U.S. patent application number 15/064492 was filed with the patent office on 2016-07-28 for protein formulation.
The applicant listed for this patent is MedImmune, LLC. Invention is credited to Scott HAMMOND, Ambarish SHAH, Monika S. SHARMA.
Application Number | 20160215051 15/064492 |
Document ID | / |
Family ID | 40678967 |
Filed Date | 2016-07-28 |
United States Patent
Application |
20160215051 |
Kind Code |
A1 |
SHARMA; Monika S. ; et
al. |
July 28, 2016 |
PROTEIN FORMULATION
Abstract
The present invention relates to methods of minimizing the
aggregation of BiTE.RTM. molecules in solution. In one embodiment,
a method of the invention is used to minimize the aggregation of
BiTE.RTM. molecules in solution. In another embodiment, a method of
the invention is used to minimize the aggregation of BiTE.RTM.
molecules comprising a first and a second binding site specific for
the CD3 and CD19 antigens, respectively, wherein said molecule is
in solution. In a specific embodiment, the present invention
provides methods to minimize the aggregation of the MT103.TM.
BiTE.RTM. molecule in solution.
Inventors: |
SHARMA; Monika S.; (Potomac,
MD) ; SHAH; Ambarish; (Boyds, MD) ; HAMMOND;
Scott; (Olney, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MedImmune, LLC |
Gaithersburg |
MD |
US |
|
|
Family ID: |
40678967 |
Appl. No.: |
15/064492 |
Filed: |
March 8, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12744745 |
May 26, 2010 |
9308257 |
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PCT/US2008/084796 |
Nov 26, 2008 |
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15064492 |
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60990772 |
Nov 28, 2007 |
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61039985 |
Mar 27, 2008 |
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61040384 |
Mar 28, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 16/2809 20130101; A61P 37/06 20180101; C07K 2317/31 20130101;
A61K 39/39591 20130101; C07K 16/2803 20130101; A61K 2039/507
20130101; A61P 35/00 20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1-76. (canceled)
77. A method of preventing, managing, treating or ameliorating B
cell disorders or diseases in a human subject in need thereof, said
method comprising administering to the subject a prophylactically
or therapeutically effective amount of a liquid pharmaceutical
formulation comprising a bispecific antibody, wherein the
bispecific antibody comprises a first binding domain that
specifically binds the CD3 T cell surface antigen and a second
binding domain that specifically binds an antigen selected from
CD19, CD20, CD22, EphA2, EphA4, INFR, ICOS, Ep-CAM, CEA, and IL-5
receptor.
78. The method of claim 77, wherein the B cell disorder or disease
is selected from B cell malignancies, autoimmune diseases,
graft-versus-host disease (GVHD), humoral rejection, and
post-transplantation lymphoproliferative disorder.
79. The method of claim 77, wherein the B cell disorder includes B
cell malignancies.
80. The method of claim 79, wherein the B cell malignancy is
selected from B cell subtype non-Hodgkin's lymphoma (NHL);
Burkitt's lymphoma; multiple myeloma; pre-B acute lymphoblastic
leukemia; malignancies that derive from early B cell precursors;
common acute lymphocytic leukemia (ALL); chronic lymphocytic
leukemia (CLL); hairy cell leukemia; Null-acute lymphoblastic
leukemia; Waldenstrom's Macroglobulinemia; diffuse large B cell
lymphoma (DLBCL); pro-lymphocytic leukemia; light chain disease;
plasmacytoma; osteosclerotic myeloma; plasma cell leukemia;
monoclonal gammopathy of undetermined significance (MGUS);
smoldering multiple myeloma (SMM); indolent multiple myeloma (IMM);
Hodgkin's lymphoma including classical and nodular lymphocyte
pre-dominant type; lymphoplasmacytic lymphoma (LPL); and
marginal-zone lymphoma including gastric mucosal-associated
lymphoid tissue (MALT) lymphoma.
