active ingredients can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and polymethylmethacrylate-microcapsules), colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or microemulsions, respectively. This technique is disclosed in Remington's Pharmaceutical Sciences 16 th edition, Osol, A. edition (1980).

Sustained release formulations can be prepared. Examples of suitable sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly 2-hydroxyethyl methacrylate, or polyethylene)Enol), polylactide (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, nondegradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRONDEPOT^TM(injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) -3-hydroxybutyric acid.

Formulations for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

Preferably, as determined above, the formulations of the invention comprise one or more isotonic agents in an amount of about 5mM to about 350 mM.

Preferably, as determined above, the formulations of the invention comprise sugar in an amount of about 25mM to about 500 mM.

Also preferably, the formulation of the present invention further comprises one or more of the following ingredients: antioxidants, ascorbic acid, glutathione, preservatives, such as m-cresol, phenol, benzyl alcohol, methyl paraben, propyl paraben, chlorobutanol, thimerosal, benzalkonium chloride, polyethylene glycols, such as PEG 3000, 3350, 4000, 6000, Human Serum Albumin (HSA), Bovine Serum Albumin (BSA), polyols, glycerol, ethanol, mannitol, salts, acetates (e.g., sodium acetate), magnesium chloride, calcium chloride, tromethamine, EDTA (e.g., Na-EDTA).

Also preferably, the formulation of the invention further comprises one or more stabilizers as defined above and components also known in the art as "lyoprotectants" (such as sugars, sugar alcohols, amino acids and dextrans known in the art).

In a certain embodiment, the formulation of the invention comprises a liquid, lyophilized, or liquid formulation reconstituted from a lyophilized form of:

15mg/mL of a type II anti-CD 20 antibody, preferably a humanized B-Ly1 antibody, most preferably HuMab < CD20>,

0.01% w/v polysorbate 20,

20mM of L-histidine, and

140mM of sodium chloride, in the form of sodium chloride,

pH 6.0；

or

10mg/mL of a type II anti-CD 20 antibody, preferably a humanized B-Ly1 antibody, most preferably HuMab < CD20>,

0.01% w/v polysorbate 20,

20mM of L-histidine, and

140mM of sodium chloride, in the form of sodium chloride,

pH 6.0；

or

optionally 0.001 to 1% w/v of a surfactant,

20mM of L-histidine, in the form of a peptide,

pH 6.0；

or

0.02% w/v polysorbate 20,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

25mg/mL of a type II anti-CD 20 antibody, preferably a humanized B-Ly1 antibody, most preferably HuMab < CD20>,

0.02% w/v polysorbate 20,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

0.02% w/v poloxamer 188^TM，

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

0.01% w/v poloxamer 188^TM，

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

0.1% w/v poloxamer 188^TM，

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

0.02% w/v polysorbate 80,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

0.1% w/v polysorbate 80,

20mM of acetate, and

a concentration of 240mM of trehalose in the sample,

pH 5.5；

or

0.1% w/v polysorbate 80,

20mM of acetate, and

140mM of sodium chloride, in the form of sodium chloride,

pH 5.5；

or

30mg/mL of a type II anti-CD 20 antibody, preferably a humanized B-Ly1 antibody, most preferably HuMab < CD20>,

0.01% w/v poloxamer 188^TM，

20mM of L-histidine, and

200mM of trehalose in a medium containing a sugar-free polysaccharide,

pH 6.5；

in a preferred embodiment of the formulation according to the invention, said formulation is in lyophilized form and comprises, after reconstitution with an appropriate amount of water for injection:

0.02% w/v polysorbate 20,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

such formulations exhibit good stability with sufficient stability towards physical end points (e.g. aggregation) and chemical end points (e.g. fragmentation) when stored at 2-8 ℃ and 25 ℃.

In a preferred embodiment of the formulation according to the invention, the formulation is in liquid form:

0.2% w/v poloxamer 188^TM，

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

in a preferred embodiment, the formulation is used to prevent or reduce metastasis or further spread in a patient suffering from a CD20 expressing cancer. The formulations are useful for increasing survival in such patients, increasing progression-free survival in such patients, increasing the duration of response, resulting in a statistically significant and clinically meaningful improvement in the treated patients, as measured by survival, progression-free survival, response rate, or duration of response. In a preferred embodiment, the formulation is used to increase the response rate in a group of patients.

In the context of the present invention, other additional cytotoxic, chemotherapeutic or anti-cancer agents, or compounds that enhance the effects of such therapeutic agents, may be used in combination with the anti-CD 20 antibody formulation according to the present invention.

