U.S. patent application number 12/879486 was filed with the patent office on 2011-03-31 for highly concentrated pharmaceutical formulations.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Michael Adler, Hanns-Christian Mahler, Oliver Boris Stauch.
Application Number | 20110076273 12/879486 |
Document ID | / |
Family ID | 43732866 |
Filed Date | 2011-03-31 |
United States Patent
Application |
20110076273 |
Kind Code |
A1 |
Adler; Michael ; et
al. |
March 31, 2011 |
Highly Concentrated Pharmaceutical Formulations
Abstract
The present invention relates to a highly concentrated, stable
pharmaceutical formulation of a pharmaceutically active anti-CD20
antibody, such as e.g. Rituximab, Ocrelizumab, or
HuMab<CD20>, or a mixture of such antibody molecules for
subcutaneous injection. In particular, the present invention
relates to formulations comprising, in addition to a suitable
amount of the anti-CD20 antibody, an effective amount of at least
one hyaluronidase enzyme as a combined formulation or for use in
form of a co-formulation. The said formulations comprise
additionally at least one buffering agent, such as e.g. a histidine
buffer, a stabilizer or a mixture of two or more stabilizers (e.g.
a saccharide, such as e.g. .alpha.,.alpha.-trehalose dihydrate or
sucrose, and optionally methionine as a second stabilizer), a
nonionic surfactant and an effective amount of at least one
hyaluronidase enzyme. Methods for preparing such formulations and
their uses thereof are also provided.
Inventors: |
Adler; Michael; (Riehen,
CH) ; Mahler; Hanns-Christian; (Basel, CH) ;
Stauch; Oliver Boris; (Freiburg, DE) |
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
43732866 |
Appl. No.: |
12/879486 |
Filed: |
September 10, 2010 |
Current U.S.
Class: |
424/133.1 ;
424/173.1 |
Current CPC
Class: |
A61K 47/10 20130101;
A61P 37/02 20180101; A61K 47/22 20130101; C07K 2317/24 20130101;
A61K 9/0019 20130101; C07K 2317/94 20130101; A61K 47/42 20130101;
C07K 2317/734 20130101; A61K 39/3955 20130101; A61K 45/06 20130101;
A61K 2039/505 20130101; A61K 38/47 20130101; A61K 9/19 20130101;
A61P 43/00 20180101; C07K 2317/21 20130101; A61K 47/26 20130101;
A61K 47/20 20130101; A61K 47/183 20130101; C07K 16/2887 20130101;
C07K 16/3061 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/133.1 ;
424/173.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 11, 2009 |
EP |
09170110.2 |
Claims
1. A highly concentrated, stable pharmaceutical formulation of a
pharmaceutically active anti-CD20 antibody comprising: a. about 50
to 350 mg/ml anti-CD20 antibody; b. about 1 to 100 mM of a
buffering agent providing a pH of 5.5.+-.2.0; c. about 1 to 500 mM
of a stabilizer or a mixture of two or more stabilizers; d. about
0.01 to 0.1% of a nonionic surfactant; and e. optionally an
effective amount of at least one hyaluronidase enzyme.
2. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the anti-CD20 antibody
concentration is 100 to 150 mg/ml, preferably 120.+-.20 mg/ml.
3. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, comprising about 1,000 to 16,000
U/ml of a hyaluronidase enzyme, preferably about 2,000 U/ml or
12,000 U/ml.
4. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the buffering agent is at
a concentration of 1 to 50 mM.
5. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the buffering agent
provides a pH of 5.5 to 6.5, preferably selected from the group
consisting of 5.3, 5.5, 6.0, 6.1 and 6.5.
6. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the buffering agent is a
histidine buffer.
7. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the stabilizer is a
saccharide, such as e.g. .alpha.,.alpha.-trehalose dihydrate or
sucrose.
8. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the stabilizer is at a
concentration of 15 to 250 mM.
9. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 7, wherein methionine is used as a
second stabilizer.
10. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 9, wherein methionine is present in
a concentration of 5 to 25 mM.
11. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the nonionic surfactant
is a polysorbate, preferably selected from the group consisting of
polysorbate 20, polysorbate 80, and polyethylene-polypropylene
copolymer.
12. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 11, wherein the concentration of the
polysorbate is 0.02% (w/v) to 0.08% (w/v).
13. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the hyaluronidase enzyme
is rHuPH20.
14. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the anti-CD20 antibody is
Rituximab.
15. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the anti-CD20 antibody is
Ocrelizumab.
16. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 15, wherein the anti-CD20 antibody
is 2H7.v16.
17. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, wherein the anti-CD20 antibody is
HuMab<CD20>.
18. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, which is stable upon freezing and
thawing.
19. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1, which is for subcutaneous or
intramuscular administration.
20. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1 in liquid form.
21. A highly concentrated, stable pharmaceutical anti-CD20 antibody
formulation according to claim 1 in lyophilized form.
22. A method of treating a disease or disorder which is amenable to
treatment with an anti-CD20 antibody, such as cancer or a
non-malignant disease, in a subject comprising administering the
formulation of claim 1 to a subject in an amount effective to treat
the said disease or disorder.
23. The method according to claim 22 wherein the anti-CD20 antibody
comprises Rituximab.
24. The method according to claim 23 wherein the anti-CD20 antibody
is administered subcutaneously at a fixed dose of about 1400
mg.
25. The method according to claim 24 wherein the fixed dose is
administered approximately every 2 months or approximately every 3
months.
26. The method according to claim 24 wherein the disease or
disorder is non-Hodgkin's lymphoma.
27. The method according to claim 22 wherein the formulation is
co-administered concomitantly or sequentially with
chemotherapy.
28. The method according to claim 27 wherein the chemotherapy is
selected from the group consisting of CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) and CVP
(cyclophosphamide, vincristine, and predisone).
29. A method of treating non-Hodkin's lymphoma (NHL) in a human
patient comprising administering a formulation comprising Rituximab
subcutaneously to the subject, wherein the Rituximab is
administered at a fixed dose of 1400 mg thereof.
30. The method according to claim 29 wherein the formulation
comprising rituximab is administered approximately every 2 months
or approximately every 3 months.
31. The method according to claim 30 further comprising
administering chemotherapy to the patient.
32. The method according to claim 31 wherein the chemotherapy is
selected from the group consisting of CHOP (cyclophosphamide,
doxorubicin, vincristine, and prednisone) and CVP
(cyclophosphamide, vincristine, and predisone).
33. The method according to claim 29 wherein the formulation
comprises about 50 to 350 mg/ml rituximab and at least one
hyaluronidase enzyme.
Description
[0001] The present invention relates to highly concentrated, stable
pharmaceutical formulations of a pharmaceutically active anti-CD20
antibody or a mixture of such antibody molecules for subcutaneous
injection. Such formulations comprise, in addition to the high
amounts of anti-CD20 antibody or mixture thereof, a buffering
agent, a stabilizer or a mixture of two ore more stabilizing
agents, a nonionic surfactant and an effective amount of at least
one hyaluronidase enzyme. The invention also relates to a process
for the preparation of the formulation and to the uses of such
formulation.
BACKGROUND OF THE INVENTION
[0002] The pharmaceutical use of antibodies has increased over the
past years. In many instances such antibodies are either injected
or infused via the intravenous (IV) route. Unfortunately the amount
of antibody that can be administered via the intravenous route is
limited by the physico-chemical properties of the antibody, in
particularly by its solubility and stability in a suitable liquid
formulation and by the volume of the infusion fluid. Alternative
administration pathways are subcutaneous or intramuscular
injection. These injection pathways require high protein
concentration in the final solution to be injected (Shire et al.,
"Challenges in the development of high protein concentration
formulations", J. Pharm. Sci. 2004; 93(6): 1390-1402; Roskos 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 it has been proposed to use
glycosaminoglycanase enzyme(s) in order to increase the
interstitial space into which the antibody formulation can be
injected (WO2006/091871).
[0003] Examples of stable formulations of pharmaceutically active
antibodies for therapeutic use currently on the market are as
follows:
[0004] RITUXAN.RTM./MABTHERA.RTM. (Rituximab) is a chimeric
antibody which 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 (10 mL) in either 100 mg
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 for Rituximab suitable
for IV administration is disclosed in U.S. Pat. No. 6,991,790.
[0005] HERCEPTIN.RTM. (Trastuzumab) is a monoclonal antibody
directed against the HER2 receptor (anti-HER2) which is currently
marketed in Europe in form of a 150 mg lyophilized powder
(containing the antibody, .alpha.,.alpha.-trehalose dihydrate,
L-histidine and L-histidine hydrochloride and polysorbate 20) which
should be reconstituted for infusions with water for injection to
yield injection dose of approximately 21 mg/ml. In the USA and many
other countries a multiple dosage vial containing 440 mg
Trastuzumab is marketed.
[0006] AVASTIN.RTM. (Bevacizumab) is a monoclonal antibody directed
against the vascular endothelial growth factor (VEGF) which is
currently marketed in Europe as a liquid formulation in two types
of 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.
[0007] While the above antibody formulations have been found
suitable for use for intravenous administration there is a desire
to provide highly concentrated, stable pharmaceutical formulations
of therapeutically active antibodies for subcutaneous injection.
The advantage of subcutaneous injections is that it allows the
medical practitioner to perform it in a rather short intervention
with the patient. Moreover the patient can be trained to perform
the subcutaneous injection by himself. Usually injections via the
subcutaneous route are limited to approximately 2 ml. For patients
requiring multiple doses, several unit dose formulations can be
injected at multiple sites of the body surface.
[0008] The following two antibody products for subcutaneous
administration are already on the market.
[0009] HUMIRA.RTM. (Adalimumab) is a monoclonal antibody directed
against tumor necrosis factor alpha (TNF alpha) which is currently
marketed in Europe in form of a 40 mg dose in 0.8 ml injection
volume for subcutaneous application (concentration: 50 mg
antibody/ml injection volume).
[0010] XOLAIR.RTM. (Omalizumab) a monoclonal antibody directed
against immunoglobulin E (anti-IgE) which is currently marketed in
form of a 150 mg lyophilized powder (containing the antibody,
sucrose, histidine and histidine hydrochloride monohydrate and
polysorbate 20) which should be reconstituted with water for
subcutaneous injection to yield a 125 mg/ml injection dose.
[0011] No highly concentrated, stable pharmaceutical anti-CD20
antibody formulation suitable for subcutaneous administration is
currently available on the market. There is therefore a desire to
provide such highly concentrated, stable pharmaceutical
formulations of therapeutically active antibodies for subcutaneous
injection.
[0012] The injection of parenteral drugs into the hypodermis is
generally limited to volumes of less than 2 ml due to this
viscoelastic resistance to hydraulic conductance in the
subcutaneous (SC) tissue and generated backpressure upon injection
(Aukland K. and Reed R., "Interstitial-Lymphatic Mechanisms in the
control of Extracellular Fluid Volume", Physiology Reviews",
73:1-78 (1993)) as well as due to the perceptions of pain.
[0013] The preparation of high concentration protein formulations
is very challenging and there is a need to adapt each formulation
to the particular proteins used because each protein has a
different aggregation behavior. Aggregates are suspected to cause
immunogenicity of therapeutic proteins in at least some of the
cases. Immunogenic reaction against protein or antibody aggregates
may lead to neutralizing antibodies which may render the
therapeutic protein or antibody ineffective. It appears that the
immunogenicity of protein aggregates is most problematic in
connection with subcutaneous injections, whereby repeated
administration increases the risk of an immune response.
[0014] While antibodies have a very similar overall structure, such
antibodies differ in the amino acid composition (in particular in
the CDR regions responsible for the binding to the antigen) and the
glycosylation pattern. Moreover there may additionally be
post-translational modifications such as charge and glycosylation
variants.
SUMMARY OF THE INVENTION
[0015] The present invention provides a highly concentrated, stable
pharmaceutical formulation of a pharmaceutically active anti-CD20
antibody or a mixture of such antibody molecules, preferably for
subcutaneous injection.
[0016] More particularly the highly concentrated, stable
pharmaceutical formulation of a pharmaceutically active anti-CD20
antibody formulation of the present invention comprises: [0017]
about 20 to 350 mg/ml anti-CD20 antibody; [0018] about 1 to 100 mM
of a buffering agent providing a pH of 5.5.+-.2.0; [0019] about 1
to 500 mM of a stabilizer or a mixture of two or more stabilizers,
whereby optionally methionine is used as a secondary stabilizer,
preferably in a concentration of 5 to 25 mM; [0020] 0.01 to 0.1% of
a nonionic surfactant; and [0021] Preferably an effective amount of
at least one hyaluronidase enzyme.
DETAILED DESCRIPTION OF THE INVENTION
[0022] The term "antibody" herein is used in the broadest sense and
specifically covers full length antibodies, genetically engineered
antibodies like monoclonal antibodies, or recombinant antibodies,
polyclonal antibodies, multispecific antibodies (e.g. bispecific
antibodies) formed from at least two full length antibodies,
chimeric antibodies, humanized antibodies, fully human antibodies,
and as well as fragments of such antibodies as long as they exhibit
the desired biological activity.
[0023] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variants that may arise during production of the
monoclonal antibody, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations that
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they are uncontaminated by other immunoglobulins. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al, Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567). The "monoclonal antibodies" may 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). The terms "monoclonal
antibody" or "monoclonal antibody composition" as used herein refer
to a preparation of antibody molecules of a single amino acid
composition. Accordingly, the term "human monoclonal antibody"
refers to antibodies displaying a single binding specificity which
have variable and constant regions derived from human germline
immunoglobulin sequences. In one embodiment, the human monoclonal
antibodies are produced by a hybridoma which includes 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 light human chain transgene fused to an immortalized cell. The
term "monoclonal antibodies" herein specifically include the
so-called chimeric antibodies in which a portion of the heavy
and/or light chain is identical with or homologous to corresponding
sequences in antibodies derived from a particular species or
belonging to a particular antibody class or subclass, while the
remainder of the chain(s) is identical with or homologous to
corresponding sequences in antibodies derived from another species
or belonging to another antibody class or subclass, as well as
fragments of such antibodies, so long as they exhibit the desired
biological activity (U.S. Pat. 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
comprising variable domain antigen-binding sequences derived from a
non-human primate (e.g. Old World Monkey, Ape etc) and human
constant region sequences.
[0024] "Antibody fragments" comprise a portion of a full length
antibody, generally at least the antigen binding portion or the
variable region thereof. Examples of antibody fragments include
Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies, single-chain
antibody molecules, immunotoxins, and multispecific antibodies
formed from antibody fragments. In addition, antibody fragments
comprise single chain polypeptides having the characteristics of a
VH chain, namely being able to assemble together with a VL chain
binding to the CD20 antigen. "Antibody fragments" also comprise
such fragments which per se are not able to provide effector
functions (ADCC/CDC) but provide this function in a manner
according to the invention after being combined with appropriate
antibody constant domain(s).
[0025] A "full length antibody" is one which comprises an
antigen-binding variable region as well as a light chain constant
domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The
constant domains may be native sequence constant domains (e.g.
human native sequence constant domains) or amino acid sequence
variants thereof. Preferably, the full length antibody has one or
more effector functions.
[0026] An "amino acid sequence variant" antibody herein is an
antibody with an amino acid sequence which differs from a main
species antibody. Ordinarily, amino acid sequence variants will
possess at least about 70% homology with the main species antibody,
and preferably, they will be at least about 80%, more preferably at
least about 90% homologous with the main species antibody. The
amino acid sequence variants possess substitutions, deletions,
and/or additions at certain positions within or adjacent to the
amino acid sequence of the main species antibody. Examples of amino
acid sequence variants herein include acidic variant (e.g.
deamidated antibody variant), basic variant, the antibody with an
amino-terminal leader extension (e.g. VHS--) on one or two light
chains thereof, antibody with a C-terminal lysine residue on one or
two heavy chains thereof, etc, and includes combinations of
variations to the amino acid sequences of heavy and/or light
chains. The antibody variant of particular interest herein is the
antibody comprising an amino-terminal leader extension on one or
two light chains thereof, optionally further comprising other amino
acid sequence and/or glycosylation differences relative to the main
species antibody.
[0027] A "glycosylation variant" antibody herein is an antibody
with one or more carbohydrate moieties attached thereto which
differ from one or more carbohydrate moieties attached to a main
species antibody. Examples of glycosylation variants herein include
antibody with a G1 or G2 oligosaccharide structure, instead a G0
oligosaccharide structure, attached to an Fc region thereof,
antibody with one or two carbohydrate moieties attached to one or
two light chains thereof, antibody with no carbohydrate attached to
one or two heavy chains of the antibody, etc, and combinations of
glycosylation alterations. Moreover the term "glycosylation
variant" includes also glycoengineered antibodies such as those
described in WO 1,331,266 and U.S. Pat. No. 7,517,670.
[0028] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody.
Examples of antibody effector functions include C1q binding;
complement dependent cytotoxicity (CDC); Fc receptor binding;
antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;
down regulation of cell surface receptors (e.g. B cell receptor;
BCR), etc.
[0029] Depending on the amino acid sequence of the constant domain
of their heavy chains, full length antibodies can be assigned to
different "classes". There are five major classes of full length
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that
correspond to the different classes of antibodies are called
.alpha. (alpha), .delta. (delta), .epsilon. (epsilon), .gamma.
(gamma), and .mu. (mu), respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0030] Herein, "biological activity" of a monoclonal antibody
refers to the ability of the antibody to bind to antigen and result
in a measurable biological response which can be measured in vitro
or in vivo. Such activity may be antagonistic (for example where
the antibody is a CD20 antibody) or agonistic.
[0031] The term "humanized antibody" refers to antibodies in which
the framework or "complementarity determining regions" (CDR) have
been modified to comprise the CDR of an immunoglobulin of different
specificity as compared to that of the parent immunoglobulin. In a
preferred embodiment, a murine CDR is grafted into the framework
region of a human antibody to prepare the "humanized antibody."
Particularly preferred CDRs correspond to those representing
sequences recognizing the antigens noted below for chimeric and
bifunctional antibodies. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will 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.
[0032] The term "chimeric antibody" refers to a monoclonal antibody
comprising a variable region, i.e., binding region, from one source
or species and at least a portion of a constant region derived from
a different source or species, usually prepared by recombinant DNA
techniques. Chimeric antibodies comprising a murine variable region
and a human constant region are especially 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 producing chimeric
antibodies involve conventional recombinant DNA and gene
transfection techniques now well known in the art. See, e.g.,
Morrison, S. L., et al., Proc. Natl. Acad. Sci. USA 81 (1984)
6851-6855; U.S. Pat. No. 5,202,238 and U.S. Pat. No. 5,204,244.
[0033] 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 state of 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 against a great variety of
targets can be produced. Examples of human antibodies are for
example described in Kellermann, S. A., et al., Curr Opin
Biotechnol. 13 (2002) 593-597.
[0034] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies isolated from a host cell such as a NS0 or CHO cell or
from an animal (e.g. a mouse) that is transgenic for human
immunoglobulin genes or antibodies expressed using a recombinant
expression vector transfected into a host cell. Such recombinant
human antibodies have variable and constant regions derived from
human germline immunoglobulin sequences in a rearranged form. The
recombinant human antibodies according to the 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, while derived from and related to human
germline VH and VL sequences, may not naturally exist within the
human antibody germline repertoire in vivo.
[0035] As used herein, "specifically binding" or "binds
specifically to" refers to an antibody specifically binding to the
CD20 antigen. Preferably the binding affinity is of Kd value of
10.sup.-9 mol/l or lower (e.g. 10.sup.-10 mol/l), preferably with a
Kd value of 10.sup.-10 mol/l or lower (e.g. 10.sup.-12 mol/l). The
binding affinity is determined with a standard binding assay, such
as surface plasmon resonance technique (BIACORE.RTM.).
[0036] The term "nucleic acid molecule", as used herein, is
intended to include DNA molecules and RNA molecules. A nucleic acid
molecule may be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0037] The "constant domains" are not involved directly in binding
the antibody to an antigen but are involved in the effector
functions (ADCC, complement binding, and CDC).
[0038] The "variable region" (variable region of a light chain
(VL), variable region of a heavy chain (VH)) as used herein denotes
each of the pair of light and heavy chains which is involved
directly in binding the antibody to the antigen. The domains of
variable human light and heavy chains have the same general
structure and each domain comprises four framework (FR) regions
whose sequences are widely conserved, connected by three
"hypervariable regions" (or complementarity determining regions,
CDRs). The framework regions adopt a .beta.-sheet conformation and
the CDRs may form loops connecting the b-sheet structure. The CDRs
in each chain are held in their three-dimensional structure by the
framework regions and form together with the CDRs from the other
chain the 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 invention
and therefore provide a further object of the invention.