81. The method of claim 80, wherein the B cell malignancy is
selected from: (a) non-Hodgkin's lymphoma selected from low
grade/follicular NHL, small lymphocytic (SL) NHL, intermediate
grade/follicular NHL, intermediate grade diffuse NHL, high grade
immunoblastic NHL, high grade lymphoblastic NHL, high grade small
non-cleaved cell NHL; mantle-cell lymphoma, and bulky disease NHL;
(b) chronic lymphocytic leukemia selected from
immunoglobulin-mutated CLL and immunoglobulin-unmutated CLL; and
(c) diffuse large B cell lymphoma (DLBCL) selected from germinal
center B cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL,
and type 3 DLBCL.
82. The method of claim 77, wherein administering the formulation
results in the depletion of pre-B cells that do not express surface
immunoglobulin by at least 50% in comparison to B cell levels in
the subject before administration of the formulation.
83. The method of claim 77, wherein administering the formulation
results in the depletion of mature B cells that express surface
immunoglobulin by at least 50% in comparison to B cell levels in
the subject before administration of the formulation.
84. The method of claim 77, wherein administering the formulation
results in the depletion of all non-malignant B cell types by at
least 50% in comparison to B cell levels in the subject before
administration of the formulation.
85. The method of claim 77, comprising administering the
formulation to the subject for a period of at least about 1, 2, 3,
5, 7, 10, 14, 20, 30, 45, 60, 75, 90, 120, 150 or 180 days or
longer.
86. The method of claim 77, wherein the formulation is administered
as a continuous infusion.
87. The method of claim 77, wherein the first binding domain
comprises an amino acid sequence of residues 283-525 of SEQ ID
NO:5.
88. The method of claim 77, wherein the second binding domain
comprises an amino acid sequence of residues 28-277 of SEQ ID
NO:5.
89. The method of claim 77, wherein the bispecific antibody
comprises an amino acid sequence of residues 28-525 of SEQ ID
NO:5.
90. The method of claim 77, wherein the formulation comprises
between about 1 microgram/ml and about 500 micrograms/ml of the
bispecific antibody.
91. The method of claim 77, wherein the formulation comprises
lysine.
92. The method of claim 91, wherein the formulation further
comprises: (a) a buffering agent selected from the group consisting
of: histidine, citrate, phosphate, glycine, and acetate; (b) a
carbohydrate excipient selected from the group consisting of:
sucrose, trehalose, lactose, mannitol, and raffinose; and (c) a
surfactant selected from the group consisting of: Polysorbate 20,
Polysorbate 40, Polysorbate 60, and Polysorbate 80.
93. The method of claim 92, wherein the formulation comprises
citrate, trehalose, and Polysorbate 80.
94. The method of claim 77, wherein the formulation has a pH of
between about 4.0 and about 8.0 and comprises: (a) between about 1
micrograms/ml and about 500 micrograms/ml of the bispecific
antibody; (c) between about 5 mM and about 125 mM citrate, (d)
between about 25 mM and about 400 mM lysine, (e) between about 3%
and about 50% trehalose, and (f) between about 0.01% and about 1%
Polysorbate 80.
Description
1. INTRODUCTION
[0001] The present invention provides methods to minimize
aggregation of bispecific antibodies or fragments thereof in
solution and to minimize antibody loss through absorption to
contact surfaces.
[0002] The present invention relates to formulations of bispecific
antibodies or fragments thereof, which formulations exhibit
stability, low to undetectable levels of antibody fragmentation,
low to undetectable levels of aggregation, very little to no loss
of the biological activities of the bispecific antibodies and very
little to no loss of antibody through absorption to contact
surfaces during manufacture, preparation, storage and use.