Such therapeutic agents include, for example: alkylating agents or drugs having an alkylating action, such as cyclophosphamide (CTX; e.g. cyclophosphamide; cyclophosph) Chlorambucil (CHL; for example) Cisplatin (CisP; for example) Busulfan (e.g. for) Melphalan, carmustine (BCNU), streptozotocin, Tritamine (TEM), mitomycin C, and the like; antimetabolites, e.g. Methotrexate (MTX), etoposide (VP 16; e.g.) 6-mercaptopurine (6MP), 6-thioguanidine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g., L-cysteine, L-) Dacarbazine (DTIC), and the like; antibiotics, e.g. actinomycin D, doxorubicin (DXR; e.g. Proben, Proben) Daunorubicin (daunomycin), bleomycin, daunomycin and the like; alkaloids, such as vinca alkaloids, e.g., Vincristine (VCR), vinblastine, and the like; and other antineoplastic agents, e.g., paclitaxel (e.g., paclitaxel)) And paclitaxel derivatives, cytostatics, such as dexamethasone (DEX; for example) And a glucocorticoid andcorticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid consuming enzymes such as asparaginase, folinic acid and other folic acid derivatives, and similar, diverse antineoplastic agents. The following drugs may also be used as additional drugs: amifostine (e.g. amifostine)) Dactinomycin, mechlorethamine, streptozotocin, cyclophosphamide, lomustine (CCNU), liposomal doxorubicin (e.g.) Gemcitabine (e.g. Gemcitabine)) Daunorubicin liposomes (e.g. daunorubicin liposomes)) Procarbazine, mitomycin, docetaxel (e.g. procarbazine, mitomycin)) Aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy-7-ethylcamptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferon alpha, mitoxantrone, topotecan, leuprorelin acetate, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, asparaginase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil. Preferably, the anti-CD 20 antibody is used in combination therapy without such additional drugs.

The use of the above cytotoxic and anticancer agents and antiproliferative target-specific anticancer drugs (such as protein kinase inhibitors) in chemotherapeutic regimens is well characterized in the field of cancer therapy, and their use herein is subject to the same considerations to monitor tolerability and efficacy and to control administration route and dose, with some adjustments. For example, the actual dose of cytotoxic agent may vary depending on the patient's cultured cell response as determined by using tissue culture methods. Typically, the dosage will be reduced relative to the amount used in the absence of the other additional drug.

Typical dosages of effective cytotoxic agents may be within the manufacturer's recommended ranges and, in cases dictated by in vitro responses or responses in animal models, can be reduced by up to about an order of magnitude concentration or amount. Thus, the actual dosage will depend on the judgment of the practitioner, the condition of the patient, and the effectiveness of the treatment method (based on the in vitro reactivity of the primary cultured malignant cells or tissue cultured tissue samples, or the response observed in an appropriate animal model).

In the context of the present invention, in addition to the anti-CD 20 antibody preparation according to the present invention, an effective amount of ionizing radiation may be performed and/or a radiopharmaceutical may be used. The radiation source may be external or internal to the patient under treatment. When the radiation source is external to the patient, the treatment is called External Beam Radiotherapy (EBRT). When the radiation source is internal to the patient, the treatment is called Brachytherapy (BT). The radioactive atom used in the context of the present invention may be selected from, but is not limited to, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and indium-111. It is also possible to label the antibody with such a radioisotope. Preferably, the anti-CD 20 antibody preparation according to the invention is used in the absence of such ionizing radiation.

Radiation therapy is the standard treatment for controlling unresectable or inoperable tumors and/or tumor metastases. Improved results have been seen when radiation therapy is combined with chemotherapy. Radiotherapy is based on the principle that high doses of radiation delivered to a target area can lead to the death of proliferating cells in tumor and normal tissues. Radiation dose schedules are usually determined in terms of dose absorbed by radiation (Gy), time and grading and must be carefully determined by oncological specialists. The amount of radiation a patient receives will depend on various considerations, but the two most important points are the location of the tumor relative to other important 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 is a 1 to 6 week cycle, administered to the patient in a single daily divided dose of about 1.8 to 2.0Gy for 5 days a week, for a total dose of between 10 and 80 Gy. In a preferred embodiment of the invention, there is a synergistic effect when treating a tumor in a human patient with a formulation according to the invention and radiation. In other words, the inhibition of tumor growth by a medicament comprising a combination of the invention is enhanced when combined with radiation, optionally with additional chemotherapeutic or anti-cancer agents. Parameters for adjuvant radiation therapy are for example contained in WO 99/60023.

The antibody formulation is administered to the patient according to known methods by intravenous injection as a bolus injection or by continuous infusion over a period of time by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial or intrathecal routes. Intravenous or subcutaneous administration of the antibody is preferred.

The invention further comprises a kit characterized by comprising a container, a composition comprising the anti-CD 20 antibody formulation within the container, and a package insert directing the user of the formulation to administer the anti-CD 20 antibody formulation to a patient having a CD 20-expressing cancer.

The term "package insert" refers to instructions typically included in commercial packaging for therapeutic products, which may include information regarding the indications, usage, dosage, administration, contraindications, and/or precautions for the use of such therapeutic products.

In a preferred embodiment, the finished container may further comprise a pharmaceutically acceptable carrier. The finished product may further comprise a sterile diluent, which is preferably stored in a separate container.

As used herein, "pharmaceutically acceptable carrier" is intended to include any and all materials compatible with pharmaceutical administration, including solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and other materials and compounds compatible with pharmaceutical administration. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions of the invention is contemplated. Auxiliary active compounds may also be incorporated into the compositions.

In a further embodiment of the invention, the formulation according to the invention comprises a type I anti-CD 20 antibody, which is co-administered with a type II anti-CD 20 antibody according to the invention. The formulation according to the invention may be two separate formulations for each anti-CD 20 antibody. Alternatively, the formulations herein may also contain both antibodies in one formulation.