[0039] The terms "hypervariable region" or "antigen-binding portion
of an antibody" when used herein refer to the amino acid residues
of an antibody which are responsible for antigen-binding. The
hypervariable region comprises amino acid residues from the
"complementarity determining regions" or "CDRs". "Framework" or
"FR" regions are those variable domain regions other than the
hypervariable region residues as herein defined. Therefore, the
light and heavy chains of an antibody comprise from N- to
C-terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
Especially, CDR3 of the heavy chain is the region which contributes
most to antigen binding. CDR and FR regions are determined
according to the standard definition 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 a "hypervariable loop".
[0040] The terms "CD20" and "CD20 antigen" are used interchangeably
herein, and include any variants, isoforms and species homologs of
human CD20 which are naturally expressed by cells or are expressed
on cells transfected with the CD20 gene. Binding of an antibody of
the invention to the CD20 antigen mediate the killing of cells
expressing CD20 (e.g., a tumor cell) by inactivating CD20. The
killing of the cells expressing CD20 may occur by one or more of
the following mechanisms: antibody dependent cellular cytotoxicity
(ADCC), complement-dependent cytotoxity (CDC), inducing cell death
and/or apoptosis, homotypic aggregation etc.
[0041] Synonyms of CD20, as recognized in the art, include
B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Leu-16,
and Bp35.
[0042] The term "anti-CD20 antibody" according to the invention is
an antibody that binds specifically to CD20 antigen. Depending on
binding properties and biological activities of anti-CD20
antibodies to 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-US-00001 TABLE 1 Properties of Type I and Type II anti-CD20
Antibodies Type I anti-CD20 antibodies Type II anti-CD20 antibodies
Type I CD20 epitope Type II CD20 epitope Localize CD20 to lipid
rafts Do not localize CD20 to lipid rafts Increased CDC (if IgG1
isotype) Decreased 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 Apoptosis induction upon cross-linking Strong cell
death induction without cross-linking
[0043] One essential property of type I and type II anti-CD20
antibody is their mode of binding. Thus type I and type II
anti-CD20 antibody can be classified by the ratio of the binding
capacities to CD20 on Raji cells (ATCC-No. CCL-86) of said
anti-CD20 antibody compared to rituximab.
[0044] As used herein, "anti-CD20 antibody" can be either a type I
or type II antibody. Preferably, it is a type I antibody, most
preferred it is rituximab.
[0045] The type I anti-CD20 antibodies have a ratio of the binding
capacities to CD20 on Raji cells (ATCC No. CCL-86) of said
anti-CD20 antibody compared to rituximab of 0.8 to 1.2, preferably
of 0.9 to 1.1. Examples of such type I anti-CD20 antibodies include
e.g. Rituximab, in U.S. Pat. No. 7,381,560 (Anderson et. al., see
e.g. FIGS. 4 and 5), 1F5 IgG2a (ECACC, hybridoma; Press et al.,
Blood 69/2:584-591 (1987)), H147 IgG3 (ECACC, hybridoma), 2C6 IgG1
(as disclosed in WO2005/103081), 2F2 IgG1 or ofatumumab (as
disclosed and WO 2004/035607 and WO2005/103081) and 2H7 IgG1 (as
disclosed in WO 2004/056312) and WO 2006/084264 (e.g. the variants
disclosed in tables 1 and 2). Preferably said type I anti-CD20
antibody is a monoclonal antibody that binds to the same epitope as
rituximab.
[0046] The type II anti-CD20 antibodies have a ratio of the binding
capacities to CD20 on Raji cells (ATCC No. CCL-86) of said
anti-CD20 antibody compared to Rituximab of 0.3 to 0.6, preferably
of 0.35 to 0.55, more preferably 0.4 to 0.5. Examples of such type
II anti-CD20 antibodies include e.g. tositumomab (B1 IgG2a),
humanized B-Ly1 antibody IgG1 (a chimeric humanized IgG1 antibody
as disclosed in WO2005/044859), 11B8 IgG1 (as disclosed in WO
2004/035607), and AT80 IgG1. Preferably said type II anti-CD20
antibody is a monoclonal antibody that binds to the same epitope as
humanized B-Ly1 antibody (as disclosed in WO2005/044859).
[0047] The "ratio of the binding capacities to CD20 on Raji cells
(ATCC-No. CCL-86) of an anti-CD20 antibodies compared to rituximab"
is determined by direct immunofluorescence measurement (the mean
fluorescent intensities (MFI) is measured) using said anti-CD20
antibody conjugated with Cy5 and rituximab conjugated with Cy5 in a
FACSArray (Becton Dickinson) with Raji cells (ATCC-No. CCL-86), and
calculated as follows:
Ratio of the binding capacities to CD 20 on Raji cells ( ATCC - No
. CCL - 86 ) = MFI ( Cy 5 - anti - CD 20 antibody ) MFI ( Cy 5 -
rituximab ) .times. Cy 5 - labeling ratio ( Cy 5 - rituximab ) Cy5
- labeling ratio ( Cy 5 - anti - CD 20 antibody ) ##EQU00001##
[0048] MFI is the mean fluorescent intensity. The "Cy5-labeling
ratio" as used herein means number of Cy5-label molecules per
molecule antibody.
[0049] Typically said type I anti-CD20 antibody has a ratio of the
binding capacities to CD20 on Raji cells (ATCC-No. CCL-86) of said
first anti-CD20 antibody compared to rituximab of 0.8 to 1.2,
preferably 0.9 to 1.1.
[0050] Typically said type II anti-CD20 antibody has a ratio of the
binding capacities to CD20 on Raji cells (ATCC-No. CCL-86) of said
second anti-CD20 antibody compared to rituximab of 0.3 to 0.6,
preferably 0.35 to 0.55, more preferably 0.4 to 0.5.
[0051] In a preferred embodiment said type II anti-CD20 antibody,
preferably a humanized B-Ly1 antibody, has increased antibody
dependent cellular cytotoxicity (ADCC).
[0052] By "antibody having increased antibody dependent cellular
cytotoxicity (ADCC)" is meant an antibody, as that term is defined
herein, having increased ADCC as determined by any suitable method
known to those of ordinary skill in the art. One accepted in vitro
ADCC assay is as follows:
[0053] 1) the assay uses target cells that are known to express the
target antigen recognized by the antigen-binding region of the
antibody;
[0054] 2) the assay uses human peripheral blood mononuclear cells
(PBMCs), isolated from blood of a randomly chosen healthy donor, as
effector cells;
[0055] 3) the assay is carried out according to following
protocol:
[0056] i) the PBMCs are isolated using standard density
centrifugation procedures and are suspended at 5.times.10.sup.6
cells/ml in RPMI cell culture medium;
[0057] ii) the target cells are grown by standard tissue culture
methods, harvested from the exponential growth phase with a
viability higher than 90%, washed in RPMI cell culture medium,
labeled with 100 micro-Curies of .sup.51CI washed twice with cell
culture medium, and resuspended in cell culture medium at a density
of 10.sup.5 cells/ml;
[0058] iii) 100 microliters of the final target cell suspension
above are transferred to each well of a 96-well microtiter
plate;
[0059] iv) the antibody is serially-diluted from 4000 ng/ml to 0.04
ng/ml in cell culture medium and 50 microliters of the resulting
antibody solutions are added to the target cells in the 96-well
microtiter plate, testing in triplicate various antibody
concentrations covering the whole concentration range above;
[0060] v) for the maximum release (MR) controls, 3 additional wells
in the plate containing the labeled target cells, receive 50
microliters of a 2% (VN) aqueous solution of non-ionic detergent
(Nonidet, Sigma, St. Louis), instead of the antibody solution
(point iv above);
[0061] vi) for the spontaneous release (SR) controls, 3 additional
wells in the plate containing the labeled target cells, receive 50
microliters of RPMI cell culture medium instead of the antibody
solution (point iv above); vii) the 96-well microtiter plate is
then centrifuged at 50.times.g for 1 minute and incubated for 1
hour at 4.degree. C.;
[0062] viii) 50 microliters of the PBMC suspension (point i above)
are added to each well to yield an effector:target cell ratio of
25:1 and the plates are placed in an incubator under 5% CO2
atmosphere at 37 C for 4 hours;
[0063] ix) the cell-free supernatant from each well is harvested
and the experimentally released radioactivity (ER) is quantified
using a gamma counter;
[0064] x) the percentage of specific lysis is calculated for each
antibody concentration according to the formula
(ER-MR)/(MR-SR).times.100, where ER is the average radioactivity
quantified (see point ix above) for that antibody concentration, MR
is the average radioactivity quantified (see point ix above) for
the MR controls (see point V above), and SR is the average
radioactivity quantified (see point ix above) for the SR controls
(see point vi above);
[0065] 4) "increased ADCC" is defined as either an increase in the
maximum percentage of specific lysis observed within the antibody
concentration range tested above, and/or a reduction in the
concentration of antibody required to achieve one half of the
maximum percentage of specific lysis observed within the antibody
concentration range tested above. The increase in ADCC is relative
to the ADCC, measured with the above assay, mediated by the same
antibody, produced by the same type of host cells, using the same
standard production, purification, formulation and storage methods,
which are known to those skilled in the art, but that has not been
produced by host cells engineered to overexpress GnTIII.
[0066] Said "increased ADCC" can be obtained by glycoengineering of
said antibodies, that means enhance said natural, cell-mediated
effector functions of monoclonal antibodies by engineering their
oligosaccharide component as described in Umana, P. et al., Nature
Biotechnol. 17:176-180 (1999) and U.S. Pat. No. 6,602,684.
[0067] The term "complement-dependent cytotoxicity (CDC)" refers to
lysis of human tumor target cells by the antibody according to the
invention in the presence of complement. CDC is measured preferably
by the treatment of a preparation of CD20 expressing cells with an
anti-CD20 antibody according to the invention in the presence of
complement. CDC is found if the antibody induces at a concentration
of 100 nM the lysis (cell death) of 20% or more of the tumor cells
after 4 hours. The assay is performed preferably with .sup.51Cr or
Eu labeled tumor cells and measurement of released .sup.51Cr or Eu.
Controls include the incubation of the tumor target cells with
complement but without the antibody.
[0068] Typically type I and type II anti-CD20 antibodies of the
IgG1 isotype show characteristic CDC properties. Type I anti-CD20
antibodies have and increased CDC (if IgG1 isotype) and type II
anti-CD20 antibodies have a decreased CDC (if IgG1 isotype)
compared to each other. Preferably both type I and type II
anti-CD20 antibodies are IgG1 isotype antibodies.
[0069] The "rituximab" antibody is a genetically engineered
chimeric human gamma 1 murine constant domain containing monoclonal
antibody directed against the human CD20 antigen. This chimeric
antibody contains human gamma 1 constant domains and is identified
by the name "C2B8" in EP2000149B1 and U.S. Pat. No. 7,381,560
(Anderson et. al., see e.g. FIGS. 4 and 5). Rituximab is approved
for the treatment of patients with relapsed or refracting 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)). Additionally, it exhibits
significant activity in assays that measure antibody-dependent
cellular cytotoxicity (ADCC).
[0070] The term "humanized B-Ly1 antibody" refers to humanized
B-Ly1 antibody as disclosed in WO2005/044859, which were obtained
from the murine monoclonal anti-CD20 antibody B-Ly1 (variable
region of the murine heavy chain (VH): SEQ ID NO: 1; variable
region of the murine light chain (VL): SEQ ID NO: 2; see Poppema,
S, and Visser, L., Biotest Bulletin 3: 131-139 (1987)) by
chimerization with a human constant domain from IgG1 and following
humanization (see WO2005/044859). These "humanized B-Ly1
antibodies" are disclosed in detail in WO2005/044859.
[0071] Preferably the "humanized B-Ly1 antibody" has variable
region of the heavy chain (VH) selected from group of SEQ ID No: 3
to SEQ ID No: 20 (B-HH2 to B-HH9 and B-HL8 to B-HL17 of
WO2005/044859). Especially preferred are Seq. ID Nos: 3, 4, 7, 9,
11, 13 and 15 (B-HH2, BHH-3, B-HH6, B-HH8, B-HL8, B-HL11 and B-HL13
of WO2005/044859). Most preferably, said VH is BHH6. Preferably the
"humanized B-Ly1 antibody" has variable region of the light chain
(VL) of SEQ ID No: 20 (B-KV1) of WO2005/044859. Furthermore the
humanized B-Ly1 antibody is preferably an IgG1 antibody. Preferably
such humanized B-Ly1 antibodies are glycoengineered (GE) in the Fc
region according to the procedures described in WO2005/044859, WO
2004/065540, Umana, P. et al., Nature Biotechnol. 17:176-180 (1999)
and WO 99/154342. Most glycoengineered humanized B-Ly1 antibodies
have an altered pattern of glycosylation in the Fc region,
preferably having a reduced level of fucose residues. Preferably at
least 40% or more (in one embodiment between 40% and 60%, in
another embodiment at least 50%, and in still another embodiment at
least 70% or more) of the oligosaccharides of the Fc region are
non-fucosylated. Furthermore the oligosaccharides of the Fc region
are preferably bisected. Most preferably, the "humanized B-Ly1
antibody" comprises VH B-HH6 and VL B-KV1 of WO2005/044859. As used
herein, said antibody is also referred to as "HuMab<CD20>".
Said antibody was designated with the INN Afutuzumab. In another
most preferable embodiment, said antibody has a reduced level of
fucose residues as defined above and/or the oligosaccharides of the
Fc region are most preferably bisected. In yet another most
preferable embodiment, said antibody displays increased ADCC as
defined herein.
[0072] The oligosaccharide component can significantly affect
properties relevant to the efficacy of a therapeutic glycoprotein,
including physical stability, resistance to protease attack,
interactions with the immune system, pharmacokinetics, and specific
biological activity. Such properties may depend not only on the
presence or absence, but also on the specific structures, of
oligosaccharides. Some generalizations between oligosaccharide
structure and glycoprotein function can be made. For example,
certain oligosaccharide structures mediate rapid clearance of the
glycoprotein from the bloodstream through interactions with
specific carbohydrate binding proteins, while others can be bound
by antibodies and trigger undesired immune reactions. (Jenkins et
al., Nature Biotechnol. 14:975-81 (1996)).
[0073] Mammalian cells are the preferred hosts for production of
therapeutic glycoproteins, due to their capability to glycosylate
proteins in the most compatible form for human application.
(Cumming et al., Glycobiology 1:115-30 (1991); Jenkins et al.,
Nature Biotechnol. 14:975-81 (1996)). Bacteria very rarely
glycosylate proteins, and like other types of common hosts, such as
yeasts, filamentous fungi, insect and plant cells, yield
glycosylation patterns associated with rapid clearance from the
blood stream, undesirable immune interactions, and in some specific
cases, reduced biological activity. Among mammalian cells, Chinese
hamster ovary (CHO) cells have been most commonly used during the
last two decades. In addition to giving suitable glycosylation
patterns, these cells allow consistent generation of genetically
stable, highly productive clonal cell lines. They can be cultured
to high densities in simple bioreactors using serumfree media, and
permit the development of safe and reproducible bioprocesses. Other
commonly used animal cells include baby hamster kidney (BHK) cells,
NS0-- and SP2/0-mouse myeloma cells. More recently, production from
transgenic animals has also been tested. (Jenkins et al., Nature
Biotechnol. 14: 975-981 (1996)).
[0074] All antibodies contain carbohydrate structures at conserved
positions in the heavy chain constant regions, with each isotype
possessing a distinct array of N-linked carbohydrate structures,
which 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 carbohydrate
varies considerably, depending on the degree of processing, and can
include highmannose, multiply-branched as well as biantennary
complex oligosaccharides. (Wright, A., and Morrison, S. L., Trends
Biotech. 15: 26-32 (1997)). Typically, there is heterogeneous
processing of the core oligosaccharide structures attached at a
particular glycosylation site such that even monoclonal antibodies
exist as multiple glycoforms. Likewise, it has been shown that
major differences in antibody glycosylation occur between cell
lines, and even minor differences are seen for a given cell line
grown under different culture conditions. (Lifely, M. R. et al.,
Glycobiology 5(8):813-22 (1995)).
[0075] One way to obtain large increases in potency, while
maintaining a simple production process and potentially avoiding
significant, undesirable side effects, is to enhance the natural,
cell-mediated effector functions of monoclonal antibodies by
engineering their oligosaccharide component as described in Umana,
P. et al., Nature Biotechnol. 17:176-180 (1999) and U.S. Pat. No.
6,602,684. IgG1 type antibodies, the most commonly used antibodies
in cancer immunotherapy, are glycoproteins that have a conserved
N-linked glycosylation site at Asn297 in each CH2 domain. The two
complex biantennary oligosaccharides attached to Asn297 are buried
between the CH2 domains, forming extensive contacts with the
polypeptide backbone, and their presence is essential for the
antibody to mediate 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)).
[0076] It was previously shown that overexpression in Chinese
hamster ovary (CHO) cells of
.beta.(1,4)-N-acetylglucosaminyltransferase III ("GnTIII") a
glycosyltransferase catalyzing the formation of bisected
oligosaccharides, significantly increases the in vitro ADCC
activity of an antineuroblastoma chimeric monoclonal antibody
(chCE7) produced by the 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 which 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 (Umana,
P., et al., Nature Biotechnol. 17: 176-180 (1999)). That study was
the first to show that large increases of ADCC activity could be
obtained by engineering the antibody producing cells to express
GnTIII, which also led to an increase in the proportion of constant
region (Fc)-associated, bisected oligosaccharides, including
bisected, non-fucosylated oligosaccharides, above the levels found
in naturally-occurring antibodies.
[0077] The term "expression of the CD20" antigen is intended to
indicate an significant level of expression of the CD20 antigen in
a cell, preferably on the cell surface of a B-Cell, more preferably
a B-cell, from a tumor or cancer, respectively, preferably a
non-solid tumor. Patients having a "CD20 expressing cancer" can be
determined by standard assays known in the art. "Expression of the
CD20" antigen is also preferable intended to indicate an
significant level of expression of the CD20 antigen in a cell,
preferably on the cell surface of a B-Cell, more preferably a
B-cell, in an autoimmune disease, e.g. CD20 antigen expression is
measured using immunohistochemical (IHC) detection, FACS or via
PCR-based detection of the corresponding mRNA.
[0078] The term "CD20 expressing cancer" as used herein refers
preferably to lymphomas (preferably B-Cell Non-Hodgkin's lymphomas
(NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic
leukemias include e.g.: (a) follicular lymphomas, (b) Small
Non-Cleaved Cell Lymphomas/Burkitt's lymphoma (including endemic
Burkitt's lymphoma, sporadic Burkitt's lymphoma and Non-Burkitt's
lymphoma), (c) marginal zone lymphomas (including extranodal
marginal zone B cell lymphoma (Mucosa-associated lymphatic tissue
lymphomas, 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, Angiocentric Lymphoma-Pulmonary B-Cell
Lymphoma), (f) hairy cell leukemia, (g) lymphocytic lymphoma,
Waldenstrom's macroglobulinemia, (h) acute lymphocytic leukemia
(ALL), chronic lymphocytic leukemia (CLL), small lymphocytic
lymphoma (SLL), B-cell prolymphocytic leukemia, (i) plasma cell
neoplasms, plasma cell myeloma, multiple myeloma, plasmacytoma, and
(j) Hodgkin's disease.
[0079] Preferably the CD20 expressing cancer is a B-Cell
Non-Hodgkin's lymphomas (NHL). Other examples of CD20 expressing
cancers include: Mantle cell lymphoma (MCL), acute lymphocytic
leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell diffuse
large cell lymphoma (DLCL), Burkitt's lymphoma, 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.