[0003] In further embodiments, formulations of the invention
comprise BiTE.RTM. molecules. In certain embodiments, a formulation
of the invention is a lyophilized formulation, which lyophilized
formulation maximizes BiTE.RTM. molecule recovery from the
lyophilized product upon reconstitution. In further embodiments,
formulations described herein are injectable formulations suitable
for intravenous, subcutaneous, or intramuscular administration. In
other embodiments, formulations of the invention comprise a
BiTE.RTM. molecule having at least a first and a second binding
site specific for the CD3 and CD19 antigens, respectively. In one
embodiment, treatment is effected by use of a BiTE.RTM. molecule
specific for CD19. In yet another embodiment, treatment is effected
by use of MT103 or by the bispecific antibody encoded by SEQ ID
NO:5. In yet other embodiments, formulations of the invention
comprise a BiTE.RTM. molecule having at least a first and a second
binding site specific for CD3 and the EphA2 antigens, respectively.
In yet other embodiments, formulations of the invention comprise a
BiTE.RTM. molecule having at least a first and a second binding
site specific for CD3 and the carcinoembryonic antigen (CEA)
antigens, respectively. In further embodiments, BiTE.RTM. molecules
formulated according to the methods of the invention are highly
bioavailable upon subcutaneous administration to a subject.
[0004] The invention also provides methods for preparing
formulations of bispecific antibodies or fragments thereof, which
formulations exhibit stability, low to undetectable levels of
antibody fragmentation, low to undetectable levels of aggregation,
very little to no loss of the biological activities of the
bispecific antibodies and very little to no loss of antibody
through absorption to contact surfaces during manufacture,
preparation, storage and use. In certain embodiments, a formulation
described herein is an injectable formulation suitable for
intravenous, subcutaneous, or intramuscular administration.
[0005] The present invention is further directed to methods for the
treatment of B cell disorders or diseases in human subjects,
including B cell malignancies, using the lyophilized formulations
of bispecific antibodies or fragments thereof that comprise at
least a first and a second binding site specific for the CD3 and
CD19 antigens, respectively. In one embodiment, treatment is
effected by use of a BiTE.RTM. molecule specific for CD19. In yet
another embodiment, treatment is effected by use of MT103 or by the
bispecific antibody encoded by SEQ ID NO:5. The present invention
is directed to methods for the treatment and prevention of
autoimmune disease as well as the treatment and prevention of
graft-versus-host disease (GVHD), humoral rejection, and
post-transplantation lymphoproliferative disorder in human
transplant recipients using lyophilized formulations of bispecific
antibodies or fragments thereof that comprise at least a first and
a second binding site specific for the CD3 and CD19 antigens,
respectively. In one embodiment, treatment is effected by use of a
BiTE.RTM. molecule specific for CD19. In yet another embodiment,
treatment is effected by use of MT103 or by the bispecific antibody
encoded by SEQ ID NO:5.
2. BACKGROUND
[0006] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Bispecific
antibodies may bind to two different epitopes of a single cell
surface marker. Other such antibodies may bind a first cell surface
marker and further bind a second cell surface marker on the same
cell. A binding arm specific for a surface antigen of a target cell
may also be combined with an arm which binds to a triggering
molecule on a leukocyte such as a T cell receptor molecule (e.g.,
CD2 or CD3), or Fc receptors for IgG (Fc.gamma.R), so as to focus
cellular defense mechanisms to the target cell. Bispecific
antibodies may also be used to localize cytotoxic agents to a
target cell. These antibodies possess a target cell marker-binding
arm and an arm which binds the cytotoxic agent (e.g., saporin,
anti-interferon-.alpha., vinca alkaloid, ricin A chain,
methola-exate or radioactive isotope hapten). Bispecific antibodies
can be prepared as full-length antibodies or antibody fragments
(e.g., F(ab')).
[0007] Methods for making bispecific antibodies are known in the
art. (See, for example, Millstein et al., Nature, 305:537-539
(1983); Traunecker et al., EMBO J., 10:3655-3659 (1991); Suresh et
al., Methods in Enzymology, 121:210 (1986); Kostelny et al., J.
Immunol., 148(5):1547-1553 (1992); Hollinger et al., Proc. Natl
Acad. Sci. USA, 90:6444-6448 (1993); Gruber et al., J. Immunol.,
152:5368 (1994); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920; 5,601,81; 95,731,168; 4,676,980; and 4,676,980, WO
94/04690; WO 91/00360; WO 92/200373; WO 93/17715; WO 92/08802; and
EP 03089).