In a further embodiment of the invention, the formulation according to the invention comprises an anti-CD 20 antibody, in that the anti-CD 20 antibody is co-administered with an anti-Bcl-2 active agent. The term "Bcl-2" as used herein refers to the Bcl-2 protein (Swiss Prot ID No. P10415), which is a member of the Bcl-2 protein family. The term "anti-Bcl-2 active agent" encompasses "anti-Bcl-2 antisense nucleotides" and "Bcl-2 inhibitors". An "anti-Bcl-2 antisense nucleotide" down-regulates Bcl-2mRNA levels and reduces Bcl-2 protein expression. Examples of such anti-Bcl-2 antisense nucleotides include Obblimmersen and SPC-2996. ABT-737 as used herein means N- [4- [4- (4' -chlorobiphenyl-2-ylmethyl) piperazin-1-yl]Benzoyl radical]-3- [3- (dimethylamino) -1(R) - (phenylthiomethyl) propylamino]-4-nitrobenzenesulfonamide; 4- [4- (4' -chlorobiphenyl-2-ylmethyl) piperazin-1-yl]-N- [3- [3- (dimethylamino) -1(R) - (phenylthioalkyl) propylamino]-4-Nitrobenzenesulfonyl]Benzamide (Bcl-2 inhibitor), which is described in WO 2006/099667 or Corey, s., et al, Cancer Cell (2005) 5-6. BT-263 as used herein means Bcl-2 inhibitors, which are described in US 2007027135. Preferably, the anti-Bcl-2 active agent is selected from the group consisting of Obllisers, SPC-2996, TA-402, gossypol, AT-101, Obatoclax mesylate, A-371191, A-385358, A-438744, ABT-737, AT-101, BL-11, BL-193, GX-15-003, 2-methoxyantimycin A₃HA-14-1, KF-67544, red purple gallogen, TP-TW-37, YC-137 and Z-24. Preferably, the anti-Bcl-2 active agent is a Bcl-2 protein binding inhibitor having an IC50 for anti-Bcl-2 inhibitory activity of 5 μ M or less. Such Bcl-2 protein binding inhibitors are preferably selected from the group consisting of gossypol, AT-101, Obatoclax mesylate, ABT-263 and ABT-737, more preferablyIs selected from ABT-263 or ABT-737.

In a further embodiment of the invention, the formulation according to the invention comprises an anti-CD 20 antibody, since the anti-CD 20 antibody is co-administered with a proteasome inhibitor. The term "proteasome inhibitor" as used herein refers to a drug that inhibits the activity of the 26S proteasome. Such proteasome inhibitors include, inter alia, for example, peptide derivatives, such as peptide aldehydes (e.g. MG132, MG115, CEP-1615, PSI or immunoproteasome-specific inhibitors IPSI-001(Cbz-LnL-CHO ═ N-benzyloxycarbonyl-leucyl-norleucine, see US 20060241056), peptide borates (e.g. bortezomib (bortezomib, PS-341) or DFLB), peptide epoxyketones (e.g. epoxomicin, dihydroepemomycin or epomicin derivatives carfilzomib (PR-171)) or peptide vinylsulfones (e.g. NLVS) and non-peptide derivatives (e.g. salinosporamide A (NPI-0052), salinosporamide A derivatives, lactasin or lactasin derivatives (e.g. claspin-lactaside-L-lactone (lactoside) or PS-519), different types and structures of said proteasome inhibitors are described, for example, in Kissell et al, Biossevir A, WO 35758, et al, Res 66(16) (2006)7840 and 7842; kanagasabaphy, et al, Curr Opin Investig Drugs 8(2007) 447-51; adams, J., Nat Rev Cancer 4(2004)349-360 and US 20060241056.

Preferably, such proteasome inhibitors are selected from peptide aldehydes (preferably N-benzyloxycarbonyl-leucyl-norleucine (IPSI-001)), peptide borates (preferably bortezomib (PS-341)), peptide epoxy ketones (preferably the epoxyicin derivative carfilzomib (PR-171)) or salinosporamideA (NPI-0052). More preferably, such proteasome inhibitors are chosen from bortezomib (PS-341), carfilzomib (PR-171), salinosporamide A (NPI-0052) or N-benzyloxycarbonyl-leucyl-norleucine (IPSI-001).

In a preferred embodiment, the proteasome inhibitor is a peptide derivative selected from the group consisting of a peptide aldehyde (preferably N-benzyloxycarbonyl-leucyl-norleucine (IPSI-001)), a peptide borate (preferably bortezomib (PS-341)), or a peptide epoxy ketone. In another preferred embodiment, the proteasome inhibitor is a peptide borate (preferably bortezomib (PS-341); see, e.g., Adams, Cur. Opin. chem biol.6(2002)493-500 and U.S. Pat. No. 5,780,454).

Preferably, the IC50 for the anti-proteasome inhibitory activity of the proteasome inhibitor is 5 μ M or less, more preferably 1 μ M or less. Cell-based assays for identifying such Proteasome inhibitors and IC50 for determining anti-Proteasome inhibitory activity (by serial dilution and calculation using nonlinear curve fitting (XLfit software (ID Business Solution ltd., Guilford, Surrey, UK)) are described in Moravec, et al, Cell Notes 15(2006)4-7, using Proteasome-GloTM Cell-based assay reagents from Promega with U266 cells (human plasma myeloma).