[0080] As used herein, "autoimmune disease" relates to a disease or
disorder arising from and directed against an individual's own
tissues. Examples of autoimmune diseases or disorders include, but
are not limited to arthritis (rheumatoid arthritis, juvenile
rheumatoid arthritis, osteoarthritis, psoriatic arthritis),
psoriasis, dermatitis, polymyositis/dermatomyositis, toxic
epidermal necrolysis, systemic scleroderma and sclerosis, responses
associated with 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 infiltration of T cells and chronic
inflammatory responses, atherosclerosis, autoimmune myocarditis,
leukocyte adhesion deficiency, systemic lupus erythematosus (SLE),
juvenile onset diabetes, multiple sclerosis, allergic
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 leukocyte diapedesis, central nervous system (CNS)
inflammatory disorders, multiple organ injury syndrome, mysathenia
gravis, antigen-antibody complex mediated diseases, anti-glomerular
basement membrane disease, anti-phospholipid antibody syndrome,
allergic neuritis, Bechet disease, Castleman's syndrome,
Goodpasture's syndrome, Lambert-Eaton Myasthenic Syndrome,
Reynaud's syndrome, Sjorgen's syndrome, Stevens Johnson syndrome,
pemphigoid bullous, pemphigus, autoimmune polyendocrinopathies, s
nephropathy, IgM polyneuropathies or IgM mediated neuropathy,
idiopathic thrombocytopenic purpura (ITP), thrombotic
throbocytopenic purpura (TTP), autoimmune thrombocytopenia,
autoimmune disease of the testis and ovary including autoimune
orchitis and oophoritis, primary hypothyroidism; autoimmune
endocrine diseases including autoimmune thyroiditis, chronic
thyroiditis (Hashimoto's Thyroiditis), subacute thyroiditis,
idiopathic hypothyroidism, Addison's disease, Grave's disease,
autoimmune polyglandular syndromes (or polyglandular I
endocrinopathy syndromes), Type I diabetes also referred to as
insulin-dependent diabetes i mellitus (IDDM) and Sheehan's
syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis
(HIV), bronchiolitis obliterans (non-transplant) vs NSIP,
Guillain-Barre' 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, primary
biliary cirrhosis, Celiac sprue (gluten enteropathy),
cryoglobulinemia, amyotrophic lateral sclerosis (ALS), coronary
artery disease etc.
[0081] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
a CD20 expressing cancer cell either in vitro or in vivo. Thus, the
growth inhibitory agent may be one which significantly reduces the
percentage of CD20 expressing cells in S phase. Examples of growth
inhibitory agents include agents that block cell cycle progression
(at a place other than S phase), such as agents that induce G1
arrest and M-phase arrest. Classical M-phase blockers include the
vincas (vincristine and vinblastine), taxanes, and topo 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. Further information can be
found in "The Molecular Basis of Cancer", Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (WB Saunders:
Philadelphia, 1995), especially p. 13.
[0082] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disease as well as those in which
the disease is to be prevented. Hence, the patient to be treated
herein may have been diagnosed as having the disease or may be
predisposed or susceptible to the disease.
[0083] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof.
[0084] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.TM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, trietylenephosphoramide,
triethiylenethiophosphoramide and trimethylolomelarnine;
acetogenins (especially bullatacin and bullatacinone);
delta-9-tetrahydrocannabinol (dronabinol, MARENOL.TM.);
beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.TM.), CPT-11 (irinotecan, CAMPTOSAR.TM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptoplhycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranirnnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma 11 and calicheamicin omega 11 (see, e.g., Angew, Chemie Intl.
Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A;
an esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antiobiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including ADRIAMYCIN.TM., morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin
HCl liposome injection (DOXIL.TM.), liposomal doxorubicin TLC D-99
(MYOCET.TM.), peglylated liposomal doxorubicin (CAELYX.TM.), and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate, gemcitabine (GEMZAR.TM.), tegafur (UFTORAL.TM.),
capecitabine (XELODA.TM.), an epothilone, and 5-fluorouracil
(5-FU); folic acid analogues such as denopterin, methotrexate,
pteropterin, trimetrexate; purine analogs such as fludarabine,
6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such
as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,
dideoxyuridine, doxifluridine, enocitabine, floxuridine;
anti-adrenals such as aminoglutethimide, mitotane, trilostane;
folic acid replenisher such as frolinic acid; aceglatone;
aldophosphamide glycoside; aminol evulinic acid; eniluracil;
amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;
demecolcine; diaziquone; elformithine; elliptinium acetate;
etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine;
maytansinoids such as maytansine and ansamitocins; mitoguazone;
mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;
pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSKL.TM.
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofuran; spirogermaraium; tenuazonic acid;
triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid,
e.g., paclitaxel (TAXOL.TM.), albumin-engineered nanoparticle
formulation of paclitaxel (ABRAXANE.TM.), and docetaxel
(TAXOTERE.TM.); chloranbucil; 6-thioguanine; mercaptopurine;
metliotrexate; platinum agents such as cisplatin, oxaliplatin, and
carboplatin; vincas, which prevent tubulin polymerization from
forming microtubules, including vinblastine (VELBAN.TM.),
vincristine (ONCOVIN.TM.), vindesine (ELDISINE.TM.), FILDESIN.TM.),
and vinorelbine (NAVELBINE.TM.)); etoposide (VP-16); ifosfamide;
mitoxantrone; leucovovin; novantrone; edatrexate; daunomycin;
aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;
difluoromethylornithine (DMFO); retinoids such as retinoic acid,
including bexarotene (TARGRETIN.TM.); bisphosphonates such as
clodronate (for example, BONEFOS.TM. or OSTAC.TM.), etidronate
(DIDROCAL.TM.), NE-58095, zoledronic acid/zoledronate (ZOMETA.TM.),
alendronate (FOSAMAJX.TM.), pamidronate (AREDIA.TM.), tiludronate
(SKELID.TM.), or risedronate (ACTONEL.TM.); troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); antisense
oligonucleotides, particularly those that inhibit expression of
genes in signaling pathways implicated in aberrant cell
proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and
epidermal growth factor receptor (EGF-R); vaccines such as
THERATOPE.TM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.TM. vaccine, LEUVECTIN.TM. vaccine, and VAXID.TM.
vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN.TM.); rmRH
(e.g., ABARELIX.TM.); BAY439006 (sorafenib; Bayer); SU-11248
(Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or
etoricoxib), proteosome inhibitor (e.g. PS341); bortezomib
(VELCADE.TM.); CCI-779; tipifarnib (R11577); orafenib, ABT510;
Bc1-2 inhibitor such as oblimersen sodium (GENASENSE.TM.);
pixantrone; EGFR inhibitors (see definition below); tyrosine kinase
inhibitors (see definition below); and pharmaceutically acceptable
salts, acids or derivatives of any of the above; as well as
combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone (optionally further
comprising interferon-.alpha. (CHVP/interferon-.alpha.), FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin, CVP
(cyclophosphamide, vincristine, and prednisolone), MCP
(mitozantrone, chlorambucil and prednisolone), FC (fludarabine and
cyclophosphamide), ICE (ifosfamide, carboplatin, and etoposide),
and dexamethasone, cytarabine, and cisplatin (DHAP), dexamethasone,
doxorubicin liposomal, and vincristine (DVD) etc.
[0085] An "anti-angiogenic agent" refers to a compound which
blocks, or interferes with to some degree, the development of blood
vessels. The anti-angiogenic factor may, for instance, be a small
molecule or antibody that binds to a growth factor or growth factor
receptor involved in promoting angiogenesis. The preferred
anti-angiogenic factor herein is an antibody that binds to Vascular
Endothelial Growth Factor (VEGF), such as Bevacizumab
(AVASTIN.TM.).
[0086] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone, parathyroid hormone,
thyroxine, insulin, proinsulin, relaxin; prorelaxin, glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH), hepatic
growth factor; fibroblast growth factor, prolactin, placental
lactogen, tumor necrosis factor .alpha. and .beta.,
mullerian-inhibiting substance, mouse gonadotropin-associated
peptide, inhibin; activin, vascular endothelial growth factor,
integrin, thrombopoietin (TPO), nerve growth factors such as
NGF-13, platelet-growth factor; transforming growth factors (TGFs)
such as TGF-.alpha. and TGF-13, insulin-like growth factor-I and
-II, erythropoietin (EPO), osteoinductive factors; interferons such
as interferon-.alpha., -.beta., and -.gamma., colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF),
granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF),
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, a tumor necrosis
factor such as TNF-a or TNF-13, and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0087] The term "effective amount" refers to an amount which
provides the desired effect. In the case of a formulation
ingredient such as the hyaluronidase enzyme in accordance with the
present invention an effective amount is the amount necessary to
increase the dispersion and absorption of the co-administered
anti-CD20 antibody in such a way that the anti-CD20 antibody can
act in a therapeutically effective way as outline above. In the
case of a pharmaceutical drug substance it is the amount of active
ingredient effective to treat a disease in the patient. Where the
disease is cancer, the effective amount of the drug may reduce the
number of cancer cells; reduce the tumor size; inhibit (i.e., slow
to some extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the cancer. To the extent the drug may prevent
growth and/or kill existing cancer cells, it may be cytostatic
and/or cytotoxic. The effective amount may extend progression free
survival, result in an objective response (including a partial
response, PR, or complete response, CR), increase overall survival
time, and/or improve one or more symptoms of cancer.
[0088] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of the active ingredient to be effective, and which
contains no additional components which are unacceptably toxic to a
subject to which the formulation would be administered. Such
formulations are sterile.
[0089] A "sterile" formulation is aseptic or free from all living
microorganisms and their spores.
[0090] A "stable" formulation is one in which all the protein
therein essentially retain their physical stability and/or chemical
stability and/or biological activity upon storage at the intended
storage temperature, e.g. 2-8.degree. C. Preferably, the
formulation essentially retains its physical and chemical
stability, as well as its biological activity upon storage. The
storage period is generally selected based on the intended
shelf-life of the formulation. Furthermore, the formulation is
preferably stable following freezing (to, e.g., -20.degree. C.) and
thawing of the formulation, for example following 1 or more cycles
of freezing and thawing. Various analytical techniques for
measuring protein stability are available in the art and are
reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee
Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones,
A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. Stability
can be measured at a selected temperature for a selected time
period. Stability can be evaluated qualitatively and/or
quantitatively in a variety of different ways, including evaluation
of aggregate formation (for example using size exclusion
chromatography, by measuring turbidity, and/or by visual
inspection); by assessing charge heterogeneity using cation
exchange chromatography or capillary zone electrophoresis; SDS-PAGE
analysis to compare reduced and intact antibody; evaluating
biological activity or antigen binding function of the antibody;
etc. Instability may involve any one or more of: aggregation,
deamidation (e.g. Asn deamidation), oxidation (e.g. Met oxidation),
isomerization (e.g. Asp isomeriation),
clipping/hydrolysis/fragmentation (e.g. hinge region
fragmentation), succinimide formation, unpaired cysteine(s),
etc.
[0091] Therapeutic formulations of the antibodies used in
accordance with the present invention are prepared for storage by
mixing an antibody having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed.
[0092] The term "surfactant" as used herein denotes a
pharmaceutically acceptable surface-active agent. In the
formulation of the invention, the amount of surfactant is described
a percentage expressed in weight/volume. The most commonly used
weight/volume unit is mg/mL. Suitable examples of pharmaceutically
acceptable surfactants include polyoxyethylen-sorbitan fatty acid
esters (Tween), polyethylene-polypropylene glycols,
polyoxyethylene-stearates, polyoxyethylene alkyl ethers, e.g.
polyoxyethylene monolauryl ether, alkylphenylpolyoxy-ethylene
ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer
(Poloxamer, Pluronic), and sodium dodecyl sulphate (SDS). Most
suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate
20, (sold under the trademark Tween 20.TM.) and polysorbate 80
(sold under the trademark Tween 80.TM.). Most suitable
polyethylene-polypropylene copolymers are those sold under the
names Pluronic.RTM. F68 or Poloxamer 188.TM.. Preferred
polyoxyethylene-stearates are those sold under the trademark
Myrj.TM.. Most suitable polyoxy-ethylene alkyl ethers are those
sold under the trademark Brij.TM. Most suitable
alkylphenolpoly-oxyethylene ethers are sold under the trade name
Triton-X.
[0093] The term "buffer" as used herein denotes a pharmaceutically
acceptable buffer. As used herein the term "buffering agent
providing a pH of 5.5.+-.2.0" refers to an agent which provides
that the solution comprising it resists changes in pH by the action
of its acid/base conjugate components. Suitable pharmaceutically
acceptable buffers according to the invention comprise but are not
limited to histidine-buffers, citrate-buffers, gluconate-buffers,
succinate-buffers, acetate-buffers glycylglycine and other organic
acid buffers, and phosphate-buffers. Preferred buffers comprise
L-histidine or mixtures of L-histidine with L-histidine
hydrochloride with isotonicity agents and potentially pH adjustment
with an acid or a base known in the art. Most preferred is
L-histidine.
[0094] A "histidine buffer" is a buffer comprising the amino acid
histidine. Examples of histidine buffers include histidine
chloride, histidine acetate, histidine phosphate, histidine
sulfate. The preferred histidine buffer identified in the examples
herein was found to be histidine chloride. In the preferred
embodiment, the histidine chloride buffer is prepared by titrating
L-histidine (free base, solid) with diluted hydrochloric acid or by
dissolving L-histidine and L-histidine hydrochloride (e.g. as
monohydrate) in a defined amount and ratio.
[0095] By "isotonic" is meant that the formulation of interest has
essentially the same osmotic pressure as human blood. Isotonic
formulations will generally have an osmolality of .about.300
mOsm/kg. Isotonicity can be measured using a vapor pressure or
freezing-point depression type osmometer.
[0096] The term "isotonicity agents" as used herein denotes
pharmaceutically acceptable isotonicity agents. Isotonicity agents
are used to provide an isotonic formulation. An isotonic
formulation is liquid or liquid reconstituted from a solid form,
e.g. a lyophilized form and denotes a solution having the same
tonicity as some other solution with which it is compared, such as
physiologic salt solution and the blood serum. Suitable isotonicity
agents comprise but are not limited to salts, including but not
limited to sodium chloride (NaCl) or potassium chloride, sugars and
sugar alcohols including but not limited to glucose, sucrose,
trehalose or glycerol and any component from the group of amino
acids, sugars, salts and combinations thereof. Isotonicity agents
are generally used in a total amount of about 5 mM to about 350
mM.
[0097] The term "liquid" as used herein in connection with the
formulation according to the invention denotes a formulation which
is liquid at a temperature of at least about 2 to about 8.degree.
C.
[0098] The term "lyophilized" as used herein in connection with the
formulation according to the invention denotes a formulation which
is dried by freezing the formulation and subsequently subliming the
ice from the frozen content by any freeze-drying methods known in
the art, for example commercially available freeze-drying
devices.
[0099] The term "salts" as used herein denotes a salt in an amount
of about 1 mM to about 500 mM. Non-limiting examples of salts
include salts of any combinations of the cations sodium potassium,
calcium or magnesium with anions chloride, phosphate, citrate,
succinate, sulphate or mixtures thereof.
[0100] The term "amino acid" as used herein denotes an amino acid
in an amount of about 1 to about 100 mg/mL comprising but not
limited to arginine, glycine, ornithine, glutamine, asparagine,
lysine, histidine, glutamic acid, asparagic acid, isoleucine,
leucine, alanine, phenylalanine, tyrosine, tryptophane, methionine,
serine, proline.
[0101] A "saccharide" herein comprises the general composition
(CH.sub.2O).sub.n and derivatives thereof, including
monosaccharides, disaccharides, trisaccharides, polysaccharides,
sugar alcohols, reducing sugars, nonreducing sugars, etc. Examples
of saccharides herein include glucose, sucrose, trehalose, lactose,
fructose, maltose, dextran, glycerin, dextran, erythritol,
glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose,
melezitose, raffinose, mannotriose, stachyose, maltose, lactulose,
maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc. Also
included in the definition according to the invention are
glucosamine, N-Methylglucosamine (so-called "Meglumine"),
galactosamine and neuraminic acid and combinations of the
saccharides according to the invention. The preferred saccharide
herein is a non-reducing disaccharide, such as trehalose or
sucrose. The most preferred saccharide in accordance with the
present invention is trehalose.
[0102] The term "stabilizer" refers to pharmaceutically acceptable
stabilizers, like for example but not limited to amino acids and
sugars as described in the above sections as well as commercially
available dextrans of any kind and molecular weight as known in the
art.
[0103] The term "antioxidant" denotes a pharmaceutically acceptable
antioxidant. This may include excipients such as methionine,
benzylalcohol or any other excipient used to minimize
oxidation.
[0104] The term "a method of treating" or its equivalent, when
applied to, for example, cancer refers to a procedure or course of
action that is designed to reduce or eliminate the number of cancer
cells in a patient, or to alleviate the symptoms of a cancer. "A
method of treating" cancer or another proliferative disorder does
not necessarily mean that the cancer cells or other disorder will,
in fact, be eliminated, that the number of cells or disorder will,
in fact, be reduced, or that the symptoms of a cancer or other
disorder will, in fact, be alleviated. Often, a method of treating
cancer will be performed even with a low likelihood of success, but
which, given the medical history and estimated survival expectancy
of a patient, is nevertheless deemed to induce an overall
beneficial course of action.
[0105] The problem to be solved by the present invention is
therefore to provide novel highly concentrated, stable
pharmaceutical formulations of a pharmaceutically active anti-CD20
antibody or a mixture of such antibody molecules for subcutaneous
injection. Such formulations comprise, in addition to the high
amounts of anti-CD20 antibody or mixture thereof, a buffering
agent, a stabilizer or a mixture of two or more stabilizers, a
nonionic surfactant and preferably an effective amount of at least
one hyaluronidase enzyme. The preparation of highly-concentrated
antibody formulations is challenging because of a potential
increase in viscosity at higher protein concentration and a
potential increase in protein aggregation, a phenomenon that is per
se concentration-dependent. High viscosities negatively impact the
process ability (e.g. pumping and filtration steps) of the antibody
formulations and the administration (e.g. the syringe ability). By
the addition of excipients high viscosities could be decreased in
some cases. Control and analysis of protein aggregation is an
increasing challenge. Aggregation is potentially encountered during
various steps of the manufacturing process, which include
fermentation, purification, formulation and during storage.
Different factors, such as temperature, protein concentration,
agitation stress, freezing and thawing, solvent and surfactant
effects, and chemical modifications, might influence the
aggregation behavior of a therapeutic protein. During development
of a highly concentrated antibody formulation the aggregation
tendency of the protein has to be monitored and controlled by the
addition of various excipients and surfactants (Kiese S. et al., J.
Pharm. Sci., 2008; 97(10); 4347-4366).
[0106] 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 application. Preferably the route
of application is intravenous administration as a bolus or by
continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, or intrathecal routes.
[0107] Intravenous or subcutaneous administration of the antibodies
is preferred; subcutaneous injection is preferred most. As set out
above, it is by far not trivial to generate a highly concentrated,
stable, pharmaceutical formulation of a CD20 antibody which is
essentially free of particles. If said formulation is intended for
subcutaneous application, then in a preferred embodiment said
formulation is combined with a hyaluronidase enzyme.
[0108] More particularly the highly concentrated, stable
pharmaceutical formulation of a pharmaceutically active anti-CD20
antibody formulation of the present invention comprises: [0109]
about 20 to 350 mg/ml anti-CD20 antibody; [0110] about 1 to 100 mM
of a buffering agent providing a pH of 5.5.+-.2.0; [0111] about 1
to 500 mM of a stabilizer or a mixture of two or more stabilizers,
whereby optionally methionine is used as a secondary stabilizer,
preferably in a concentration of 5 to 25 mM; [0112] 0.01 to 0.1% of
a nonionic surfactant; and [0113] preferably an effective amount of
at least one hyaluronidase enzyme.
[0114] The highly concentrated, stable pharmaceutical formulation
of a pharmaceutically active anti-CD20 antibody formulation of the
present invention may be provided in liquid form or may be provided
in lyophilized form. The antibody concentration in the
reconstituted formulation can be increased by reconstitution of a
lyophilized formulation to provide a protein concentration in the
reconstituted formulation which is about 2-40 times greater than
the protein concentration in the mixture before the lyophilization
step.
[0115] The preferred anti-CD20 antibody concentration is 50 to 150
mg/ml, more preferred is 75 to 150 mg/ml, even more preferred is
120.+-.20 mg/ml, most preferred is about 120 mg/ml.
[0116] The preferred concentration of the buffering agent is 1 to
50 mM, more preferably 10 to 30 mM; the most preferred
concentration is about 20 mM. Various buffering agents are known to
the person skilled in the art as outlined above. The preferred
buffering agent is selected from the group consisting of a
histidine buffer, acetic acid buffer, and citric acid buffer, most
preferably a L-histidine/HCl buffer. The histidine-buffer according
to the invention is used in an amount of about 1 mM to about 50 mM,
preferably of about 10 mM to about 30 mM and still more preferably
of about 20 mM. The acetic acid buffer according to the invention
is preferably of about 10 mM to about 30 mM and most preferably of
about 20 mM. The citric acid buffer according to the invention is
preferably of about 10 mM to about 30 mM and most preferably of
about 20 mM.