[0008] MT103 is a recombinant, bispecific single-chain antibody or
BiTE.RTM. molecule, which binds to CD19 on target cells and the CD3
complex on T effector cells. See Hoffmann P et. al., Int J Cancer.
2005 May 20; 115(1):98-104. It is a single polypeptide chain of 55
kDa with the variable binding domains of two murine monoclonal
antibodies. By simultaneously binding to target and effector cells,
MT103 triggers the T effector cell to form an immunological
synapse, and to release perforin and granzymes which induce cell
death of target cells. Target cells comprise human B cells of all
development stages (with the exception of plasma cells) and
malignant cells of most B cell-derived lymphoma and leukemia.
Therefore, MT103 is currently in development for treatment of
B-cell cancers.
[0009] Initial toxicology studies and clinical trials utilized a
frozen liquid formulation which consisted of 1 .mu.g/ml MT103
monomer in isotonic phosphate-buffered saline (pH 7.0-7.5)
stabilized with 0.1% human serum albumin (HSA), sodium chloride,
disodium monohydrogen phosphate, potassium dihydrogen phosphate and
potassium chloride. A second lyophilized formulation was later
developed to eliminate the use of HSA and enhanced the stability
and recovery of MT103. This formulation consists of MT103 at 50
.mu.g/mL in 25 mM citrate at pH 7.0, 500 mM lysine acetate, 15%
trehalose dihydrate, 2.5% polyethylene glycol (PEG) 3350, 0.05%
polysorbate 80. However, clinical use of the second lyophilized
formulation never took place because the formulation presented
significant interference in several essential analytical assays due
to presence of the excipients lysine acetate and PEG 3350.
[0010] Therefore, further development was initiated to generate a
formulation that 1) excludes the interfering excipients, 2)
minimizes aggregate formation in the liquid bulks during storage,
3) maximizes the recovery of MT103 protein from the lyophilized
product, 4) is suitable for intravenous, subcutaneous, or
intramuscular administration, and 5) results in high
bioavailability of the BiTE.RTM. molecules upon subcutaneous
administration to a subject. Additional effort was put into
optimization of the lyophilization cycle and robustness (boundary
condition) studies.
3. SUMMARY OF THE INVENTION
[0011] The present invention relates to methods of minimizing the
aggregation of BiTE.RTM. molecules in solution. In one embodiment,
a method of the invention is used to minimize the aggregation of
BiTE.RTM. molecules in solution. In another embodiment, a method of
the invention is used to minimize the aggregation of BiTE.RTM.
molecules comprising a first and a second binding site specific for
the CD3 and CD19 antigens, respectively, wherein said molecule is
in solution. In a specific embodiment, the present invention
provides methods to minimize the aggregation of the MT103.TM.
BiTE.RTM. molecule in solution.
[0012] The present invention relates to sterile, stable, liquid
formulations of BiTE.RTM. molecules. In one embodiment, a liquid
formulation of the invention comprises a BiTE.RTM. molecule and
lysine. In a specific embodiment, a liquid formulation of the
invention comprises a BiTE.RTM. molecule and lysine HCl. In one
embodiment, a liquid formulation of the invention is suitable for
lyophilization. In a specific embodiment, a liquid formulation of
the invention comprises the MT103.TM.. In a specific embodiment, a
liquid formulation of the invention comprises between about 25
micrograms/ml and about 250 micrograms/ml MT103. In a specific
embodiment, a liquid MT103 formulation of the invention comprises
25 mM citrate, 15% (w/v) trehalose dihydrate, 200 mM lysine HCl,
0.1% (w/v) polysorbate 80 and has a pH of 7.0. In a specific
embodiment, a liquid formulation of the invention comprises MT103,
about 30 mM citrate, about 75 mM lysine HCl, about 6.5% trehalose
and about 0.02% Polysorbate 80 and has a pH of about 7.0. In a
further specific embodiment, a liquid formulation of the invention
comprises MT103, about 30 mM citrate, about 75 mM lysine HCl, about
6% trehalose and about 0.02% Polysorbate 80 and has a pH of about
7.0. In a specific embodiment, a liquid formulation of the
invention consist of between about 25 micrograms/ml and about 250
micrograms/ml MT103, 25 mM citrate, 15% (w/v) trehalose dihydrate,
200 mM lysine HCl, 0.1% (w/v) polysorbate 80 and has a pH of 7.0.