In addition to IPSI-001(Cbz-LnL-CHO ═ N-benzyloxycarbonyl-leucyl-norleucine), the following peptide derivatives in US 20060241056 are also preferred proteasome inhibitors: n-benzyloxycarbonyl-homophenylaminoacyl-phenylalanine, N-benzyloxycarbonyl-leucyl-phenylalanine, N-benzyloxycarbonyl-alanyl-phenylalanine, N-benzyloxycarbonyl-glycyl-prolyl-phenylalanyl-phenylalanine, N-benzyloxycarbonyl-glycyl-phenylalanyl-phenylalanine, N-benzyloxycarbonyl-leucyl-norleucine boronic acid, N-benzyloxycarbonyl-phenylalanyl-phenylalanine boronic acid, N-benzyloxycarbonyl-homophenylalanyl-phenylalanine boronic acid, N-benzyloxycarbonyl-leucyl-phenylalanine boronic acid, N-beta, N-benzyloxycarbonyl-glycyl-prolyl-alanyl-phenylalanine boronic acid, N-benzyloxycarbonyl-glycyl-prolyl-phenylalanyl-phenylalanine boronic acid, N-benzyloxycarbonyl-leucyl-phenylalanine boronic acid, N-benzyloxycarbonyl-glycyl-phenylalanyl-phenylalanine boronic acid, N-benzyloxycarbonyl-leucyl-norleucine methylvinyl sulfone, N-benzyloxycarbonyl-phenylalanyl-phenylalanine methylvinyl sulfone, N-benzyloxycarbonyl-homophenylalanyl-phenylalanine methylvinyl sulfone, N-benzyloxycarbonyl-leucyl-phenylalanine methylvinyl sulfone, N-benzyloxycarbonyl-phenylalanyl-phenylalanine methylvinyl sulfone, N-benzyloxycarbonyl-leucyl-phenylalanyl-phenylalanine methylvinyl sulfone, N-benzyloxycarbonyl-methylvinyl, N-benzyloxycarbonyl-alanyl-phenylalanine methylvinylsulfone, N-benzyloxycarbonyl-glycyl-prolyl-phenylalanyl-phenylalanine methylvinylsulfone, N-benzyloxycarbonyl-leucyl-phenylalanyl-phenylalanine methylvinylsulfone, N-benzyloxycarbonyl-glycyl-phenylalanyl-phenylalaninyl methylvinylsulfone, N-benzyloxycarbonyl-leucyl-norleucine epoxyketone, N-benzyloxycarbonyl-phenylalanyl-phenylalanin epoxyketone, N-benzyloxycarbonyl-homophenylalanyl-phenylalanin epoxyketone, N-benzyloxycarbonyl-phenylalan, N-benzyloxycarbonyl-leucyl-phenylalanyl-epoxyketone, N-benzyloxycarbonyl-alanyl-phenylalanyl-epoxyketone, N-benzyloxycarbonyl-glycyl-prolyl-alanyl-phenylalanyl-epoxyketone, N-benzyloxycarbonyl-glycyl-prolyl-phenylalanyl-epoxyketone, N-benzyloxycarbonyl-leucyl-phenylalanyl-epoxyketone, and N-benzyloxycarbonyl-glycyl-phenylalanyl-epoxyketone.

The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications may be made in the steps set forth without departing from the spirit of the invention.

Examples

Example 1

The following liquid, lyophilized or liquid formulations reconstituted from lyophilized form were prepared:

15mg/mL of HuMab < CD20>,

0.01% w/v polysorbate 20,

20mM of L-histidine, and

140mM of sodium chloride, in the form of sodium chloride,

pH 6.0；

10mg/mL of HuMab < CD20>,

0.01% w/v polysorbate 20,

20mM of L-histidine, and

140mM of sodium chloride, in the form of sodium chloride,

pH 6.0；

15mg/mL of HuMab < CD20>,

20mM of L-histidine, in the form of a peptide,

pH 6.0；

10mg/mL of HuMab < CD20>,

0.02% w/v polysorbate 20,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

25mg/mL of HuMab < CD20>,

0.02% w/v polysorbate 20,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0。

a lyophilized form is also prepared which, upon reconstitution with an appropriate amount of water for injection, comprises:

10mg/mL of HuMab < CD20>,

0.02% w/v polysorbate 20,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

the formulations exhibit good stability when stored at 2-8 ℃ and 25 ℃, with sufficient stability for physical end points (e.g. aggregation) and chemical end points (e.g. cleavage).

Liquid and lyophilized pharmaceutical product formulations for parenteral administration according to the invention were developed as follows:

preparation of liquid formulations

Formulations of HuMab < CD20> were prepared by homogenization of a solution of HuMab < CD20> in a preparation buffer (e.g. 20mM histidine buffer at a pH of about 6.0 or 20mM histidine buffer at a pH of about 6.0 containing 140mM sodium chloride and 0.01% (w/v) polysorbate 20). Formulations of HuMab < CD20> may also be prepared by adjusting the protein concentration to the desired concentration by dilution with a buffer. Excipients for stabilizing the protein and for tonicity adjustment are added as needed and may be added in dissolved form or alternatively as a solid. Surfactants are added to the formulation as a stock solution as needed. All formulations were sterile filtered through a 0.22 μm filter, then aseptically aliquoted into sterile glass bottles and sealed with rubber stoppers and aluminum crimp caps (alucrimp). These formulations were stored at different temperatures for different time periods and were removed for analysis at the time points indicated in the individual paragraphs. The formulations were analyzed 1) by UV spectrophotometry, 2) by Size Exclusion Chromatography (SEC), 3) by visible and sub-visible particles, 4) by Ion Exchange Chromatography (IEC) and 5) by turbidity of the solution as follows.