[0117] Independently from the buffer used, the pH will be adjusted
at a value comprising about 4.5 to about 7.0 and preferably about
5.5 to about 6.5, also preferably preferably selected from the
group consisting of 5.5, 6.0, 6.1 and 6.5. This pH can be obtained
by adjustment with an acid or base known in the art or by using
adequate mixtures of buffer components or both.
[0118] The stabilizer(s) (used synonymously with the term
"stabilizing agent" in the present patent description) is/are
preferably selected from the group consisting of a salt, a
carbohydrate, saccharide and amino acid(s), more preferably a
carbohydrate or saccharide, more preferably a sugar admitted by the
authorities as a suitable additive or excipient in pharmaceutical
formulations, most preferably selected from the group consisting of
.alpha.,.alpha.-trehalose dihydrate, NaCl and methionine. The
preferred concentration of the stabilizer is 15 to 250 mM, or more
preferably 150 to 250 mM. Most preferred is a concentration of
about 210 mM. The formulation may contain a secondary stabilizer,
whereby this secondary stabilizer is preferably methionine,
preferably in a concentration of 5 to 25 mM, more preferably in a
concentration of 5 to 15 mM. The most preferred methionine
concentration is about 10 mM.
[0119] The nonionic surfactant is preferably a polysorbate, more
preferably is selected from the group 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).
[0120] The term "sugar" as used herein denotes a pharmaceutically
acceptable sugar used in an amount of about 25 mM to about 500 mM.
Preferred is 100 to 300 mM. More preferred is 180 to 240 mM. Most
preferred is 210 mM.
[0121] The concentration of the hyaluronidase enzyme depends on the
actual hyaluronidase enzyme used in the preparation of the
formulation in accordance with the invention. An effective amount
of the hyaluronidase enzyme can easily be determined by the person
skilled in the art based on the disclosure further below. It should
be provided in sufficient amount so that an increase in the
dispersion and absorption of the co-administered anti-CD20 antibody
is possible. The effective amount of the hyaluronidase enzyme is
preferably about 1,000 to 16,000 U/ml, whereby the said amount
corresponds to about 0.01 mg to 0.15 mg protein based on an assumed
specific activity of 100,000 U/mg. The preferred concentration of
the hyaluronidase enzyme is about 1,500 to 12,000 U/ml. Most
preferred is a concentration of about 2,000 U/ml or about 12,000
U/ml. The amounts specified herein before correspond to the amount
of hyaluronidase enzyme initially added to the formulation. The
hyaluronidase enzyme is present either as a combined final
formulation or for use for co-administration, e.g. as a
co-formulation as further outlined below. The important issue for
the claimed formulation is that at the time it is ready for use
and/or is injected it has the claimed composition.
[0122] The hyaluronidase enzyme may be derived from animals, human
samples or manufactured based on the recombinant DNA technology as
described further below.
[0123] More particularly the highly concentrated, stable
pharmaceutical formulations in accordance with the present
invention have one of the following preferred compositions:
[0124] a) 100 to 150 mg/ml anti-CD20 antibody, whereby this
antibody is preferably Rituximab, Ocrelizumab or HuMab<CD20>;
1 to 50 mM of a histidine buffer, preferably L-histidine/HCl at a
pH of about 5.5; 15 to 250 mM of a stabilizer which is preferably
.alpha.,.alpha.-trehalose dihydrate and optionally methionine as a
second stabilizer at a concentration of 5 to 25 mM; a non-ionic
surfactant selected from the group of polysorbate 20 and
polysorbate 80, preferably 0.02 to 0.06% (w/v), and optionally
1,000 to 16,000 U/ml of a hyaluronidase enzyme, preferably rHuPH20,
most preferably at a concentration of 2,000 U/ml or 12,000
U/ml.
[0125] b) 120.+-.20 mg/ml anti-CD20 antibody, whereby this antibody
is preferably Rituximab, Ocrelizumab or HuMab<CD20>; 10 to 30
mM, preferably 20 mM of a histidine buffer, preferably
L-histidine/HCl at a pH of about 5.5; 150 to 250 mM, preferably 210
mM of a stabilizer which is preferably .alpha.,.alpha.-trehalose
dihydrate and optionally methionine as a second stabilizer at a
concentration of 5 to 25 mM, preferably 5 to 15 mM, most preferably
10 mM; a non-ionic surfactant selected from the group of
polysorbate 20 and polysorbate 80, preferably 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 of a hyaluronidase
enzyme, preferably rHuPH20.
[0126] c) 120 mg/ml anti-CD20 antibody, whereby this antibody is
preferably Rituximab, Ocrelizumab or HuMab<CD20>; 10 to 30
mM, preferably 20 mM of a histidine buffer, preferably
L-histidine/HCl at a pH of about 5.5; 150 to 250 mM, preferably 210
mM of a stabilizer which is preferably .alpha.,.alpha.-trehalose
dihydrate and optionally methionine as a second stabilizer at a
concentration of 5 to 25 mM, preferably 5 to 15 mM, most preferably
10 mM; a non-ionic surfactant selected from the group of
polysorbate 20 and polysorbate 80, preferably 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 of a hyaluronidase
enzyme, preferably rHuPH20.
[0127] d) 120 mg/ml anti-CD20 antibody, preferably Rituximab; 20 mM
of a histidine buffer, preferably L-histidine/HCl at a pH of about
5.5; 210 mM .alpha.,.alpha.-trehalose dihydrate and optionally 10
mM methionine as a second stabilizer; a non-ionic surfactant
selected from the group of polysorbate 20 and polysorbate 80,
preferably 0.02 to 0.06% (w/v), and optionally 2,000 U/ml or 12,000
U/ml of a hyaluronidase enzyme, preferably rHuPH20.
[0128] e) A lyophilized formulation comprising 120 mg/ml anti-CD20
antibody, preferably Rituximab; 20 mM of a histidine buffer,
preferably L-histidine/HC1 at a pH of about 5.5; 210 mM of
.alpha.,.alpha.-trehalose dihydrate and optionally 10 mM methionine
as a second stabilizer; a non-ionic surfactant selected from the
group of polysorbate 20 and polysorbate 80, preferably 0.02 to
0.06% (w/v), and optionally 2,000 U/ml or 12,000 U/ml of a
hyaluronidase enzyme, preferably rHuPH20.
[0129] A stable pharmaceutical formulation of a pharmaceutically
active anti-CD20 antibody is provided comprising about 30 mg/ml to
350 mg/ml, for example about 30 mg/ml to 100 mg/ml (including about
30 mg/ml, about 50 mg/ml or about 100 mg/ml) Ocrelizumab (e.g.
humanized 2H7.v16); about 1 to 100 mM of a buffering agent (e.g.
sodium acetate) providing a pH of 5.5.+-.2.0 (e.g. pH 5.3); about
15 to 250 mM of a 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) of a nonionic surfactant; and
optionally an effective amount of at least one hyaluronidase enzyme
(e.g. rhHUPH20), preferably in an amount from about 1,500 U/ml to
about 12,000 U/ml.
[0130] Alternative compositions of preferred formulations are given
in the examples.
[0131] It has been proposed to facilitate the subcutaneous
injection of therapeutic proteins and antibodies by using small
amounts of soluble hyaluronidase glycoproteins (sHASEGPs); see
WO2006/091871. It has been shown that the addition of such soluble
hyaluronidase glycoproteins (either as a combined formulation or by
co-administration) facilitates the administration of therapeutic
drug into the hypodermis. By rapidly depolymerizing hyaluronan HA
in the extracellular space sHASEGP reduces the viscosity of the
interstitium, thereby increasing hydraulic conductance and allowing
for larger volumes to be administered safely and comfortably into
the subcutaneous tissue. The increased hydraulic conductance
induced by sHASEGP through reduced interstitial viscosity allows
for greater dispersion, potentially increasing the systemic
bioavailability of SC administered therapeutic drug.
[0132] The highly concentrated, stable pharmaceutical formulations
of the present invention comprising a soluble hyaluronidase
glycoprotein are therefore particularly suited for subcutaneous
injection. It is clearly understood by the person skilled in the
art that such a formulation comprising an anti-CD20 antibody and a
soluble hyaluronidase glycoprotein can be provided for
administration in form of one single combined formulation or
alternatively in form of two separate formulations which can be
mixed just prior to the subcutaneous injection. Alternatively the
anti-CD20 antibody and the soluble hyaluronidase glycoprotein can
be administered as separate injections at different sites of the
body, preferably at sites which are immediately adjacent to each
other. It is also possible to inject the therapeutic agents present
in the formulation in accordance with the present invention as
consecutive injections, e.g. first the soluble hyaluronidase
glycoprotein followed by the injection of the anti-CD20 antibody
formulation. These injections can also be performed in the reversed
order, viz. by first injecting the anti-CD20 antibody formulation
followed by injecting the soluble hyaluronidase glycoprotein. In
case the anti-CD20 antibody and the soluble hyaluronidase
glycoprotein are administered as separate injections, one or both
of the proteins have to be provided with the buffering agent, the
stabilizer(s) and the nonionic surfactant in the concentrations as
specified in the appended claims but excluding the hyaluronidase
enzyme. The hyaluronidase enzyme can then be provided e.g. in a
L-histidine/HCl buffer at pH of 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 the buffering
agent, the stabilizer(s) and the nonionic surfactant in the
concentrations as specified in the appended claims.
[0133] As noted above the soluble hyaluronidase glycoprotein may be
considered to be a further excipient in the anti-CD20 formulation.
The soluble hyaluronidase glycoprotein may be added to the
anti-CD20 formulation at the time of manufacturing the anti-CD20
formulation or may be added shortly before the injection.
Alternatively the soluble hyaluronidase glycoprotein may be
provided as a separate injection. In the latter case the soluble
hyaluronidase glycoprotein may be provided in a separate vial
either in lyophilized form which must be reconstituted with
suitable diluents before the subcutaneous injection takes place, or
my be provided as a liquid formulation by the manufacturer. The
anti-CD20 formulation and the soluble hyaluronidase glycoprotein
may be procured as separate entities or may also be provided as
kits comprising both injection components and suitable instructions
for their subcutaneous administration. Suitable instructions for
the reconstitution and/or administration of one or both of the
formulations may also be provided.
[0134] Therefore the present invention also provides pharmaceutical
compositions consisting of an a highly concentrated, stable
pharmaceutical formulation of a pharmaceutically active anti-CD20
antibody or a mixture of such antibody and a suitable amount of at
least one hyaluronidase enzyme in the form of a kit comprising both
injection components and suitable instructions for their
subcutaneous administration.
[0135] A further aspect of the present invention relates to
injection devices comprising a highly concentrated, stable
pharmaceutical formulation in accordance with the present
invention. Such formulation may consist of a pharmaceutically
active anti-CD20 antibody or a mixture of such antibody molecules
and suitable excipients as outlined below and may additionally
comprise a soluble hyaluronidase glycoprotein either as a combined
formulation or as a separate formulation for co-administration.
[0136] A variety of anti-CD20 antibodies are known in the prior
art. Such antibodies are preferably monoclonal antibodies. The may
either be so-called chimaeric antibodies, humanized antibodies or
fully human antibodies. They may either be full length anti-CD20
antibodies; anti-CD20 antibody fragments having the same biological
activity; including amino acid sequence variants and/or
glycosylation variants of such antibodies or fragments. Examples of
humanized anti-CD20 antibodies are known under the INN names
Rituximab, 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 name
MABTHERA.RTM. or RITUXAN.RTM..
[0137] The anti-CD20 antibody as defined herein is preferably
selected from the group of Rituximab (see e.g. U.S. Pat. No.
7,381,560, and EP2000149B1 Anderson et. al., see e.g. FIGS. 4 and
5), Ocrelizumab (as disclosed in WO 2004/056312) and WO 2006/084264
(e.g. the variants disclosed in tables 1 and 2), preferably the
variant v.16 or v.114 or v.511 and Afutuzumab (HuMab<CD20>;
see WO2005/044859). The most preferred anti-CD20 antibody is
Rituximab. The terms "Rituximab", "Ocrelizumab" and "Afutuzumab"
(HuMab<CD20>) encompass all corresponding anti-CD20
antibodies that fullfill the requirements necessary for obtaining a
marketing authorization as an identical or biosimilar product in a
country or territory selected from the group of countries
consisting of the USA, Europe and Japan. Rituximab has the CDR
regions defined in U.S. Pat. No. 7,381,560 and EP2000149B1.
[0138] A number of a soluble hyaluronidase glycoproteins are known
in the prior art. In order to further define the function, the
mechanism of action and the properties of such soluble
hyaluronidase glycoproteins the following background information is
provided.
[0139] The SC (hypodermal) interstitial matrix is comprised of a
network of fibrous proteins embedded within a viscoelastic gel of
glycosaminoglycans. Hyaluronan (HA), a non-sulfated repeating
linear disaccharide, is the prominent glycosaminoglycan of the SC
tissue. HA is secreted into the interstitium by fibroblasts as a
high molecular weight, megadalton viscous polymer that is
subsequently degraded locally, in the lymph, and in the liver,
through the action of lysosomal hyaluronidases and exoglycosidases.
Approximately 50% of the hyaluronan in the body is produced by the
SC tissue, where it is found at approximately 0.8 mg/gm wet weight
tissue (Aukland K. and Reed R., "Interstitial-Lymphatic Mechanisms
in the control of Extracellular Fluid Volume", Physiology Reviews",
73:1-78 (1993)). It is estimated that the average 70 kg adult
contains 15 grams of HA, of which 30 percent 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 a
major constituent of the gel-like component of the hypodermal
matrix, HA contributes significantly to its viscosity.
[0140] Glycosaminoglycans (GAGs) are complex linear polysaccharides
of the extracellular matrix (ECM). GAGs are characterized by
repeating disaccharide structures of an N-substituted hexosamine
and an uronic acid (in the case of hyaluronan (HA), chondroitin
sulfate (CS), chondroitin (C), dermatan sulfate (DS), heparan
sulfate (HS), and heparin (H)), or a galactose (in the case of
keratan sulfate (KS)). Except for HA, all exist covalently bound to
core proteins. The GAGs with their core proteins are structurally
referred to as proteoglycans (PGs).
[0141] Hyaluronan (HA) is found in mammals predominantly in
connective tissues, skin, cartilage, and in synovial fluid.
Hyaluronan is also the main constituent of the vitreous of the eye.
In connective tissue, the water of hydration associated with
hyaluronan creates hydrated matrices between tissues. Hyaluronan
plays a key role in biological phenomena associated with 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 correlate
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).
[0142] HA is found in the extracellular matrix of many cells,
especially in soft connective tissues. HA has been assigned various
physiological functions, 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 locomotion and cell
migration. HA seems to play important roles in cell regulation,
development, and differentiation (Laurent et al., supra).
[0143] HA has widely been used in clinical medicine. Its tissue
protective and rheological properties have proved 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 accumulation of HA may cause
dysfunction in various organs (Laurent et al., supra).
[0144] Hyaluronan protein interactions also are involved in the
structure of the extracellular matrix or "ground substance".
[0145] Hyaluronidases are a group of generally neutral- or
acid-active enzymes found throughout the animal kingdom.
Hyaluronidases vary with respect to substrate specificity, and
mechanism of action (WO 2004/078140). There are three general
classes of hyaluronidases:
1. Mammalian-type hyaluronidases, (EC 3.2.1.35) which are
endo-beta-N-acetylhexosaminidases with tetrasaccharides and
hexasaccharides as the major end products. They have both
hydrolytic and transglycosidase activities, and can degrade
hyaluronan and chondroitin sulfates (CS), generally C4-S and C6-S.
2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan and,
and to various extents, CS and DS. They are
endo-beta-N-acetylhexosaminidases that operate by a beta
elimination reaction that yields primarily disaccharide end
products. 3. Hyaluronidases (EC 3.2.1.36) from leeches, other
parasites, and crustaceans are endo-beta-glucuronidases that
generate tetrasaccharide and hexasaccharide end products through
hydrolysis of the beta 1-3 linkage.
[0146] Mammalian hyaluronidases can be further divided into two
groups: neutral-active and acid-active enzymes. There are six
hyaluronidase-like genes in the human genome, HYAL1, HYAL2, HYAL3,
HYAL4, HYALP1 and PH20/SPAM1. HYALP1 is a pseudogene, and HYAL3 has
not been shown to possess enzyme activity toward any known
substrates. HYAL4 is a chondroitinase and exhibits little activity
towards hyaluronan. HYAL1 is the prototypical acid-active enzyme
and PH20 is the prototypical neutral-active enzyme. Acid-active
hyaluronidases, such as HYAL1 and HYAL2 generally lack catalytic
activity at neutral pH (i.e. pH 7). For example, HYAL1 has little
catalytic activity in vitro over pH 4.5 (Frost I. G. and Stern, R.,
"A microtiter-based assay for hyaluronidase activity not requiring
specialized reagents", Anal. Biochemistry, 1997; 251:263-269).
HYAL2 is an acid-active enzyme with a very low specific activity in
vitro.
[0147] The hyaluronidase-like enzymes can also be characterized by
those which are generally locked to the plasma membrane via a
glycosylphosphatidyl inositol 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 which are generally soluble such as human
HYAL1 (Frost, I. G. et al., "Purification, cloning, and expression
of human plasma hyaluronidase", Biochem. Biophys. Res. Commun.
1997; 236(1):10-15). However, there are variations from species to
species: bovine PH20 for example 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 permitted the use of the
soluble bovine testes hyaluronidase enzyme as an extract for
clinical use (Wydase.TM., Hyalase.TM.) Other PH20 species are lipid
anchored enzymes that are generally not soluble without the use of
detergents or lipases. For example, human PH20 is anchored to the
plasma membrane via a GPI anchor. Attempts to make human PH20 DNA
constructs that would not introduce a lipid anchor into the
polypeptide resulted in either 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 a soluble and membrane bound form. While the 64 kDa
membrane bound form possesses enzyme activity at pH 7.0, the 54 kDa
form is only active at pH 4.0 (Cherr et al., Dev. Biol., 1996; 10;
175(1): 142-53). Thus, soluble forms of PH20 are often lacking
enzyme activity under neutral conditions.
[0148] As noted above and in accordance with the teachings in
WO2006/091871 and U.S. Pat. No. 7,767,429, small amounts of soluble
hyaluronidase glycoproteins (sHASEGPs) can be introduced into a
formulation in order to facilitate the administration of
therapeutic drug into the hypodermis. By rapidly depolymerizing HA
in the extracellular space sHASEGP reduces the viscosity of the
interstitium, thereby increasing hydraulic conductance and allowing
for larger volumes to be administered safely and comfortably into
the SC tissue. The increased hydraulic conductance induced by
sHASEGP through reduced interstitial viscosity allows for greater
dispersion, potentially increasing the systemic bioavailability of
SC administered therapeutic drug.
[0149] When injected in the hypodermis, the depolymerization of HA
by sHASEGP is localized to the injection site in the SC tissue.
Experimental evidence shows that the sHASEGP is inactivated locally
in the interstitial space with a half life of 13 to 20 minutes in
mice, without detectable systemic absorption in blood following
single intravenous dose in CD-1 mice. Within the vascular
compartment sHASEGP demonstrates a half life of 2.3 and 5 minutes
in mice and Cynomolgus monkeys, respectively, with doses up to 0.5
mg/kg. The rapid clearance of sHASEGP, combined with the continual
synthesis of the HA substrate in the SC tissue, results in a
transient and locally-active permeation enhancement for other
co-injected molecules, the effects of which are fully reversible
within 24 to 48 hours post 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).
[0150] In addition to its effects on local fluid dispersion,
sHASEGP also acts as absorption enhancer. Macromolecules greater
than 16 kilodaltons (kDa) are largely excluded from absorption
through the capillaries via diffusion and are mostly absorbed via
the draining lymph nodes. A subcutaneously administered
macromolecule such as e.g. a therapeutic antibody (molecular weight
approximately 150 kDa) must therefore traverse the interstitial
matrix before reaching the draining lymphatics for subsequent
absorption into the vascular compartment. By increasing local
dispersion, sHASEGP increases the rate (Ka) of absorption of many
macromolecules. This leads to increased peak blood levels
(C.sub.max) and potentially to increased bioavailability relative
to SC administration in the absence of sHASEGP (Bookbinder L. H.,
et al., "A recombinant human enzyme for enhanced interstitial
transport of therapeutics", J. Control. Release 2006; 114:
230-241).
[0151] Hyaluronidase products of animal origin have been used
clinically for over 60 years, primarily to increase the dispersion
and absorption of other co-administered drugs and for
hypodermoclysis (SC injection/infusion of fluid in large volume)
(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). The details on
the mechanism of action of hyaluronidases have been 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, A. M., Maibach, H. I., 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, U. B. G., Dahl, L. B., Reed,
R. K., "Catabolism of hyaluronan in rabbit skin takes place
locally, in lymph nodes and liver", Exp. Physiol. 1991; 76:
695-703; Laurent, T. C. and Fraser, J. R. E., "Degradation of
Bioactive Substances: Physiology and Pathophysiology", Henriksen,
J. H. (Ed) CRC Press, Boca Raton, Fla.; 1991. pp. 249-265; Harris,
E. N., et al., "Endocytic function, glycosaminoglycan specificity,
and antibody sensitivity of the recombinant human 190-kDa
hyaluronan receptor for endocytosis (HARE)", J. Biol. Chem. 2004;
279:36201-36209; 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.
Hyaluronidase products approved in EU countries include Hylase.RTM.
"Dessau" and Hyalase.RTM.. Hyaluronidase products of animal origin
approved in the US include Vitrase.TM., Hydase.TM., and
Amphadase.TM.
[0152] The safety and efficacy of hyaluronidase products have been
widely established. The most significant safety risk identified is
hypersensitivity and/or allergenicity, which is thought to be
related to the lack of purity of the 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 with respect to the approved
dosages of animal-derived hyaluronidases between the UK, Germany
and the US. In the UK, the usual dose as an adjuvant to
subcutaneous or intramuscular injection is 1500 units, added
directly to the injection. In the US, the usual dose used for this
purpose is 150 units. In hypodermoclysis, hyaluronidase is used to
aid the subcutaneous administration of relatively large volumes of
fluids. In the UK, 1500 units of hyaluronidase are generally given
with each 500 to 1000 ml of fluid for subcutaneous use. In the US,
150 units are considered adequate for each liter of hypodermoclysis
solution. In Germany, 150 to 300 units are considered adequate for
this purpose. In the UK, the diffusion of local anesthetics is
accelerated by the addition of 1500 units. In Germany and the US
150 units are considered adequate for this purpose. The dosage
differences notwithstanding (the dosage in the UK is ten times
higher than in the US), no apparent differences in the safety
profiles of animal-derived hyaluronidase products marketed in the
US and UK, respectively, have been reported.
[0153] On Dec. 2, 2005, Halozyme Therapeutics Inc. received
approval from the FDA for an injectable formulation of the
recombinant human hyaluronidase, rHuPH20(HYLENEX.TM.). The FDA
approved HYLENEX.TM. at a dose of 150 units for SC administration
of the following indications: [0154] as an adjuvant to increase the
absorption and dispersion of other injected drugs [0155] for
hypodermoclysis [0156] as an adjunct in SC urography for improving
resorption of radiopaque agents.
[0157] As part of that regulatory review it was established that
rHuPH20 possesses the same properties of enhancing the dispersion
and absorption of other injected drugs as the previously approved
animal-derived hyaluronidase preparations, but with an improved
safety profile. In particular, the use of recombinant human
hyaluronidase (rHuPH20) compared with animal-derived hyaluronidases
minimizes the potential risk of contamination with animal pathogens
and transmissible spongiform encephalopathies.
[0158] Soluble Hyaloronidase glycoproteins (sHASEGP), a process for
preparing the same and their use in pharmaceutical compositions
have been described in WO 2004/078140.
[0159] The detailed experimental work as outlined further below has
shown that the claimed formulation surprisingly has favorable
storage stability and fulfils all necessary requirements for
approval by the health authorities.
[0160] The hyaluronidase enzyme in the formulation in accordance
with the present invention is believed to enhance the delivery of
the anti-CD20 antibody to the systemic circulation, e.g. by
increasing the absorption of the active substance (it acts as a
permeation enhancer). The hyaluronidase enzyme is also believed to
increases the delivery of the therapeutic anti-CD20 antibody into
the systemic circulation via the subcutaneous application route by
the reversible hydrolyzation of hyaluronan, an extracellular
component of the SC interstitial tissue. The hydrolysis of
hyaluronan in the hypodermis temporarily opens channels in the
interstitial space of the SC tissue and thereby improves the
delivery of the therapeutic anti-CD20 antibody into the systemic
circulation. In addition, the administration shows reduced pain in
humans and less volume-derived swelling of the SC tissue.
[0161] Hyaluronidase, when administered locally has its entire
effect locally. In other word hyaluronidase is inactivated and
metabolized locally in minutes and has not been noted to have
systemic or long term effects. The rapid inactivation of
hyaluronidase within minutes when it enters the blood stream
precludes a realistic ability to perform comparable biodistribution
studies between different hyaluronidase products. This property
also minimizes any potential systemic safety concerns because the
hyaluronidase product cannot act at distant sites.
[0162] The unifying feature of all hyaluronidase enzymes in
accordance with the present invention is their ability to
depolymerize hyaluronan, regardless of differences in chemical
structure, in species source, in tissue sources, or in the batches
of drug product sourced from the same species and tissue. They are
unusual in the fact that their activity is the same (except for
potency) in spite of having different structures.
[0163] The hyaluronidase enzyme in accordance with the formulation
of the present invention is characterized by having no adverse
effect on the molecular integrity of the anti-CD20 antibody in the
stable pharmaceutical formulation described herein. Furthermore,
the hyaluronidase enzyme merely modifies the delivery of the
anti-CD20 antibody to the systemic circulation but does not possess
any properties that could provide or contribute to the therapeutic
effects of systemically absorbed anti-CD20 antibody. The
hyaluronidase enzyme is not systemically bioavailable and does not
adversely affect the molecular integrity of the anti-CD20 antibody
at the recommended storage conditions of the stable pharmaceutical
formulation in accordance with the invention. It is therefore to be
considered as an excipient in the anti-CD20 antibody formulation in
accordance with this invention. As it exerts no therapeutic effect
it represents a constituent of the pharmaceutical form apart from
the therapeutically active anti-CD20 antibody.
[0164] A number of suitable hyaluronidase enzymes in accordance
with the present invention are known from the prior art. The
preferred enzyme is a human hyaluronidase enzyme, most preferably
the enzyme known as rHuPH20. rHuPH20 is a member of the family of
neutral and acid-active .beta.-1,4 glycosyl hydrolases that
depolymerize hyaluronan by the hydrolysis of the .beta.-1,4 linkage
between the C.sub.1 position of N-acetyl glucosamine and the
C.sub.4 position of glucuronic acid. Hyaluronan is a polysaccharide
found in the intracellular ground substance of connective tissue,
such as the subcutaneous interstitial tissue, and of certain
specialized tissues, such as the umbilical cord and vitreous humor.
The hydrolysis of hyaluronan temporarily decreases the viscosity of
the interstitial tissue and promotes the dispersion of injected
fluids or of localized transudates or exudates, thus facilitating
their absorption. The effects of hyaluronidase are local and
reversible with complete reconstitution of the tissue hyaluronan
occurring within 24 to 48 hours (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). The increase in the permeability of connective tissue
through the hydrolysis of hyaluronan correlates with the efficacy
of hyaluronidase for their capability to increase the dispersion
and absorption of co-administered molecules.
[0165] The human genome contains several hyaluronidase genes. Only
the PH20 gene product possesses effective hyaluronidase activity
under physiologic extracellular conditions and acts as a spreading
agent, whereas acid-active hyaluronidases do not have this
property.
[0166] 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
physiologic extracellular conditions and acts as a spreading agent.
Naturally occurring human PH20 protein has a lipid anchor attached
to the carboxy terminal amino acid that anchors it to the plasma
membrane. The rHuPH20 enzyme developed by Halozyme is a truncated
deletion variant that lacks such amino acids in the carboxy
terminus responsible for the lipid attachment. This gives rise to a
soluble, neutral pH-active enzyme similar to the protein found in
bovine testes preparations. The rHuPH20 protein is synthesized with
a 35 amino acid signal peptide that is removed from the N-terminus
during the process of secretion. The mature rHuPH20 protein
contains an authentic N-terminal amino acid sequence orthologous to
that found in some bovine hyaluronidase preparations.
[0167] The PH20 hyaluronidases, including the animal derived PH20
and recombinant human rHuPH20, depolymerize hyaluronan by the
hydrolysis of the .beta.-1,4 linkage between the C.sub.1 position
of N-acetyl glucosamine and the C.sub.4 position of glucuronic
acid. The tetrasaccharide is the smallest digestion product
(Weissmann, B., "The transglycosylative action of testicular
hyaluronidase", J. Biol. Chem., 1955; 216: 783-94). This N-acetyl
glucosamine/glucuronic acid structure is not found in N-linked
glycans of recombinant biological products and therefore rHuPH20
will not affect the glycosylation of antibodies it is formulated
with, such as e.g. Rituximab. The rHuPH20 enzyme itself possesses
six N-linked glycans per molecule with core structures similar to
that found in monoclonal antibodies. As anticipated, 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 constant synthesis of
hyaluronan lead to a short and local action of the enzyme on
tissues.
[0168] The hyaluronidase enzyme which is an excipient in the
subcutaneous formulation in accordance with the present invention
is preferably prepared by using recombinant DNA technology. In this
way it is ensured that the same protein (identical amino acid
sequence) is obtained all the time and that allergic reactions
caused by contaminating proteins co-purified during extraction from
a tissue is avoided. The hyaluronidase enzyme used in the
formulation in accordance with the present invention is preferably
a human enzyme, most preferably rHuPH20.
[0169] The amino acid sequence of rHuPH20(HYLENEX.TM.) is well
known and available under CAS Registry No. 757971-58-7. The
approximate molecular weight is 61 kDa. See, also, U.S. Pat. No.
7,767,429.
[0170] Multiple structural and functional comparisons have been
performed between naturally sourced mammalian hyaluronidase and
PH-20 cDNA clones from humans and other mammals. The PH-20 gene is
the gene used for the recombinant product rHuPH20; however the
recombinant drug product is a 447 amino acid truncated version of
the full protein encoded by the PH-20 gene. Structural similarities
with respect to amino acid sequences rarely exceed 60% in any
comparison. Functional comparisons show that the activity of
rHuPH20 is very similar to that of previously approved
hyaluronidase products. This information is consistent with the
clinical findings during the past 50 years that regardless of the
source of the hyaluronidase, the clinical safety and efficacy of
units of hyaluronidase are equivalent.
[0171] The use of rHuPH20 in the anti-CD20 antibody SC formulation
in accordance with the present invention allows the administration
of higher volumes of drug product and to potentially enhance the
absorption of subcutaneously administered CD20 antibody, preferably
Rituximab into the systemic circulation.
[0172] The osmolality of the stable pharmaceutical formulation in
accordance with the invention is 350.+-.50 mOsm/kg.
[0173] The stable pharmaceutical formulation in accordance with the
invention is essentially free from visible (human eye inspection)
particles. The sub-visible particles (as measured by light
obscuration) should preferably fulfill the following criteria:
maximum number of particles.gtoreq.10.mu.m per vial->6000
maximum number of particles.gtoreq.25.mu.m per vial->600
[0174] In a further aspect the present invention provides a use of
a formulation for the preparation of a medicament useful for
treating a disease or disorder amenable to treatment with an
anti-CD20 antibody such as preferably cancer or a non-malignant
disease in a subject comprising administering the formulation
described herein to a subject in an amount effective to treat the
said disease or disorder. Preferably the anti-CD20 antibody is
co-administered concomitantly or sequentially with a
chemotherapeutic agent.
[0175] In a further aspect the present invention provides a method
of treating a disease or disorder which is amenable to treatment
with an anti-CD20 antibody (e.g. cancer (preferred) or a
non-malignant disease) in a subject comprising administering the
formulation described herein to a subject in an amount effective to
treat the said disease or disorder. The cancer or a non-malignant
disease will generally involve CD20-expressing cells, such that the
CD20 antibody in the therapeutic pharmaceutical SC formulation in
accordance with the present invention is able to bind to the
affected cells. The cancer is preferably a CD20 expressing cancer.
The non-malignant disease that can be treated with the composition
in accordance with the present invention is preferably an
autoimmune disease as defined herein. Preferably the anti-CD20
antibody is co-administered concomitantly or sequentially with a
chemotherapeutic agent.
[0176] The addition of the hyaluronidase to the formulation allows
increasing the injection volume which can be safely and comfortably
administered subcutaneously. The preferred injection volume is 1 to
15 ml. It has been observed that the administration of the
formulation in accordance with the present invention increases the
dispersion, absorption and the bioavailability of the therapeutic
antibody. Large molecules (i.e. >16 kDa) that are administered
via the SC route are preferentially absorbed into the vascular
compartment through the draining lymphatic fluids (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). The rate of
introduction of these large molecules into the systemic circulation
is thus slowed relative to intravenous infusion, therefore
potentially resulting in reduced frequency/intensity of infusion
related reactions.
[0177] For the production of the subcutaneous CD20 antibody
(preferably Rituximab) formulation in accordance with the invention
requires high antibody concentrations (approx. 120 mg/ml) in the
final step of purification of the manufacturing process. Therefore
an additional process step (ultrafiltration/diafiltration) is added
to the conventional manufacturing process of the CD20 antibody,
preferably Rituximab. The highly concentrated, stable
pharmaceutical anti-CD20 antibody formulation in accordance with
the present invention can also be provided as stabilized protein
formulation which can reconstituted with a suitable diluent to
generate a high anti-CD20 antibody concentration reconstituted
formulation.
[0178] The CD20 antibody SC formulation in accordance with this
invention is preferably used to treat cancer, preferably a CD20
expressing cancer.
[0179] The term "about" as used in the present patent specification
is meant to specify that the specific value provided may vary to a
certain extent, such as e.g. means that variations in the range of
.+-.10%, preferably .+-.5%, most preferably .+-.2% are included in
the given value.
[0180] Aside from the above assays, various in vivo assays are
available to the skilled practitioner. For example, one may expose
cells within the body of the patient to an antibody which is
optionally labeled with a detectable label, e.g. a radioactive
isotope, and binding of the antibody to cells in the patient can be
evaluated, e.g. by external scanning for radioactivity or by
analyzing a biopsy taken from a patient previously exposed to the
antibody.
[0181] It is contemplated that the CD20 antibody SC formulation in
accordance with this invention may also be used to treat various
non-malignant diseases or disorders, such a include autoimmune
disease as defined herein; endometriosis; scleroderma; restenosis;
polyps such as colon polyps, nasal polyps or gastrointestinal
polyps; fibroadenoma; respiratory disease; cholecystitis;
neurofibromatosis; polycystic kidney disease; inflammatory
diseases; skin disorders including psoriasis and dermatitis;
vascular disease; conditions involving abnormal proliferation of
vascular epithelial cells; gastrointestinal ulcers; Menetrier's
disease, secreting adenomas or protein loss syndrome; renal
disorders; angiogenic disorders; ocular disease 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 associated pathologies such as
osteoarthritis, rickets and osteoporosis; damage following a
cerebral ischemic event; fibrotic or edemia diseases such as
hepatic cirrhosis, lung fibrosis, carcoidosis, throiditis,
hyperviscosity syndrome systemic, Osier Weber-Rendu disease,
chronic occlusive pulmonary disease, or edema following burns,
trauma, radiation, stroke, hypoxia or ischemia; hypersensitivity
reaction of the skin; diabetic retinopathy and diabetic
nephropathy; Guillain-Barre syndrome; graft versus host disease or
transplant rejection; Paget's disease; bone or joint inflammation;
photoaging (e.g. caused by UV radiation of human skin); benign
prostatic hypertrophy; certain microbial infections including
microbial pathogens selected from adenovirus, hantaviruses,
Borrelia burgdorferi, Yersinia spp. and Bordetella pertussis;
thrombus caused by platelet aggregation; reproductive conditions
such as endometriosis, ovarian hyperstimulation syndrome,
preeclampsia, dysfunctional uterine bleeding, or menometrorrhagia;
synovitis; atheroma; acute and chronic nephropathies (including
proliferative glomerulonephritis and diabetes-induced renal
disease); eczema; hypertrophic scar formation; endotoxic shock and
fungal infection; familial adenomatosis polyposis;
neurodedenerative diseases (e.g. Alzheimer's disease, AIDS-related
dementia, Parkinson's disease, amyotrophic lateral sclerosis,
retinitis pigmentosa, spinal muscular atrophy and cerebellar
degeneration); myelodysplastic syndromes; aplastic anemia; ischemic
injury; fibrosis of the lung, kidney or liver; T-cell mediated
hypersensitivity disease; infantile hypertrophic pyloric stenosis;
urinary obstructive syndrome; psoriatic arthritis; and Hasimoto's
thyroiditis. Preferred non-malignant indications for therapy are as
defined herein.
[0182] Where the indication is cancer, the patient may be treated
with a combination of the antibody formulation, and a
chemotherapeutic agent. The combined administration includes
co-administration or concurrent administration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Thus, the
chemotherapeutic agent may be administered prior to, or following,
administration of the antibody formulation in accordance with the
present invention. In this embodiment, the timing between at least
one administration of the chemotherapeutic agent and at least one
administration of the antibody formulation in accordance with the
present invention is preferably approximately 1 month or less, and
most preferably approximately 2 weeks or less. Alternatively, the
chemotherapeutic agent and the antibody formulation in accordance
with the present invention are administered concurrently to the
patient, in a single formulation or separate formulations.
[0183] Treatment with the said antibody formulation will result in
an improvement in the signs or symptoms of cancer or disease. For
instance, where the disease being treated is cancer, such therapy
may result in an improvement in survival (overall survival and/or
progression free survival) and/or may result in an objective
clinical response (partial or complete). Moreover, treatment with
the combination of the chemotherapeutic agent and the antibody
formulation may result in a synergistic or greater than additive,
therapeutic benefit to the patient.
[0184] Preferably, the antibody in the formulation administered is
a naked antibody. However, the antibody administered may be
conjugated with a cytotoxic agent. Preferably, the immunoconjugate
and/or antigen to which it is bound is/are internalized by the
cell, resulting in increased therapeutic efficacy of the
immunoconjugate in killing the cancer cell to which it binds. In a
preferred embodiment, the cytotoxic agent targets or interferes
with nucleic acid in the cancer cell. Examples of such cytotoxic
agents include maytansinoids, calioheamicins, ribonucleases and DNA
endonucleases. The preferred immunoconjugates are
Rituximab-maytansinoid immunoconjugates similarly to
Trastuzumab-DM1 (T-DM1) as they are described in WO 2003/037992,
more preferably the immunoconjugate T-MCC-DM1.
[0185] For subcutaneous delivery, the formulation may be
administered via a suitable device, such as (but not limited to) a
syringe; an injection device (e.g. the INJECT-EASE.TM. and
GENJECT.TM. device); an infusion pump (such as e.g. Accu-Chek.TM.);
an injector pen (such as the GENPEN.TM.; an needleless device (e.g.
MEDDECTOR.TM. and BIOJECTOR.TM.); or via a subcutaneous patch
delivery system.
[0186] The amount of administration of said anti-CD20 antibody
formulation for the prevention or treatment of disease, and the
timing of administration will depend on the type (species, gender,
age, weight, etc.) and condition of the patient being treated and
the severity of the disease or condition being treated. Also
important for the appropriate dose determination are the course of
the disease, whether the antibody is administered for preventive or
therapeutic purposes, the previous therapy, the patient's clinical
history and his response to the antibody. The ultimate dose
determination is at the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of
said anti-CD20 antibody is an initial candidate dosage for
administration to the patient.
[0187] The preferred dosage of said anti-CD20 antibody will be in
the range from 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) may be
administered to the patient. Depending on the on the type (species,
gender, age, weight, etc.) and condition of the patient and on the
type of anti-CD20 antibody, the dosage of said first can differ
from the dosage of the second anti-CD20 antibody. Such doses may be
administered daily or intermittently, e.g. every third to six day
or even every one to three weeks. An initial higher loading dose,
followed by one or more lower doses may be administered. Based on
clinical studies (see also Examples 3 and 4 for non-limiting
exemplification for rituximab), the preferred dosage range is 300
mg/m.sup.2 to 900 mg/m.sup.2. More preferred, the preferred dosage
range of said anti-CD20 antibody is about 375 mg/m.sup.2 to about
800 mg/m.sup.2. Preferred specific dosages of said anti-CD20
antibody are dosages of about 375 mg/m.sup.2, about 625 mg/m.sup.2
and about 800 mg/m.sup.2. Also preferred are fixed doses of said
anti-CD20 antibody.