Preparation of lyophilized preparation

A solution of HuMab < CD20> was prepared as described above for the liquid formulation, or by homogenisation of a HuMab < CD20> 20mM histidine in HuMab < CD20> solution at a pH of approximately 6.0, which contained sugar and surfactant. All formulations were sterile filtered through a 0.22 μm filter and then aseptically aliquoted into sterile glass bottles. The vials were partially sealed with rubber stoppers suitable for the lyophilization process and then transferred to the drying chamber of the freeze dryer. Any lyophilization method known in the art is intended to be included within the scope of the present invention. For example, the lyophilization process employed in this study included cooling the formulation from room temperature to about 5 ℃ (pre-cooling), followed by freezing at-40 ℃ at a cooling rate of about 1 ℃/minute to 5 ℃/minute (freeze I). The first drying step may be performed at a ramp rate of 0.3 to 0.5 deg.c/minute from-40 deg.c to-30 deg.c and then held at-30 deg.c for at least 50 hours at a chamber pressure of about 75 to 80 millitorr. The second drying step may be performed at a ramp rate of 0.1 to 0.3 deg.c/minute from-30 deg.c to 25 deg.c and then held at 25 deg.c for at least 5 hours at a chamber pressure of about 50 to 80 millitorr (the drying protocol used is shown in table 1). It was found that the HuMab < CD20> formulation dried using the described lyophilization process conveniently had a rapid reconstitution time of about 2-3 minutes. According to the Karl-Fischer method (Karl-Fischer method), the residual moisture content of all the lyophilised cakes in this study was about 0.1 to 1.0%. Freeze-dried vials were stored at different temperatures for different time intervals. The lyophilized formulations were reconstituted with respective volumes of water for injection (WFI) and then 1) analyzed by UV spectrophotometry, 2) the reconstitution time was determined, 3) analyzed by Size Exclusion Chromatography (SEC), 4) the sub-visible and visible particles were determined by Ion Exchange Chromatography (IEC), 5) the turbidity through the solution was determined.

Size Exclusion Chromatography (SEC) was performed to detect high molecular weight species (aggregates) and low molecular weight hydrolysis products in the formulation. The method uses a suitable HPLC apparatus equipped with a UV detector (detection wavelength: 280nm) and a Zorbax GF-250 column (9.4x250mm, Agilent); the method employs 200mM sodium phosphate pH 7.0 as the mobile phase.

Ion Exchange Chromatography (IEC) was performed to detect chemical degradation products that alter the net charge of HuMab < CD20> in the formulation. The method uses a suitable HPLC apparatus equipped with a UV detector (detection wavelengths of 220 and 280nm) and a Dionex ProPac WCX-10 column (4mmx250 mm). As mobile phases A and B, 10mM sodium phosphate buffer in water at pH 6.0 and 10mM sodium phosphate buffer at pH 6.0 +0.75M NaCl were used, respectively, at a flow rate of 1.0 mL/min.

UV spectrophotometry for determining protein concentration was performed at 280nm on an arian Cary Bio UV spectrophotometer.

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

The samples were analyzed for sub-visible particles by using a HIAC Royco PharmaSpec (HRLD-150) and for visible particles by using a Seidenader V90-T visual detector.

TABLE 1 Freeze-drying cycle

Step (ii) of	Storage temperature (. degree.C.)	Rate of temperature rise (. degree. C./minute)	Retention time (minutes)	Vacuum setpoint (mTorr)
					Pre-cooling	5℃	0.0	60	-
Freezing I	-40℃	1.0	120	-
					Primary drying	-30℃	0.5	3720	80
Secondary drying	+25℃	0.2	300	80

Liquid HuMab according to the invention<CD20>Stability data for pharmaceutical product formulations

Formulation 15mg/mL HuMab < CD20>, 20mM L-histidine, pH 6.0,

n/d: not determined

And (4) qualification: substantially free of visible particles, with a maximum of 6000 particles ≥ 10 μm/vessel and a maximum of 600 particles ≥ 25 μm/vessel

Formulation 10mg/mL HuMab < CD20>, 20mM L-histidine, 240mM trehalose, 0.02% w/v polysorbate 20, pH 6.0,

n/d: not determined

Lyophilized HuMab according to the invention<CD20>Stability data for pharmaceutical product formulations

n/d: not determined

Example 2

The following liquid, lyophilized or liquid reconstituted from lyophilized form formulations were prepared:

15mg/mL of HuMab < CD20>,

0.02% w/v poloxamer 188TM,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

25mg/mL of HuMab < CD20>,

0.01% w/v poloxamer 188^TM，

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

25mg/mL of HuMab < CD20>,

0.1% w/v poloxamer 188^TM，

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

25mg/mL of HuMab < CD20>,

0.02% w/v polysorbate 80,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

25mg/mL of HuMab < CD20>,

0.1% w/v polysorbate 80,

20mM of acetate, and

a concentration of 240mM of trehalose in the sample,

pH 5.5；

or

25mg/mL of HuMab < CD20>,

0.1% w/v polysorbate 80,

20mM of acetate, and

140mM of sodium chloride, in the form of sodium chloride,

pH 5.5；

or

30mg/mL of HuMab < CD20>,

0.01% w/v poloxamer 188^TM，

20mM of L-histidine, and

200mM of trehalose in a medium containing a sugar-free polysaccharide,

pH 6.5；

liquid and lyophilized pharmaceutical product formulations for parenteral administration are prepared as follows:

preparation of liquid formulations

By mixing HuMab<CD20>In a preparation buffer (e.g., containing 240mM trehalose and 0.02% (w/v) Poloxamer 188)^TM20mM histidine buffer at a pH of about 6.0) to prepare HuMab<CD20>The formulation of (1). It is also possible to filter approximately 10-40mg/ml HuMab by tangential flow filtration<CD20>Preparation of HuMab by diafiltration of the solution in preparation buffer (e.g., 20mM histidine buffer at pH about 6.0) to increase the protein concentration above the target protein concentration and exchange of the buffer<CD20>The formulation of (1). HuMab may also be prepared by adjusting the protein concentration to the desired concentration by dilution with a buffer<CD20>The formulation of (1). Excipients for stabilizing proteins and for tonicity adjustment may be added in dissolved form or alternatively as solids. A surfactant is added to the preparation as a stock solution as needed. All formulations were sterile filtered through a 0.22 μm filter, then aseptically aliquoted into sterile glass vials and sealed with rubber stoppers and aluminum jaw caps. These formulations were stored at different temperatures for different time periods and were removed for analysis at the time points indicated in the individual paragraphs. The formulations were analyzed 1) by UV spectrophotometry, 2) by Size Exclusion Chromatography (SEC), 3) visible and sub-visible particles, 4) by Ion Exchange Chromatography (IEC) and 5) by turbidity of the solution as follows.

Preparation of lyophilized preparation

A solution of < CD20> was prepared as described above for the liquid formulation. All formulations were sterile filtered through a 0.22 μm filter and then aseptically aliquoted into sterile glass bottles. The vials were partially sealed with rubber stoppers suitable for the lyophilization process and then transferred to the drying chamber of the freeze dryer. Any lyophilization method known in the art is intended to be included within the scope of the present invention. For example, the lyophilization process employed in this study included cooling the formulation from room temperature to about 5 ℃ (pre-cooling), followed by freezing at-40 ℃ at a cooling rate of about 1 ℃/minute to 5 ℃/minute (freeze I). The first drying step may be performed at a ramp rate of 0.3 to 0.5 deg.c/minute from-40 deg.c to-30 deg.c and then held at-30 deg.c for at least 50 hours at a chamber pressure of about 75 to 80 millitorr. The second drying step may be performed at a ramp rate of 0.1 to 0.3 deg.c/minute from-30 deg.c to 25 deg.c and then held at 25 deg.c for at least 5 hours at a chamber pressure of about 50 to 80 millitorr (the drying protocol used is shown in table 1). The residual moisture content of the HuMab < CD20> formulation dried using the described lyophilization process was found to be about 0.1 to 1.0% according to the karl-fischer method. Freeze-dried vials were stored at different temperatures for different time intervals. The lyophilized formulation was reconstituted with the corresponding volume of water for injection (WFI) and then 1) analyzed by UV spectrophotometry, 2) analyzed by Size Exclusion Chromatography (SEC), 3) analyzed by Ion Exchange Chromatography (IEC), 4) the sub-visible and visible particles were determined and 5) analyzed by turbidity of the solution.

Size Exclusion Chromatography (SEC) was performed to detect high molecular weight species (aggregates) and low molecular weight hydrolysis products in the formulation. The method uses a suitable HPLC apparatus equipped with a UV detector (detection wavelength 280nm) and a Zorbax GF-250 column (9.4x250mm, agilent) or TSKgel G3000SWXL (7.8x300 mm); the process employs 200mM sodium phosphate pH 7.0 or 250mM potassium chloride in 200mM potassium phosphate pH 7.0 as the mobile phase.

UV spectrophotometry for determining protein concentration was performed at 280nm on an arian Cary Bio or Perkin Elmer UV spectrophotometer.

The samples were analyzed for sub-visible particles by using a HIAC Royco PharmaSpec (HRLD-150) and for visible particles by using a Seideneader V90-T visual detector.

TABLE 1 Freeze-drying cycle

Liquid HuMab<CD20>Stability data for pharmaceutical product formulations

Formulation 25mg/mL HuMab<CD20>20mM L-histidine, 240mM trehalose, 0.02% w/v poloxamer 188^TM、pH 6.0，

Storage conditions	Storage time (moon)	Protein (mg/mL)	SEC monomer (%)	SECHMW(％)	Turbidity of water	IEC Main Peak (%)	Visible particles	Sub-visible particles
									Initially, the process is started		25.7	98.7	1.3	6.4	61.6	Qualified	Qualified
2-8℃	1	25.6	98.7	1.3	6.0	62.0	Qualified	Qualified
										3	26.1	98.6	1.3	5.9	61.0	Qualified	Qualified
25℃	1	25.7	98.5	1.4	6.5	62.0	Qualified	Qualified
										3	26.0	97.9	1.5	5.9	58.5	Qualified	Qualified
40℃	1	25.7	97.9	1.6	6.6	52.6	Qualified	Qualified
										3	25.7	91.0	2.4	6.7	33.6	Qualified	Qualified
-80℃	3	25.6	98.7	1.3	6.2	60.9	Qualified	Qualified
									-20℃	3	25.5	98.7	1.3	6.3	60.8	Qualified	Qualified