[0188] In one embodiment, fixed dosages for B-cell lymphomas,
preferably Non-Hodgkin's lymphoma, are as follows. Preferred are
about 1200 mg to about 1800 mg of said anti-CD20 antibody per dose.
More preferred are dosages selected from the group of about 1300
mg, about 1500 mg, about 1600 mg, and about 1700 mg of said
anti-CD20 antibody per dose. Most preferred, the fixed dosage for
B-cell lymphoma patients, preferably Non-Hodgkin's lymphoma
patients, is about 1400 mg of said anti-CD20 antibody (e.g.
Rituximab) per dose which may be administered according to various
schedules including approximately every 2 months (including
approximately every 8 weeks), approximately every 3 months
(including approximately every 12 weeks), for about 2 years (or
more), etc (see also Examples 3 and 4 for non-limiting
exemplification for rituximab).
[0189] In another embodiment, fixed dosages for leukemia patients,
preferably chronic lymphocytic leukemia (CLL) patients, are as
follows. Preferred are about 1600 mg to about 2200 mg of said
anti-CD20 antibody per dose. More preferred are dosages selected
from the group of about 1700 mg, about 1800 mg, about 1900 mg, and
about 2100 mg of said anti-CD20 antibody per dose. In one
embodiment, the fixed dosage for leukemia patients, preferably CLL
patients, is about 1870 mg of said anti-CD20 antibody (e.g.
Rituximab) per dose.
[0190] In yet another embodiment, fixed dosages for patients with
autoimmune disease, such as rheumatoid arthritis, multiple
sclerosis, lupus nephritis, diabetes, ITP, and vasculitis are as
follows. Preferred are about 1200 mg to about 2200 mg of said
anti-CD20 antibody per dose, for example about 1500 mg of said
anti-CD20 antibody (e.g. Rituximab) per dose.
[0191] If a chemotherapeutic agent is administered, it is usually
administered at dosages known therefore, or optionally lowered due
to combined action of the drugs or negative side effects
attributable to administration of the chemotherapeutic agent.
Preparation and dosing schedules for such chemotherapeutic agents
may be used according to manufacturers' instructions or as
determined empirically by the skilled practitioner. Preparation and
dosing schedules for such chemotherapy are also described in
Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,
Baltimore, Md. (1992).
[0192] The stable pharmaceutical formulation of the
pharmaceutically active anti-CD20 antibody in accordance with the
invention is preferably administered as subcutaneous injection,
whereby the administration is preferably repeated several times
with time intervals of 3 weeks (q3w). Most preferably the full
volume of the injection fluid is administered within a time period
of 1 to 10 minutes, preferably 2 to 6 minutes, most preferably
3.+-.1 minutes. Most preferably, 2 ml/minute are administered, i.e.
for example approx. 240 mg/min. For many patients where no other
intravenous (IV) chemotherapeutic agents are given, such
subcutaneous administration leads to increased patient convenience
with the potential for self-administration at home. This leads to
improved compliance and would reduce/eliminate costs associated
with IV administration (viz., nursing costs for IV administration,
rental of day-beds, patient travel etc). Subcutaneous
administration in accordance with the present invention will most
likely be associated with a reduced frequency and/or intensity of
infusion-related reactions.
[0193] In a preferred embodiment, the medicament is useful for
preventing or reducing metastasis or further dissemination in such
a patient suffering from CD20 expressing cancer. The medicament is
useful for increasing the duration of survival of such a patient,
increasing the progression free survival of such a patient,
increasing the duration of response, resulting in a statistically
significant and clinically meaningful improvement of the treated
patient as measured by the duration of survival, progression free
survival, response rate or duration of response. In a preferred
embodiment, the medicament is useful for increasing the response
rate in a group of patients.
[0194] In the context of this invention, one or more additional
other growth-inhibitory, cytotoxic, chemotherapeutic,
anti-angiogenic, anti-cancer agents or cytokine(s), or compounds
that enhance the effects of such agents may be used in the
anti-CD20 antibody treatment of CD20 expressing cancer. Preferably
the anti-CD20 antibody treatment is used without such additional
cytotoxic, chemotherapeutic or anti-cancer agents, or compounds
that enhance the effects of such agents.
[0195] Such agents include, for example: alkylating agents or
agents with an alkylating action, such as cyclophosphamide (CTX;
e.g. Cytoxan.RTM.), chlorambucil (CHL; e.g. Leukeran.RTM.),
cisplatin (C is P; e.g. Platinol.RTM.) busulfan (e.g.
Myleran.RTM.), melphalan, carmustine (BCNU), streptozotocin,
triethylenemelamine (TEM), mitomycin C, and the like;
anti-metabolites, such as methotrexate (MTX), etoposide (VP16; e.g.
Vepesid.RTM.), 6-mercaptopurine (6 MP), 6-thiocguanine (6TG),
cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g.
Xeloda.RTM.), dacarbazine (DTIC), and the like; antibiotics, such
as actinomycin D, doxorubicin (DXR; e.g. Adriamycin.RTM.),
daunorubicin (daunomycin), bleomycin, mithramycin and the like;
alkaloids, such as vinca alkaloids such as vincristine (VCR),
vinblastine, and the like; and other antitumor agents, such as
paclitaxel (e.g. Taxol.RTM.) and paclitaxel derivatives, the
cytostatic agents, glucocorticoids such as dexamethasone (DEX; e.g.
Decadron.RTM.) and corticosteroids such as prednisone, nucleoside
enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes
such as asparaginase, leucovorin and other folic acid derivatives,
and similar, diverse antitumor agents. The following agents may
also be used as additional agents: arnifostine (e.g. Ethyol.RTM.),
dactinomycin, mechlorethamine (nitrogen mustard), streptozocin,
cyclophosphamide, lomustine (CCNU), doxorubicin lipo (e.g.
Doxil.RTM.), gemcitabine (e.g. Gemzar.RTM.), daunorubicin lipo
(e.g. Daunoxome.RTM.), procarbazine, mitomycin, docetaxel (e.g.
Taxotere.RTM.), 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, pegaspargase, pentostatin, pipobroman, plicamycin,
tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil
mustard, vinorelbine, chlorambucil. Preferably the anti-CD20
antibody treatment is used without such additional agents.
[0196] The use of the cytotoxic and anticancer agents described
above as well as antiproliferative target-specific anticancer drug
like protein kinase inhibitors in chemotherapeutic regimens is
generally well characterized in the cancer therapy arts, and their
use herein falls under the same considerations for monitoring
tolerance and effectiveness and for controlling administration
routes and dosages, with some adjustments. For example, the actual
dosages of the cytotoxic agents may vary depending upon the
patient's cultured cell response determined by using histoculture
methods. Generally, the dosage will be reduced compared to the
amount used in the absence of additional other agents.
[0197] Typical dosages of an effective cytotoxic agent can be in
the ranges recommended by the manufacturer, and where indicated by
in vitro responses or responses in animal models, can be reduced by
up to about one order of magnitude concentration or amount. Thus,
the actual dosage will depend upon the judgment of the physician,
the condition of the patient, and the effectiveness of the
therapeutic method based on the in vitro responsiveness of the
primary cultured malignant cells or histocultured tissue sample, or
the responses observed in the appropriate animal models.
[0198] In the context of this invention, an effective amount of
ionizing radiation may be carried out and/or a radiopharmaceutical
may be used in addition to the anti-CD20 antibody treatment of CD20
expressing cancer. The source of radiation can be either external
or internal to the patient being treated. When the source is
external to the patient, the therapy is known as external beam
radiation therapy (EBRT). When the source of radiation is internal
to the patient, the treatment is called brachytherapy (BT).
Radioactive atoms for use in the context of this invention can be
selected from the group including, but not limited to, radium,
cesium-137, iridium-192, americium-241, gold-198, cobalt-57,
copper-67, technetium-99, iodine-123, iodine-131, and indium-111.
Is also possible to label the antibody with such radioactive
isotopes. Preferably the anti-CD20 antibody treatment is used
without such ionizing radiation.
[0199] Radiation therapy is a standard treatment for controlling
unresectable or inoperable tumors and/or tumor metastases. Improved
results have been seen when radiation therapy has been combined
with chemotherapy. Radiation therapy is based on the principle that
high-dose radiation delivered to a target area will result in the
death of reproductive cells in both tumor and normal tissues. The
radiation dosage regimen is generally defined in terms of radiation
absorbed dose (Gy), time and fractionation, and must be carefully
defined by the oncologist. The amount of radiation a patient
receives will depend on various considerations, but the two most
important are the location of the tumor in relation to other
critical 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 will be a treatment schedule over a 1
to 6 week period, with a total dose of between 10 and 80 Gy
administered to the patient in a single daily fraction of about 1.8
to 2.0 Gy, 5 days a week. In a preferred embodiment of this
invention there is synergy when tumors in human patients are
treated with the combination treatment of the invention and
radiation. In other words, the inhibition of tumor growth by means
of the agents comprising the CD20 antibody formulation of the
invention is enhanced when combined with radiation, optionally with
additional chemotherapeutic or anticancer agents. Parameters of
adjuvant radiation therapies are, for example, contained in WO
99/60023.
[0200] Other therapeutic regimens may be combined with the antibody
including, but not limited to a second (third, fourth, etc)
chemotherapeutic agent(s) (in another word a "cocktail" of
different chemotherapeutic agents); another monoclonal antibody; a
growth inhibitory agent; a cytotoxic agent; a chemotherapeutic
agent; an anti-angiogenic agent; and/or cytokine, etc.; or any
suitable combination thereof.
[0201] In addition to the above therapeutic regimes, the patient
may be subjected to surgical removal of cancer cells and/or
radiation therapy.
[0202] In another embodiment of the invention, an article of
manufacture is provided which contains the pharmaceutical
formulation of the present invention and provides instructions for
its use. This article of manufacture comprises a container.
Suitable containers include, for example, bottles, vials (e.g.
multiple or dual chamber vials), syringes (such as multiple or dual
chamber syringes) and test tubes. The container may be formed from
a variety of materials such as glass or plastic. The container
holds the formulation and the label on, or associated with, the
container may indicate directions for use. The container holding
the formulation may be a multi-use vial, which allows for repeat
administrations (e.g. from 2 to 6 administrations) of the
reconstituted formulation. The article of manufacture may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0203] The antibody which is formulated in accordance with the
present invention is preferably essentially pure and desirably
essentially homogeneous (i.e. free from contaminating proteins etc,
whereby the hyaluronidase enzyme in the formulation in accordance
of this invention is not to be considered to be a contaminating
protein of the anti-CD20 monoclonal antibody in accordance of the
present invention).
[0204] The invention will be more fully understood by reference to
the following Examples. They should not, however, be construed as
limiting the scope of the invention. All literature and patent
citations are incorporated herein by reference.
EXAMPLES
[0205] The anti-CD20 formulations for subcutaneous administration
according to the invention were developed based on the experimental
results as provided below using the general preparatory and
analytical methods and assays as outlined below.
Example 1
Preparation of Highly Concentrated Liquid Formulations
[0206] Rituximab is manufactured by techniques generally known from
the production of recombinant proteins. A genetically engineered
Chinese hamster ovary cell line (CHO) prepared as described in U.S.
Pat. No. 7,381,560 is expanded in cell culture from a master cell
bank. The 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 contaminations, followed by anion exchange
chromatography and an ultrafiltration/diafiltration step.
[0207] rHuPH20 is manufactured by techniques generally known from
the production of recombinant proteins. The process begins with
thawing of cells from the working cell bank (WCB) or master cell
bank (MCB) and expansion through cell culture in a series of
spinner flasks. The cell culture up to 6 liters is used to provide
a continuous source of cells maintained under selective pressure
with methotrexate. When expanded to approximately 36 liters the
culture is transferred to a 400 liters bioreactor for a final batch
volume of approximately 300 liters. The production bioreactor is
operated in the fed-batch mode, with no selection pressure, and the
duration of the production phase is approximately two weeks.
rHuPH20 is secreted into the culture fluid. A 1000 liters
bioreactor can also be used for a final batch volume of 500 liters.
After completion of the production phase, the harvest is clarified
by filtration, and is then treated with solvent/detergent to
inactivate viruses. The protein is then purified by a series of
four column chromatography processes to remove process and product
related impurities. A viral filtration step is performed, and the
filtered bulk is then concentrated, 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 is stored
below -70.degree. C.
[0208] The other excipients of the formulation in accordance with
the present invention are widely used in the practice and known to
the person skilled in the art. There is therefore no need to be
explained them here in detail.
[0209] Liquid drug product formulations for subcutaneous
administration according to the invention were developed as
follows.
[0210] For the preparation of the liquid formulations Rituximab was
buffer exchanged against a diafiltration buffer containing the
anticipated buffer composition and where required, concentrated by
diafiltration to an antibody concentration of approx. 200 mg/ml.
After completion of the diafiltration operation, the excipients
(e.g. trehalose, rHuPH20, surfactant) were added as stock solutions
to the antibody solution. Finally the protein concentration was
adjusted with a buffer to the final Rituximab concentration of
approx. 120 mg/ml.
[0211] All formulations were sterile-filtered through 0.22 .mu.m
low protein binding filters and aseptically filled into sterile 6
ml glass vials closed with ETFE (Copolymer of ethylene and
tetrafluoroethylene)-coated rubber stoppers and alucrimp caps. The
fill volume was approx. 3.0 ml. These formulations were stored at
different climate conditions (5.degree. C., 25.degree. C. and
40.degree. C.) for different intervals of time and stressed by
shaking (1 week at a shaking frequency of 200 rpm at 5.degree. C.
and 25.degree. C.) and freeze-thaw stress methods. The samples were
analyzed before and after applying the stress tests by the
following analytical methods:
[0212] 1) UV spectrophotometry;
[0213] 2) Size Exclusion Chromatography (SEC);
[0214] 3) by Ion exchange chromatography (IEC);
[0215] 4) by turbidity of the solution;
[0216] 5) for visible particles; and
[0217] 6) for rHuPH20 activity.
[0218] UV spectroscopy, used for determination of protein content,
was performed on a Perkin Elmer .lamda.35 UV spectrophotometer in a
wavelength range from 240 nm to 400 nm. Neat protein samples were
diluted to approx. 0.5 mg/ml with the corresponding formulation
buffer. The protein concentration was calculated according to
Equation 1.
Protein content = A ( 280 ) - A ( 320 ) .times. dil . factor cm 2 /
mg .times. d cm Equation 1 ##EQU00002##
[0219] The UV light absorption at 280 nm was corrected for light
scattering at 320 nm and multiplied with the dilution factor, which
was determined from the weighed masses and densities of the neat
sample and the dilution buffer. The numerator was divided by the
product of the cuvette's path length d and the extinction
coefficient E.
[0220] Size Exclusion Chromatography (SEC) was used to detect
soluble high molecular weight species (aggregates) and low
molecular weight hydrolysis products (LMW) in the formulations. The
method used a suitable HPLC instrument equipped with a UV detector
(detection wave length 280 nm) and a TosoHaas TSK G3000SWXL column
(7.8.times.300 mm). Intact monomer, aggregates and hydrolysis
products were separated by an isocratic elution profile, using 0.2M
di-potassium hydrogen phosphate, 025 M potassium chloride, pH 7.0
with a flow rate of 0.5 ml/min,
[0221] Ion Exchange Chromatography (IEC) was performed to detect
chemical degradation products altering the net charge of Rituximab
in the formulations. For this purpose Rituximab was digested with
Papain. The method used a suitable HPLC instrument equipped with a
UV detector (detection wavelength 280 nm) and a Polymer Labs PL-SCX
1000A analytical cation-exchange column. 10 mM MES, pH 6.0 and 10
mM MES, 0.2 M sodium chloride, pH 6.0, were used as mobile phases A
and B, respectively, with a flow rate of 1 ml/min.
[0222] For the determination of the turbidity, opalescence was
measured in FTU (turbidity units) using a HACH 2100AN turbidimeter
at room temperature.
[0223] Samples were analyzed for visible particles by using a
Seidenader V90-T visual inspection instrument.
[0224] An in vitro enzyme assay of rHuPH20 as hyaluronidase was
used as activity assay. The assay is based on the formation of an
insoluble precipitate when hyaluronan (sodium hyaluronate) binds to
a cationic precipitant. Enzyme activity was measured by incubating
rHuPH20 with hyaluronan substrate and then precipitating the
undigested hyaluronan with acidified serum albumin (horse serum).
The turbidity was measured at a wavelength of 640 nm and the
decrease in turbidity resulting from enzyme activity on the
hyaluronan substrate is a measure of the enzyme activity. The
procedure is run using a standard curve generated with dilutions of
rHuPH20 assay reference standard, and sample activity is read from
the curve.
[0225] The results of the stability testing for the Formulations A
to J are provided in the tables below.
Compositions and Stability Data of Liquid Rituximab Drug Product
Formulations According to this Invention Formulation A is a liquid
formulation with the composition 120 mg/ml Rituximab, 20 mM
L-histidine, 210 mM trehalose dihydrate, 10 mM methionine, 0.06%
polysorbate 80, 2,000 U/ml rHuPH20, at pH 5.5.
TABLE-US-00002 Storage Protein Size Exclusion-HPLC Ion
Exchange-HPLC Enzyme temperature/ Storage concentration HMW Monomer
LMW Fc-Lys Fab pE/Q Turbidity Visible activity stress condition
Time (mg/ml) (%) (%) (%) (%) (%) (FTU) particles (U/ml) -- Initial
114 2.2 97.8 0.1 24 65 4.2 Free from 1970 particles Shaking
5.degree. C. 1 week 114 2.4 97.5 0.1 n.d. n.d. 4.2 Free from 1760
particles Shaking 25.degree. C. 1 week 115 2.3 97.6 0.1 n.d. n.d.
4.4 Free from 1676 particles Freezing/ 5 cycles 114 2.4 97.5 0.1
n.d. n.d. 4.4 Free from 2123 Thawing particles 5.degree. C. 4 weeks
114 2.2 97.8 0.1 25 65 4.7 Free from 1979 particles 13 weeks 114
2.1 97.8 0.1 26 62 4.6 Free from 2219 particles 26 weeks 114 2.1
97.8 0.2 25 63 4.4 Free from 2412 particles 25.degree. C. 4 weeks
114 2.0 97.9 0.1 25 64 4.7 Free from 1975 particles 13 weeks n.d.
1.9 97.8 0.3 24 61 4.8 Free from 2215 particles 26 weeks n.d. 1.9
97.6 0.5 22 60 4.3 Free from 2409 particles 40.degree. C. 4 weeks
114 2.1 97.3 0.6 19 61 5.5 Free from n.d. particles 13 weeks n.d.
3.2 91.4 5.4 13 55 8.1 Free from n.d. particles n.d. not
determined
Formulation B is a liquid formulation with the composition 120
mg/ml Rituximab, 20 mM L-histidine, 210 mM trehalose dihydrate, 10
mM methionine, 0.06% polysorbate 80, 2,000 U/ml rHuPH20, at pH
6.1.
TABLE-US-00003 Storage Protein Size Exclusion-HPLC Ion
Exchange-HPLC Enzyme temperature/ Storage concentration HMW Monomer
LMW Fc-Lys Fab pE/Q Turbidity Visible activity stress condition
Time (mg/ml) (%) (%) (%) (%) (%) (FTU) particles (U/ml) -- Initial
117 2.3 97.7 0.1 25 63 7.1 Free from 2463 particles Shaking
5.degree. C. 1 week 116 2.2 97.7 0.1 n.d. n.d. 7.2 Free from 2288
particles Shaking 25.degree. C. 1 week 118 2.1 97.8 0.1 n.d. n.d.
6.9 Free from 2613 particles Freezing/ (5 cycles) 116 2.3 97.7 0.1
n.d. n.d. 6.6 Essentially free 2259 Thawing from particles
5.degree. C. 4 weeks 117 2.2 97.8 0.1 25 62 6.3 Free from 2485
particles 13 weeks 114 2.3 97.6 0.1 25 62 6.3 Free from 2237
particles 26 weeks 119 2.2 97.7 0.1 25 61 6.8 Free from 2344
particles 25.degree. C. 4 weeks n.d. 2.0 97.9 0.1 25 62 6.6 Free
from 2179 particles 13 weeks n.d. 2.1 97.7 0.2 24 61 6.3 Free from
2083 particles 26 weeks n.d. 2.1 96.2 1.8 23 60 6.9 Free from 2397
particles 40.degree. C. 4 weeks n.d. 2.2 96.1 1.7 21 59 8.6 Free
from n.d. particles 13 weeks n.d. 3.3 92.2 4.5 14 53 21.0 Free from
n.d. particles n.d. not determined
Formulation C is a liquid formulation with the composition 120
mg/ml Rituximab, 20 mM L-histidine, 210 mM trehalose dihydrate, 10
mM methionine, 0.06% polysorbate 80, 12,000 U/ml rHuPH20, at pH
5.5.
TABLE-US-00004 Protein Size Exclusion-HPLC Ion Exchange-HPLC Enzyme
Storage Storage concentration HMW Monomer LMW Fc-Lys Fab pE/Q
Turbidity Visible activity condition Time (mg/ml) (%) (%) (%) (%)
(%) (FTU) particles (U/ml) -- Initial 126 1.7 98.3 0.0 27 58 4.4
Free from 11963 particles Shaking 5.degree. C. 1 week 127 1.6 98.3
0.0 n.d. n.d. 4.0 Free from 12083 particles Shaking 25.degree. C. 1
week 127 1.6 98.4 0.1 n.d. n.d. 4.5 Free from 11150 particles
Freezing/ (5 cycles) 126 1.6 98.4 0.0 n.d. n.d. 4.2 Essentially
free 11869 Thawing from particles 5.degree. C. 7 weeks 124 1.5 98.5
0.0 26 62 5.0 Free from 12206 particles 19 weeks 120 1.5 98.5 0.1
26 62 4.1 Free from 11945 particles 26 weeks n.d. n.d. n.d. n.d.
n.d. n.d. n.d. Free from n.d. particles 25.degree. C. 7 weeks n.d.
1.5 97.8 0.7 25 63 5.8 Free from 12259 particles 19 weeks n.d. 1.5
97.3 1.2 24 61 4.8 Free from 13137 particles 26 weeks n.d. 1.8 96.6
1.6 24 60 4.4 Free from 12948 particles 40.degree. C. 7 weeks n.d.
2.5 94.6 2.9 18 60 10.2 Free from n.d. particles 19 weeks n.d. 3.7
89.8 6.5 12 55 20.0 Free from n.d. particles n.d. not
determined
Formulation D is a liquid formulation with the composition 120
mg/ml Rituximab, 20 mM acetic acid, 210 mM trehalose dihydrate, 10
mM methionine, 0.06% polysorbate 20, 12,000 U/ml rHuPH20, at pH
5.5.
TABLE-US-00005 Protein Size Exclusion-HPLC Ion Exchange-HPLC Enzyme
Storage Storage concentration HMW Monomer LMW Fc-Lys Fab pE/Q
Turbidity Visible activity condition Time (mg/ml) (%) (%) (%) (%)
(%) (FTU) particles (U/ml) -- Initial 127 1.6 98.4 0.0 26 62 4.9
Free from 12619 particles Shaking 5.degree. C. 1 week 125 1.5 98.4
0.0 n.d. n.d. 4.3 Free from 12507 particles Shaking 25.degree. C. 1
week 123 1.5 98.4 0.1 n.d. n.d. 4.3 Free from 12923 particles
Freezing/ (5 cycles) 124 1.5 98.4 0.0 n.d. n.d. 4.4 Essentially
free 12394 Thawing from particles 5.degree. C. 7 weeks 125 1.5 98.4
0.0 26 63 5.0 Essentially free 10030 from particles 19 weeks 123
1.5 98.5 0.1 26 62 4.7 Free from 15324 particles 26 weeks n.d. n.d.
n.d. n.d. n.d. n.d. n.d. Free from n.d. particles 25.degree. C. 7
weeks n.d. 1.6 97.7 0.7 25 62 4.9 Free from 13099 particles 19
weeks n.d. 1.6 97.2 1.2 24 61 5.1 Free from 13031 particles 26
weeks n.d. n.d. n.d. n.d. n.d. n.d. n.d. Free from n.d. particles
40.degree. C. 7 weeks n.d. 2.6 94.8 2.6 17 60 28.7 Free from n.d.
particles 19 weeks n.d. 3.6 90.5 6.0 9 56 51.9 Free from n.d.
particles n.d. not determined
Formulation E is a liquid formulation with the composition 120
mg/ml Rituximab, 20 mM L-histidine, 210 mM trehalose dihydrate, 10
mM methionine, 0.06% polysorbate 20, 12,000 U/ml rHuPH20, at pH
5.5.
TABLE-US-00006 Protein Size Exclusion-HPLC Ion Exchange-HPLC Enzyme
Storage Storage concentration HMW Monomer LMW Fc-Lys Fab pE/Q
Turbidity Visible activity condition Time (mg/ml) (%) (%) (%) (%)
(%) (FTU) particles (U/ml) -- Initial 126 1.5 98.5 0.0 26 62 4.6
Essentially free 12231 from particles Shaking 5.degree. C. 1 week
128 1.5 98.5 0.0 n.d. n.d. 4.4 Essentially free 12524 from
particles Shaking 25.degree. C. 1 week 127 1.5 98.5 0.1 n.d. n.d.
4.2 Free from 11438 particles Freezing/ (5 cycles) 127 1.5 98.5 0.0
n.d. n.d. 4.3 Free from 14440 Thawing particles 5.degree. C. 7
weeks 125 1.5 98.5 0.0 26 62 4.7 Free from 12824 particles 19 weeks
125 1.5 98.5 0.1 26 62 4.5 Free from 13891 particles 26 weeks n.d.
n.d. n.d. n.d. n.d. n.d. n.d. Free from n.d. particles 25.degree.
C. 7 weeks n.d. 1.5 97.8 0.7 25 62 4.3 Free from 13540 particles 19
weeks n.d. 1.5 97.4 1.1 24 61 4.6 Free from 11243 particles 26
weeks n.d. n.d. n.d. n.d. n.d. n.d. n.d. Free from n.d. particles
40.degree. C. 7 weeks n.d. 2.5 94.6 2.9 18 60 10.6 Free from n.d.
particles 19 weeks n.d. 3.9 89.6 6.5 12 55 22.7 Free from n.d.
particles n.d. not determined
Formulation F is a liquid formulation with the composition 120
mg/ml Rituximab, 20 mM L-histidine, 120 mM sodium chloride, 10 mM
methionine, 0.02% polysorbate 80, 12,000 U/ml rHuPH20, at pH
5.5.
TABLE-US-00007 Ion Exchange-HPLC Protein Size Exclusion-HPLC Fab NS
Enzyme Storage Storage concentration HMW Monomer LMW clips a + b
Fc-Lys Fab pE/Q Turbidity Visible activity condition Time (mg/ml)
(%) (%) (%) (%) (%) (%) (FTU) particles (U/ml) -- Initial 124 1.6
98.3 0.0 3 26 62 28.7 Free from 12034 particles Shaking 5.degree.
C. 1 week 127 1.6 98.4 0.0 n.d. n.d. n.d. 31.0 Free from 12083
particles Shaking 25.degree. C. 1 week 125 2.4 97.5 0.1 n.d. n.d.
n.d. 31.1 Free from 11150 particles Freezing/ (5 cycles) 125 1.9
98.1 0.0 n.d. n.d. n.d. 30.4 Free from 11869 Thawing particles
5.degree. C. 7 weeks 122 1.6 98.4 0.0 3 25 63 31.4 Free from 10368
particles 19 weeks 118 1.5 98.4 0.1 3 26 62 31.8 Free from 11654
particles 26 weeks n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Free
from n.d. particles 25.degree. C. 7 weeks n.d. 1.7 97.6 0.7 3 25 63
31.1 Free from 13853 particles 19 weeks n.d. 1.7 97.1 1.2 3 24 61
31.6 Free from 12556 particles 26 weeks n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. Free from n.d. particles 40.degree. C. 7 weeks n.d.
2.9 94.1 3.1 5 19 59 87.0 Free from n.d. particles 19 weeks n.d.
4.3 88.8 6.9 6 13 55 211.0 Free from n.d. particles n.d. not
determined
Formulation G is a liquid formulation with the composition 120
mg/ml Rituximab, 20 mM citric acid, 120 mM sodium chloride, 10 mM
methionine, 0.02% polysorbate 80, 12,000 U/ml rHuPH20, at pH
6.5.
TABLE-US-00008 Ion Exchange-HPLC Protein Size Exclusion-HPLC Fab NS
Enzyme Storage Storage concentration HMW Monomer LMW clips a + b
Fc-Lys Fab pE/Q Turbidity Visible activity condition Time (mg/ml)
(%) (%) (%) (%) (%) (%) (FTU) particles (U/ml) -- Initial 126 1.8
98.1 0.0 4 26 62 40.3 Free from 10808 particles Shaking 5.degree.
C. 1 week 122 1.8 98.2 0.0 n.d. n.d. n.d. 35.6 Free from 9324
particles Shaking 25.degree. C. 1 week 125 2.3 97.7 0.1 n.d. n.d.
n.d. 35.7 Free from n.a. particles Freezing/ (5 cycles) 126 1.9
98.1 0.0 n.d. n.d. n.d. 34.5 Free from 11270 Thawing particles
5.degree. C. 7 weeks 124 1.8 98.2 0.0 3 24 63 38.5 Essentially free
12854 from particles 19 weeks 118 1.7 98.2 0.1 3 26 62 37.6 Free
from 11202 particles 26 weeks n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Free from n.d particles 25.degree. C. 7 weeks n.d. 1.9 97.4 0.7 2
24 63 40.2 Essentially free 11645 from particles 19 weeks n.d. 2.1
96.9 1.1 3 24 60 36.9 Free from 14233 particles 26 weeks n.d. n.d.
n.d. n.d. n.d. n.d. n.d. Free from n.d. particles 40.degree. C. 7
weeks n.d. 3.1 94.3 2.6 5 19 58 101.0 Essentially free n.d. from
particles 19 weeks n.d. 4.8 89.4 5.8 7 12 52 385.0 Free from n.d.
particles n.d. not determined
Formulation H is a liquid formulation with the composition 120
mg/ml Rituximab, 20 mM citric acid, 210 mM trehalose dihydrate, 10
mM methionine, 0.06% polysorbate 80, 12,000 U/ml rHuPH20, at pH
6.5.
TABLE-US-00009 Ion Exchange-HPLC Protein Size Exclusion-HPLC Fab NS
Enzyme Storage Storage concentration HMW Monomer LMW clips a + b
Fc-Lys Fab pE/Q Turbidity Visible activity condition Time (mg/ml)
(%) (%) (%) (%) (%) (%) (FTU) particles (U/ml) -- Initial 127 2.0
98.0 0.0 3 26 62 33.4 Free from 11951 particles Shaking 5.degree.
C. 1 week 128 1.9 98.1 0.0 n.d. n.d. n.d. 30.3 Free from 10936
particles Shaking 25.degree. C. 1 week 127 1.9 98.0 0.1 n.d. n.d.
n.d. 29.7 Free from 12595 particles Freezing/ (5 cycles) 127 1.9
98.1 0.0 n.d. n.d. n.d. 32.0 Free from 11442 Thawing particles
5.degree. C. 7 weeks 124 1.8 98.2 0.0 3 24 63 33.8 Free from 11723
particles 19 weeks 122 1.7 98.2 0.1 3 26 62 30.8 Free from 12180
particles 26 weeks n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Free
from n.d. particles 25.degree. C. 7 weeks n.d. 2.0 97.3 0.7 3 25 62
30.8 Free from 12328 particles 19 weeks n.d. 2.1 96.9 1.1 4 24 60
31.4 Free from 12017 particles 26 weeks n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. Free from n.d. particles 40.degree. C. 7 weeks n.d.
n.d. n.d. n.d. 5 20 58 84.8 Free from n d. particles 19 weeks n.d.
n.d. n.d. n.d. 7 11 50 295.0 Free from n.d. particles n.d. not
determined
Formulation I is a liquid formulation with the composition 120
mg/ml Rituximab, 20 mM L-histidine, 120 mM sodium chloride, 10 mM
methionine, 0.04% polysorbate 80, 12,000 U/ml rHuPH20, at pH
6.0.
TABLE-US-00010 Ion Exchange-HPLC Protein Size Exclusion-HPLC Fab NS
Enzyme Storage Storage concentration HMW Monomer LMW clips a + b
Fc-Lys Fab pE/Q Turbidity Visible activity condition Time (mg/ml)
(%) (%) (%) (%) (%) (%) (FTU) particles (U/ml) -- Initial 123 1.7
98.3 0.0 3 25 62 34.0 Free from 11022 particles Shaking 5.degree.
C. 1 week 125 1.6 98.3 0.0 n.d. n.d. n.d. 33.3 Free from 12231
particles Shaking 25.degree. C. 1 week 124 1.7 98.3 0.1 n.d. n.d.
n.d. 32.1 Free from 8371 particles Freezing/ (5 cycles) 123 1.8
98.1 0.0 n.d. n.d. n.d. 32.9 Free from 12058 Thawing particles
5.degree. C. 7 weeks 122 1.6 98.4 0.0 3 25 62 33.5 Free from 11108
particles 19 weeks 119 1.6 98.4 0.1 3 26 62 34.4 Free from 11548
particles 26 weeks n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. Free
from n.d. particles 25.degree. C. 7 weeks n.d. 1.7 97.7 0.6 3 25 62
34.8 Free from 12679 particles 19 weeks n.d. 1.8 97.2 1.1 4 24 60
34.6 Free from 13252 particles 26 weeks n.d. n.d. n.d. n.d. n.d.
n.d. n.d. n.d. Free from n.d. particles 40.degree. C. 7 weeks n.d.
2.5 94.9 2.6 5 20 58 88.1 Free from n.d. particles 19 weeks n.d.
3.7 90.4 5.9 7 14 53 292.0 Free from n.d. particles n.d. not
determined
Formulation J is a liquid formulation with the composition 25 mg/ml
GA101 (huMAb<CD20>), 20 mM L-histidine, 240 mM trehalose
dihydrate, 0.02% poloxamer 188, 2,000 U/ml rHuPH20, at pH 6.0.
TABLE-US-00011 Storage Protein Size Exclusion-HPLC Ion
Exchange-HPLC Enzyme temperature/ Storage concentration HMW Monomer
LMW Acidic Main Turbidity Visible activity stress condition Time
(mg/ml) (%) (%) (%) region (%) Peak (%) (FTU) particles (U/ml) --
Initial 25.1 0.6 98.5 0.9 18.5 68.6 6.0 Free from 1833 particles
-20.degree. C. 26 weeks 26.2 0.6 98.6 0.8 18.7 68.3 5.8 Free from
2078 particles 5.degree. C. 26 weeks 25.7 0.7 98.4 0.9 19.1 67.9
5.6 Free from 1380 particles 25.degree. C. 26 weeks 25.9 0.9 97.3
1.9 26.7 60.8 6.0 Free from 978 particles 40.degree. C. 26 weeks
26.1 3.0 86.8 9.7 40.9 19.5 7.2 Essentially free <limit of from
particles quantification n.d. not determined
Example 2
Preparation of Humanized 2H7 Anti-CD20 Liquid Formulations
[0226] For the preparation of the liquid formulations, recombinant
humanized 2H7 anti-CD20 antibody (2H7.v16 as disclosed in WO
2006/084264) was buffer exchanged against a diafiltration buffer
containing the anticipated buffer composition and where required,
concentrated to an antibody concentration of approx. 60 and 120
mg/ml. After achieving the target concentration, the excipients
(e.g. trehalose, rHuPH20, polysorbate 20) were then added as stock
solutions to the antibody solution. Finally the protein
concentration was adjusted with the final formulation buffer to a
humanized 2H7 concentration of approx. 30, 50, and 100 mg/ml.
[0227] All formulations were sterile-filtered through 0.22 .mu.m
low protein binding filters and aseptically filled into sterile 3
ml glass vials stoppered with fluoro-resin laminated butyl rubber
stoppers and capped with aluminum/plastic flip-off seals. The fill
volume was approx. 1.2 ml. These formulations were stored at
different temperatures (5.degree. C., 25.degree. C. and 40.degree.
C.) for different intervals of time. The samples were analyzed at
each stability time point by the following analytical methods:
[0228] 1) UV spectrophotometry
[0229] 2) Size exclusion chromatography (SEC);
[0230] 3) Ion exchange chromatography (IEC);
[0231] 4) Complement dependent cytotoxicity (CDC) assay for
humanized 2H7 activity
[0232] 5) Turbidometric assay for rHuPH20 activity
[0233] 1). Protein concentration was determined by ultraviolet
absorption spectroscopy using an Agilent 8453 spectrophotometer in
a wavelength range from 240 nm to 400 nm. Samples were
gravimetrically diluted to approx. 0.5 mg/ml with the corresponding
formulation buffer. The protein concentrations were calculated
using Equation 1:
Protein
concentration=((A.sub.max-A.sub.320).times.DF)/(.epsilon.(cm.sup-
.2/mg).times.d(cm)) (Equation 1)
wherein DF is the dilution factor, d is the cuvette path length and
.epsilon. is the extinction coefficient, which is 1.75
(cm.sup.2/mg.sup.-1) for 2H7 at A.sub.max. The UV light absorption
at A.sub.max (typically 278 to 280 nm) was corrected for light
scattering at 320 nm and multiplied with the dilution factor, which
was determined from the weighed masses and densities of the neat
sample and the dilution buffer. The numerator was divided by the
product of the cuvette's path length d and the extinction
coefficient .epsilon..
[0234] 2). Size Exclusion Chromatography (SEC) was used to detect
soluble high molecular weight species (aggregates) and low
molecular weight hydrolysis products (fragments) in the
formulations. SEC was carried out on an Agilent Technologies, Inc.
1100 series HPLC equipped with a UV detector (detection wave length
280 nm) and a TSK G3000SWXL column (7.8.times.300 mm). Intact
monomer, aggregates and hydrolysis products were separated by an
isocratic elution profile, using 0.20M potassium phosphate and
0.25M potassium chloride at pH 6.2 with a flow rate of 0.3
ml/min.
[0235] 3). Ion Exchange Chromatography (IEC) was performed to
detect chemical degradation products that alter the net charge of
the anti-CD20 antibody in the formulations. For this purpose, the
anti-CD20 antibody is incubated with Carboxypeptidase B to catalyze
the hydrolysis of basic amino acids. Ion exchange chromatography
was carried out on an Agilent Technologies, Inc. 1100 series HPLC
with a UV detector (detection wavelength 280 nm) and a Dionex
ProPac WCX-10 (4.times.250 mm) column. Acidic and basic variants
were separated using a linear gradient of 25 mM potassium phosphate
at pH 6.9 (mobile phase A) and 120 mM potassium chloride dissolved
in 25 mM potassium phosphate (mobile phase B) with a flow rate of
0.5 mL/min.
[0236] 4). The complement-dependent cytotoxicity assay (CDC) assay
was performed to determine the in vitro activity of the anti-CD20
antibody. The complement dependent cytotoxicity (CDC) potency assay
is used to measure the ability of the antibody to lyse human B
lymphoblastoid (WIL2-S) cells in the presence of human complement.
The assay is performed in 96 well tissue culture microtiter plates.
In this assay, varying concentrations of the anti-CD20 antibody
reference material, control, or sample(s) diluted in assay diluent
are incubated with WIL2-S cells (50,000 cells/well) in the presence
of a fixed amount of human complement. The plate is incubated at
37.degree. C./5% CO2 in a humidified incubator for 1 to 2 hours. At
the end of the incubation period, 50 .mu.L of the redox dye,
ALAMARBLUE.TM. is added to each well and the plate is incubated for
15 to 26 hours. ALAMARBLUE.TM. is a redox dye that fluoresces at an
excitation wavelength of 530 nm and an emission wavelength of 590
nm when reduced by live cells. Therefore, the changes in color and
fluorescence are proportional to the number of viable cells. The
results, expressed in relative fluorescence units (RFU), are
plotted against the anti-CD20 antibody concentrations and a
parallel line program is used to estimate the activity of anti-CD20
antibody samples relative to the reference material.
[0237] 5). A turbidimetric assay was used to determine
hyaluronidase activity and enzyme concentration. This method is
based on the formation of an insoluble precipitate when hyaluronic
acid binds with acidified serum albumin. Briefly, a dilution series
of the rhuPH20 hyaluronidase (Halozyme, Inc.) working reference
standard ranging from 2.5 U/ml to 0.25 U/ml is prepared in enzyme
diluent (70 mM NaCl, 25 mM PIPES, pH 5.5, 0.66 mg/ml gelatin
hydrolysate, 0.1% human serum albumin). The test samples are
diluted to a final concentration of 1.5 U/ml in enzyme diluent. 30
.mu.l of the standard and sample dilutions are transferred into a
"black clear bottom" 96-well plate (Nunc). The plate is then
covered and pre-warmed for 5 minutes at 37.degree. C. The reaction
is then initiated by adding 30 .mu.L of pre-warmed 0.25 mg/ml
hyaluronic acid substrate solution (70 mM NaCl, 25 mM PIPES, pH
5.5, 0.25 mg/ml dried sodium hyaluronate, Lifecore Biomedical). The
plate is shaken briefly and incubated for 10 minutes at 37.degree.