Formulation 25mg/mL HuMab<CD20>20mM L-histidine, 240mM trehalose, 0.01% w/v Poloxamer 188^TM、pH 6.0，

n/a: not analyzed

Formulation 25mg/mL HuMab<CD20>20mM L-histidine, 240mM trehalose, 0.1% w/v Poloxamer 188^TM、pH 6.0，

n/a: not analyzed

Formulation 25mg/mL HuMab < CD20>, 20mM acetate, 240mM trehalose, 0.1% w/v polysorbate 80, pH 5.5,

storage conditions	Storage time (moon)	Protein (mg/mL)	SEC monomer (%)	SECHMW(％)	Turbidity of water	IEC Main Peak (%)	Visible particles	Sub-visible particles
									Initially, the process is started		24.6	99.6	0.4	8.2	71.9	Qualified	Qualified
2-8℃	1	24.8	99.7	0.3	4.9	n/a	Qualified	Qualified
										2	24.5	99.4	0.6	5.2	72.6	Qualified	Qualified
	3	24.3	99.4	0.6	7.0	66.2	Qualified	Qualified
									25℃	1	n/a	99.6	0.4	6.7	n/a	Qualified	Qualified

	2	n/a	99.3	0.7	7.0	71.9	Qualified	Qualified
									3	n/a	99.2	0.8	7.3	65.1	Qualified	Qualified
40℃	1	n/a	97.5	1.0	26.8	52.9	Qualified	Qualified
									2	n/a	96.0	2.1	27.8	42.1	Qualified	Qualified
	3	n/a	94.4	3.2	26.8	n/a	Qualified	Qualified

n/a: not analyzed

Formulation 25mg/mL HuMab < CD20>, 20mM acetate, 140mM sodium chloride, 0.1% w/v polysorbate 80, pH 5.5,

storage conditions	Storage time (moon)	Protein (mg/mL)	SEC monomer (%)	SECHMW(％)	Turbidity of water	IEC Main Peak (%)	Visible particles	Sub-visible particles
									Initially, the process is started		23.7	99.5	0.5	11.4	70.9	Qualified	Qualified
2-8℃	1	24.4	99.6	0.4	11.4	n/a	Qualified	Qualified
										2	24.2	99.4	0.6	10.5	71.5	Qualified	Qualified
	3	24.2	99.3	0.7	12.5	71.3	Qualified	Qualified
									25℃	1	n/a	99.5	0.5	12.0	n/a	Qualified	Qualified
	2	n/a	99.1	0.9	013.0	68.2	Qualified	Qualified
										3	n/a	99.1	0.9	13.2	64.6	Qualified	Qualified
40℃	1	n/a	96.9	1.2	26.8	54.0	Qualified	Qualified
										2	n/a	95.0	2.9	26.5	46.1	Qualified	Qualified
	3	n/a	93.1	4.2	26.1	n/a	Qualified	Qualified

n/a: not analyzed

Formulation 30mg/mL HuMab<CD20>20mM L-histidine, 240mM trehalose, 0.01% w/v Poloxamer 188^TM、pH 6.5，

Storage conditions	Storage time (moon)	Protein (mg/mL)	SEC monomer (%)	SECHMW(％)	Turbidity of water	IEC main peak(％)	Visible particles	Sub-visible particles
									Initially, the process is started		32.0	98.4	1.6	6.4	61.0	Qualified	Qualified
2-8℃	1	32.2	98.3	1.6	6.6	62.3	Qualified	Qualified
										3	32.9	98.1	1.7	5.7	60.5	Qualified	Qualified
25℃	1	32.2	98.1	1.8	6.2	60.9	Qualified	Qualified
										3	32.1	97.6	2.0	6.2	56.5	Qualified	Qualified
40℃	1	32.0	97.3	2.1	6.3	50.9	Qualified	Qualified
										3	32.3	89.9	3.0	7.2	31.1	Qualified	Qualified
-80℃	3	31.8	98.3	1.6	6.2	61.0	Qualified	Qualified
									-20℃	3	32.1	98.3	1.6	6.6	60.7	Qualified	Qualified

Lyophilized HuMab<CD20>Stability data for pharmaceutical product formulations

Formulation 25mg/mL HuMab<CD20>20mM L-histidine, 240mM trehalose, 0.02% w/v poloxamer 188^TM，

Storage conditions	Storage time (moon)	Protein (mg/mL)	SEC monomer (%)	SECHMW(％)	Turbidity of water	IEC Main Peak (%)	Visible particles	Sub-visible particles
									Initially, the process is started		25.5	98.9	1.0	6.2	61.2	Qualified	Qualified
2-8℃	1	25.2	99.0	1.0	5.8	63.1	Qualified	Qualified
										3	25.3	99.0	1.0	6.1	60.4	Qualified	Qualified
25℃	1	25.4	98.9	1.0	6.0	62.9	Qualified	Qualified

	3	25.6	98.5	1.1	6.1	60.2	Qualified	Qualified
									40℃	1	25.3	98.9	1.1	6.3	60.6	Qualified	Qualified
	3	25.9	98.5	1.3	5.9	56.9	Qualified	Qualified
									-80℃	3	25.3	98.9	1.0	6.3	61.2	Qualified	Qualified
-20℃	3	25.4	98.9	1.0	6.7	60.6	Qualified	Qualified

Formulation 25mg/mL HuMab < CD20>, 20mM L-histidine, 240mM trehalose, 0.02% w/v polysorbate 80, pH 6.0,

n/a: not analyzed

Claims

1. A formulation, comprising:

about 1 to about 150mg/mL of a type II anti-CD 20 antibody,

-optionally about 0.001 to about 1% w/v of at least one surfactant, and

-from about 1 to about 100mM of a buffer,

-a pH of about 4.5 to about 7.0.

2. The formulation of claim 1, comprising:

about 1 to about 150mg/mL of a type II anti-CD 20 antibody,

-from about 0.005 to about 0.05% w/v of at least one surfactant, and

-from about 1 to about 100mM of a buffer,

-a pH of about 4.5 to about 7.0.

3. The formulation of claim 1 or 2, comprising:

about 10 to about 30mg/mL of a type II anti-CD 20 antibody,

-20mM of L-histidine,

240mM of trehalose, and

-0.02% w/v polysorbate 20,

-a pH of about 6.

4. The formulation of claim 1 or 2, comprising:

about 10 to about 30mg/mL of a type II anti-CD 20 antibody,

-0.02% w/v poloxamer 188,

-20mM L-histidine, and

-240mM of trehalose,

-a pH of about 6.

5. The formulation according to claims 1 to 4, which is in liquid form, lyophilized form or a liquid form reconstituted from a lyophilized form.

6. The formulation according to any one of claims 1 to 5, which can be administered by intravenous (i.v.) or subcutaneous (s.c.) or any other parenteral administration.

7. The formulation of any one of claims 1 to 6, further comprising one or more isotonic agents in an amount of about 5mM to about 350 mM.

8. The formulation of any one of claims 1 to 7, wherein the isotonic agent is selected from the group consisting of sodium chloride (NaC1), potassium chloride, sugars including glucose, glycerol, amino acids, and combinations thereof.

9. The formulation of any one of claims 1 to 8, further comprising a sugar in an amount of about 25mM to about 500 mM.

10. The formulation according to any one of claims 1 to 9, wherein the sugar is selected from trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucamine ("meglumine"), galactosamine, neuraminic acid.

11. The formulation of any one of claims 1 to 10, further comprising one or more pharmaceutically acceptable ingredients selected from the group consisting of: antioxidants, ascorbic acid, glutathione, preservatives, in particular m-cresol, phenol, benzyl alcohol, methyl paraben, propyl paraben, chlorobutanol, thimerosal, benzalkonium chloride, polyethylene glycols, in particular PEG 3000, 3350, 4000 or 6000, Human Serum Albumin (HSA), Bovine Serum Albumin (BSA), polyols, glycerol, ethanol, mannitol, salts, acetates, in particular sodium acetate, magnesium chloride, calcium chloride, trometamol, EDTA, in particular Na-EDTA.

12. The formulation of any one of claims 1 to 11, wherein the formulation comprises:

15mg/mL of HuMab < CD20>,

20mM of L-histidine, in the form of a peptide,

optionally 0.001 to 1% w/v of a surfactant,

pH 6.0；

or

HuMab < CD20> 10mg/mL,

0.02% w/v polysorbate 20,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

25mg/mL HuMab < CD20>,

0.02% w/v polysorbate 20,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0。

13. the formulation of any one of claims 1 to 12, wherein the formulation is in lyophilized form and comprises:

HuMab < CD20> 10mg/mL,

0.02% w/v polysorbate 20,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0。

14. the formulation of any one of claims 1 to 11, wherein the formulation comprises:

25mg/mL HuMab < CD20>,

0.02% w/v poloxamer 188^TM，

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

25mg/mL HuMab < CD20>,

0.01% w/v poloxamer 188^TM，

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

25mg/mL HuMab < CD20>,

0.1% w/v poloxamer 188^TM，

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

25mg/mL HuMab < CD20>,

0.02% w/v polysorbate 80,

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0；

or

25mg/mL HuMab < CD20>,

0.1% w/v polysorbate 80,

20mM of acetate, and

a concentration of 240mM of trehalose in the sample,

pH 5.5；

or

25mg/mL HuMab < CD20>,

0.1% w/v polysorbate 80,

20mM of acetate, and

140mM of sodium chloride, in the form of sodium chloride,

pH 5.5；

or

30mg/mL HuMab < CD20>,

0.01% w/v poloxamer 188^TM，

20mM of L-histidine, and

200mM of trehalose in a medium containing a sugar-free polysaccharide,

pH 6.5。

15. the formulation of claim 14, wherein the formulation is in liquid form and comprises:

25mg/mL HuMab < CD20>,

0.02% w/v poloxamer 188^TM，

20mM of L-histidine, and

a concentration of 240mM of trehalose in the sample,

pH 6.0。

16. use of a formulation according to any one of claims 1 to 15 for the manufacture of a medicament useful for the treatment of CD 20-related diseases.

17. The formulation of claim 16, wherein the disease is selected from breast cancer, colorectal cancer, non-small cell lung cancer (NSCLC), and prostate cancer.

18. The invention as hereinbefore described.