C. After this incubation step the reaction is stopped by adding 240
.mu.l 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 is measured at a
wavelength 640 nm on a microplate reader. The decrease in turbidity
resulting from enzyme activity on the hyaluronic acid substrate is
a measure of the hyaluronidase activity. The sample activity is
determined relative to the calibration curve generated with the
dilutions of the rhuPH20 working reference standard.
[0238] The results obtained with the various humanized 2H7 antibody
formulations are shown in the following tables:
Formulation K is a liquid formulation with the composition 30 mg/ml
humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02%
polysorbate 20, 0 U/ml of rhuPH20 at pH 5.3.
TABLE-US-00012 Protein Size Exclusion-HPLC Ion Exchange-HPLC CDC
Potency Enzyme Storage Storage concentration HMW Monomer LMW %
Acidic % Main (% Specific activity temperature Time (mg/ml) (%) (%)
(%) Variant Peak Activity) (U/ml) -- Initial 32 0.9 98.9 0.2 27.1
67.5 103 ND 5.degree. C. 12 weeks ND 0.8 98.1 1.1 27.8 65.9 110 ND
24 weeks ND 0.8 98.1 1.1 25.6 68.1 89 ND 36 weeks ND 0.9 98.6 0.5
27.0 68.3 97 ND 48 weeks ND 1.0 97.4 1.6 25.9 68.3 96 ND 72 weeks
ND 0.9 97.9 1.2 25.2 68.2 109 ND 96 weeks ND 0.9 97.8 1.3 28.1 66.1
96 ND 25.degree. C. 4 weeks ND 0.7 98.0 1.2 29.5 64.7 ND ND 8 weeks
ND 0.8 97.7 1.5 32.5 61.9 ND ND 12 weeks ND 0.8 97.4 1.8 35.0 59.1
83 ND 24 weeks ND 1.0 96.8 2.2 40.9 52.1 ND ND 40.degree. C. 2
weeks ND 0.7 97.8 1.6 36.0 57.7 ND ND 4 weeks ND 0.8 96.6 2.5 45.1
47.4 ND ND 8 weeks ND 1.0 94.9 4.1 60.3 33.3 ND ND *ND not
determined
Formulation L is a liquid formulation with the composition 30 mg/ml
humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02%
polysorbate 20, 1500 U/ml of rhuPH20 at pH 5.3.
TABLE-US-00013 Protein Size Exclusion-HPLC Ion Exchange-HPLC CDC
Potency Enzyme Storage Storage concentration HMW Monomer LMW %
Acidic % Main (% Specific activity temperature Time (mg/ml) (%) (%)
(%) Variant Peak Activity) (U/ml) -- Initial 32 0.9 98.9 0.2 27.1
67.4 114 1309 5.degree. C. 12 weeks ND 0.8 97.9 1.3 27.6 65.7 110
1520 24 weeks ND 0.8 98.1 1.2 26.1 67.1 82 1112 36 weeks ND 0.9
97.3 1.8 27.0 67.9 98 1166 48 weeks ND 0.9 97.3 1.8 26.2 67.9 100
1620 72 weeks ND 0.9 97.9 1.3 26.0 68.2 97 ND 96 weeks ND 0.9 97.8
1.3 28.1 66.0 96 ND 25.degree. C. 4 weeks ND 0.7 98.0 1.3 29.5 64.7
ND 1147 8 weeks ND 0.8 97.8 1.4 32.1 61.9 ND 847 12 weeks ND 0.8
97.3 1.9 35.1 59.3 89 892 24 weeks ND 1.0 96.8 2.2 41.3 51.9 ND 777
40.degree. C. 2 weeks ND 0.8 97.6 1.6 35.3 57.4 ND ND 4 weeks ND
0.8 96.7 2.4 45.2 46.9 ND ND 8 weeks ND 1.0 95.0 4.0 59.8 34.3 ND
ND *ND not determined
Formulation M is a liquid formulation with the composition 30 mg/ml
humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02%
polysorbate 20, 12,000 U/ml of rhuPH20 at pH 5.3.
TABLE-US-00014 Protein Size Exclusion-HPLC Ion Exchange-HPLC CDC
Potency Enzyme Storage Storage concentration HMW Monomer LMW %
Acidic % Main (% Specific activity temperature Time (mg/ml) (%) (%)
(%) Variant Peak Activity) (U/ml) -- Initial 33 0.9 98.9 0.2 27.2
67.4 103 10584 5.degree. C. 12 weeks ND 0.8 97.7 1.6 26.5 66.8 111
8864 24 weeks ND 0.8 97.8 1.4 25.8 68.1 85 14319 36 weeks ND 0.8
97.5 1.7 27.1 68.0 96 11408 48 weeks ND 0.9 96.7 2.4 26.2 67.9 95
13817 72 weeks ND 0.9 97.6 1.6 26.4 68.0 98 ND 96 weeks ND 0.9 97.4
1.6 28.3 65.8 108 ND 25.degree. C. 4 weeks ND 0.8 97.7 1.5 29.6
64.7 ND 13464 8 weeks ND 0.8 97.4 1.7 32.1 61.9 ND 10975 12 weeks
ND 0.9 97.0 2.1 35.4 58.7 92 10394 24 weeks ND 1.0 96.5 2.5 41.0
51.8 ND 819 40.degree. C. 2 weeks ND 1.1 97.3 1.6 35.7 57.2 ND ND 4
weeks ND 0.8 96.6 2.5 45.5 47.5 ND ND 8 weeks ND 1.0 94.9 4.1 59.9
33.3 ND ND *ND not determined
Formulation N is a liquid formulation with the composition 50 mg/ml
humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02%
polysorbate 20, 0 U/ml of rhuPH20 at pH 5.3.
TABLE-US-00015 Protein Size Exclusion-HPLC Ion Exchange-HPLC CDC
Potency Enzyme Storage Storage concentration HMW Monomer LMW %
Acidic % Main (% Specific activity temperature Time (mg/ml) (%) (%)
(%) Variant Peak Activity) (U/ml) -- Initial 50 0.9 98.9 0.2 27.0
67.6 107 ND 5.degree. C. 12 weeks ND 0.9 98.1 1.0 26.9 66.6 86 ND
24 weeks ND 0.9 97.9 1.1 25.8 67.7 88 ND 36 weeks ND 1.0 97.9 1.1
27.1 68.5 92 ND 48 weeks ND 1.0 97.8 1.2 26.2 67.8 95 ND 72 weeks
ND 1.0 97.8 1.2 26.5 67.9 101 ND 96 weeks ND 1.1 97.6 1.3 28.4 65.8
98 ND 25.degree. C. 4 weeks ND 0.9 97.9 1.2 29.5 64.9 ND ND 8 weeks
ND 0.9 97.6 1.5 32.1 61.8 ND ND 12 weeks ND 1.0 97.4 1.6 35.3 58.9
86 ND 24 weeks ND 0.9 97.9 1.1 40.0 52.8 ND ND 40.degree. C. 2
weeks ND 0.9 97.5 1.6 35.3 56.9 ND ND 4 weeks ND 1.1 96.2 2.7 45.2
47.8 ND ND 8 weeks ND 1.4 94.1 4.4 59.7 32.9 ND ND *ND not
determined
Formulation O is a liquid formulation with the composition 50 mg/ml
humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02%
polysorbate 20, 1500 U/ml of rhuPH20 at pH 5.3.
TABLE-US-00016 Protein Size Exclusion-HPLC Ion Exchange-HPLC CDC
Potency Enzyme Storage Storage concentration HMW Monomer LMW %
Acidic % Main (% Specific activity temperature Time (mg/ml) (%) (%)
(%) Variant Peak Activity) (U/ml) -- Initial 51 0.9 98.9 0.2 27.1
67.4 116 1537 5.degree. C. 12 weeks ND 0.9 97.8 1.3 25.8 67.4 109
1454 24 weeks ND 0.9 97.9 1.2 26.0 67.7 84 1372 36 weeks ND 1.0
97.5 1.5 27.7 67.3 93 1432 48 weeks ND 1.1 97.0 2.0 26.0 68.4 102
1356 72 weeks ND 1.1 97.6 1.4 26.5 68.0 97 ND 96 weeks ND 1.2 97.6
1.3 28.2 65.9 104 ND 25.degree. C. 4 weeks ND 0.9 97.9 1.3 29.7
64.6 ND 1269 8 weeks ND 0.9 97.4 1.6 32.1 62.0 ND 966 12 weeks ND
1.0 97.5 1.5 35.2 58.9 89 1002 24 weeks ND 1.3 96.5 2.2 40.5 52.2
ND ND 40.degree. C. 2 weeks ND 0.9 97.5 1.6 35.9 56.1 ND ND 4 weeks
ND 1.1 96.3 2.6 46.5 45.7 ND ND 8 weeks ND 1.4 94.6 4.0 60.5 31.9
ND ND *ND not determined
Formulation P is a liquid formulation with the composition 50 mg/ml
humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate, 0.02%
polysorbate 20, 12,000 U/ml of rhuPH20 at pH 5.3.
TABLE-US-00017 Protein Size Exclusion-HPLC Ion Exchange-HPLC CDC
Potency Enzyme Storage Storage concentration HMW Monomer LMW %
Acidic % Main (% Specific activity temperature Time (mg/ml) (%) (%)
(%) Variant Peak Activity) (U/ml) -- Initial 52 0.9 98.9 0.2 27.2
67.4 110 9932 5.degree. C. 12 weeks ND 0.9 97.7 1.4 26.8 67.6 102
9668 24 weeks ND 0.9 97.7 1.4 25.7 68.2 ND 11292 36 weeks ND 1.0
97.5 1.5 27.5 67.0 96 15469 48 weeks ND 1.1 97.4 1.6 26.1 68.2 100
10832 72 weeks ND 1.0 97.7 1.3 26.3 68.0 106 ND 96 weeks ND 1.2
97.4 1.5 29.3 64.8 100 ND 25.degree. C. 4 weeks ND 0.9 97.6 1.5
29.7 64.6 ND 11765 8 weeks ND 1.0 97.6 1.4 32.3 61.9 ND 11594 12
weeks ND 1.1 97.1 1.8 35.4 58.8 86 10119 24 weeks ND 1.2 96.4 2.4
41.1 51.8 ND 8960 40.degree. C. 2 weeks ND 1.1 97.3 1.6 35.9 55.5
ND ND 4 weeks ND 1.1 96.4 2.5 44.9 46.5 ND ND 8 weeks ND 1.4 94.5
4.1 60.4 33.2 ND ND *ND not determined
Formulation Q is a liquid formulation with the composition 100
mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate,
0.02% polysorbate 20, 0 U/ml of rhuPH20 at pH 5.3.
TABLE-US-00018 Protein Size Exclusion-HPLC Ion Exchange-HPLC CDC
Potency Enzyme Storage Storage concentration HMW Monomer LMW %
Acidic % Main (% Specific activity temperature Time (mg/ml) (%) (%)
(%) Variant Peak Activity) (U/ml) -- Initial 102 1.0 98.8 0.2 27.2
67.5 101 ND 5.degree. C. 12 weeks ND 1.2 97.7 1.1 26.7 68.0 106 ND
24 weeks ND 1.3 97.6 1.2 25.8 67.5 84 ND 36 weeks ND 1.3 97.2 1.5
27.4 67.4 95 ND 48 weeks ND 1.3 97.2 1.6 26.2 68.3 89 ND 72 weeks
ND 1.4 97.4 1.2 26.4 68.3 106 ND 96 weeks ND 1.5 97.2 1.2 28.1 66.4
96 ND 25.degree. C. 4 weeks ND 1.2 97.5 1.2 31.0 63.7 ND ND 8 weeks
ND 1.5 97.2 1.4 32.4 61.9 ND ND 12 weeks ND 1.6 96.7 1.7 35.4 58.9
90 ND 24 weeks ND 1.9 96.0 2.1 40.6 52.1 ND ND 40.degree. C. 2
weeks ND 1.4 97.1 1.6 35.4 57.7 ND ND 4 weeks ND 1.8 95.5 2.7 45.6
47.3 ND ND 8 weeks ND 2.3 93.5 4.3 60.3 32.9 ND ND *ND not
determined
Formulation R is a liquid formulation with the composition 100
mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate,
0.02% polysorbate 20, 1500 U/ml of rhuPH20 at pH 5.3.
TABLE-US-00019 Protein Size Exclusion-HPLC Ion Exchange-HPLC CDC
Potency Enzyme Storage Storage concentration HMW Monomer LMW %
Acidic % Main (% Specific activity temperature Time (mg/ml) (%) (%)
(%) Variant Peak Activity) (U/ml) -- Initial 101 1.0 98.8 0.2 27.3
67.5 98 1389 5.degree. C. 12 weeks ND 1.2 97.7 1.2 25.3 67.7 104
1655 24 weeks ND 1.3 97.6 1.2 26.0 67.6 82 1381 36 weeks ND 1.3
96.6 2.2 26.7 68.7 95 1644 48 weeks ND 1.3 96.5 2.2 26.2 68.2 94
1381 72 weeks ND 1.4 97.5 1.2 26.7 68.1 106 ND 96 weeks ND 1.5 97.2
1.3 28.1 66.2 95 ND 25.degree. C. 4 weeks ND 1.2 97.5 1.2 29.2 64.2
ND 1376 8 weeks ND 1.4 97.3 1.3 32.5 60.4 ND 1018 12 weeks ND 1.6
96.9 1.5 35.3 58.9 91 942 24 weeks ND 1.9 96.0 2.2 40.7 53.8 ND 616
40.degree. C. 2 weeks ND 1.3 97.1 1.5 35.8 55.8 ND ND 4 weeks ND
1.8 95.6 2.7 45.8 47.6 ND ND 8 weeks ND 2.3 93.6 4.0 59.3 32.4 ND
ND *ND not determined
Formulation S is a liquid formulation with the composition 100
mg/ml humanized 2H7, 30 mM sodium acetate, 8% trehalose dihydrate,
0.02% polysorbate 20, 12,000 U/ml of rhuPH20 at pH 5.3.
TABLE-US-00020 Protein Size Exclusion-HPLC Ion Exchange-HPLC CDC
Potency Enzyme Storage Storage concentration HMW Monomer LMW %
Acidic % Main (% Specific activity temperature Time (mg/ml) (%) (%)
(%) Variant Peak Activity) (U/ml) -- Initial 101 1.0 98.8 0.2 27.4
67.4 99 11692 5.degree. C. 12 weeks ND 1.1 97.6 1.2 25.8 68.1 97
10599 24 weeks ND 1.2 97.6 1.2 26.1 67.2 80 11946 36 weeks ND 1.2
97.1 1.7 26.1 69.3 95 14894 48 weeks ND 1.3 97.0 1.8 25.7 68.4 89
11820 72 weeks ND 1.4 97.4 1.3 26.4 68.3 102 ND 96 weeks ND 1.5
97.1 1.4 28.1 66.2 97 ND 25.degree. C. 4 weeks ND 1.3 97.4 1.3 29.8
64.6 ND 11897 8 weeks ND 1.4 97.1 1.4 32.5 61.6 ND 11117 12 weeks
ND 1.5 96.6 1.8 35.5 58.7 89 10763 24 weeks ND 1.9 95.9 2.3 41.1
51.1 ND 7783 40.degree. C. 2 weeks ND 1.4 97.0 1.6 35.6 55.9 ND ND
4 weeks ND 1.8 95.6 2.6 46.0 48.0 ND ND 8 weeks ND 2.2 93.8 4.0
61.4 32.7 ND ND *ND not determined
Example 3
Treatment of Patients with the Formulation
[0239] Rituximab-containing regimens have become the standard of
care for patients suffering from 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 related to the infusion were cited by
some patients as uncomfortable consequences of the current
therapeutic treatment. Furthermore, the required procedure to
establish intravenous access is considered invasive and can be
painful, particularly in patients with malignant diseases who are
treated repeatedly. Subcutaneous (SC) administration could
significantly simplify treatment, shortening administration to less
than 10 minutes and improving patient experience. Recombinant human
hyaluronidase (rHuPH20) has been developed and approved to improve
dispersion and absorption of co-administered drugs. It has been
combined with rituximab to allow injection volumes larger than 10
mL to be safely and comfortably administered SC. The aims of this
treatment were to select the dose of the SC rituximab formulation
with rHuPH20 prepared as described in Example 1 (Formulation A)
giving comparable exposure to IV rituximab and to assess its safety
and tolerability in male and female follicular lymphoma (FL)
patients during maintenance treatment.
[0240] This example provides stage 1 data from a randomized,
open-label, multi-centre adaptive Phase Ib study. 124 patients were
randomized to one of four rituximab maintenance treatment groups:
16 patients IV control, 34 patients SC dose 1 (375 mg/m.sup.2), 34
patients SC dose 2 (625 mg/m.sup.2) and 40 patients SC dose 3 (800
mg/m.sup.2). Prior to randomization, eligible patients were treated
with at least one IV rituximab dose at 375 mg/m.sup.2 in the
maintenance setting. For patients randomized to one of the SC
cohorts, a single IV dose was replaced by a SC dose. Patients
received rituximab either on an every 2 month (q2m) or every 3
month (q3m) regimen, as per local practice. Safety data are
available from a total of 119 patients. Rituximab SC was generally
well tolerated. No clinically significant observations or
treatment-related serious adverse events have been reported. A
total of 95 adverse events (AEs) were reported in 46 patients
(39%). The most commonly documented AE was
"administration-associated reaction" (AAR, including rash, erythema
and mild discomfort). These AARs were reversible, predominantly
mild in intensity and only 1 event necessitated any treatment
(metoclopramide for nausea). Overall, the AE profile is not
significantly different to that expected in patients treated with
rituximab IV (after AAR, the most frequent events were
gastrointestinal disorders and mild infections). Four serious
adverse events (SAES) were reported in 4 separate patients, all
reported as unrelated to study medication. There were no AEs
leading to death, withdrawal or treatment discontinuation.
[0241] The total volume administered SC in each patient ranged
between 4.4-15.0 mL. The average injection duration was 2 mL/min.
Rituximab maximum serum concentrations in the SC cohorts occurred
between Day 2 and Day 8 (48 h and 168 h). Pharmacokinetic
parameters were linear with respect to dose over the range of SC
doses administered (375, 625 and 800 mg/m.sup.2). Rituximab
concentrations on Day 28 (C28) and the extent of serum exposure
(AUC.sub.0-57) in patients administered 625 mg/m.sup.2 rituximab SC
were comparable to those in patients administered the standard
rituximab IV dose of 375 mg/m.sup.2 SC.
[0242] In conclusion, subcutaneous rituximab can be delivered
quickly, comfortably and safely while achieving serum exposure
comparable to the approved intravenous formulation in FL patients
during maintenance treatment. The patient experience was
favourable. These results support further testing of subcutaneous
rituximab and a fixed dose of 1400 mg rituximab SC has been
selected for formal C.sub.trough non-inferiority testing in stage 2
of the trial.
Example 4
Rituximab SQ Vs. Rituximab IV in Patients with Follicular
Non-Hodgkin's Lymphoma
[0243] Patients with previously untreated follicular (low grade)
lymphoma are treated with maintenance treatment with either: (a)
rituximab SC formulation (prepared according to Example 1,
Formulation A) in combination with CHOP or CVP, or (b) rituximab IV
in combination with CHOP or CVP.
[0244] Patients will be randomized to receive 375 mg/m.sup.2
Rituximab as intravenous infusion or 1400 mg Rituximab given
subcutaneously. In addition, patients will receive standard
chemotherapy (CVP or CHOP). Patients who achieved a complete or
partial response after 8 treatment cycles, will receive maintenance
treatment for a further maximum number of 12 cycles. Maintenance
treatment cycles will be repeated every 8 weeks. The anticipated
time on study treatment is 96 weeks.
[0245] Treatment with 1400 mg SQ Rituximab anti-CD20 antibody as a
maintenance treatment every 8 weeks for up to 12 cycles is expected
to be safe and efficacious in treating follicular lymphoma,
optionally in combination with chemotherapy (including CHOP or
CVP).
* * * * *