In a further embodiment, a liquid formulation of the invention
comprises about 25 mM citrate, about 6% (w/v) trehalose dihydrate,
about 50 mM lysine HCl, and about 0.1% (w/v) polysorbate 80 and has
a pH of 7.0.
[0013] The present invention further provides sterile, stable
lyophilized formulations of a BiTE.RTM. molecule. In one
embodiment, a lyophilized formulation of the invention comprises a
BiTE.RTM. molecule and lysine. In a specific embodiment, a
lyophilized formulation of the invention comprises a BiTE.RTM.
molecule and lysine HCl. In a specific embodiment, a reconstituted
formulation of the invention comprises the MT103.TM. BiTE.RTM.
molecule. In a specific embodiment, a lyophilized formulation of
the invention comprises the MT103.TM. BiTE.RTM. molecule, citrate,
trehalose, lysine HCl, and polysorbate 80.
[0014] The present invention further provides sterile, stable
reconstituted formulations of a BiTE.RTM. molecule. In one
embodiment, a reconstituted formulation of the invention comprises
a BiTE.RTM. molecule and lysine. In a specific embodiment, a
reconstituted formulation of the invention comprises a BiTE.RTM.
molecule and lysine HCl. In a specific embodiment, a lyophilized
formulation of the invention comprises the MT103.TM. BiTE.RTM.
molecule. In a specific embodiment, a lyophilized formulation of
the invention comprises the MT103.TM. BiTE.RTM. molecule, citrate,
trehalose, lysine HCl, and polysorbate 80.
[0015] The present invention further relates to processes of making
sterile, stable liquid formulations; sterile, stable lyophilized
formulations; and sterile, stable reconstituted formulations
comprising a BiTE.RTM. molecule. In a specific embodiment, the
processes of the invention may be utilized to make a formulation
comprising MT103. In further embodiments, formulations described
herein are injectable formulations suitable for intravenous,
subcutaneous, or intramuscular administration. In certain
embodiments, subcutaneous administration of a BiTE.RTM. formulation
of the invention results in high bioavailability of the BiTE.RTM.
molecule.
[0016] The present invention also encompasses methods of
preventing, managing, treating or ameliorating B cell disorders or
diseases such as, but not limited to, B cell malignancies,
autoimmune diseases, graft-versus-host disease (GVHD), humoral
rejection, and post-transplantation lymphoproliferative disorder in
a human subject, said methods comprising administering to a subject
in need thereof a prophylactically or therapeutically effective
amount of a formulation comprising BiTE.RTM. molecules that
comprises a first and a second binding site specific for the CD3
and CD19 antigens, respectively. Alternatively, the present
invention uses MT103 for the preventing, managing, treating or
ameliorating of said diseases.
3.1. Definitions
[0017] As used herein, the terms "antibody" and "antibodies"
(immunoglobulins) encompass monoclonal antibodies (including
full-length monoclonal antibodies), polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies) formed from
at least two intact antibodies, human antibodies, humanized
antibodies, camelised antibodies, chimeric antibodies, single-chain
Fvs (scFv), single-chain antibodies, single domain antibodies,
domain antibodies, Fab fragments, F(ab')2 fragments, antibody
fragments that exhibit the desired biological activity,
disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id)
antibodies (including, e.g., anti-Id antibodies to antibodies of
the invention), intrabodies, and epitope-binding fragments of any
of the above. In particular, antibodies include immunoglobulin
molecules and immunologically active fragments of immunoglobulin
molecules, i.e., molecules that contain an antigen-binding site.
Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM,
IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2) or subclass.
[0018] In some embodiments, an antibody is a bispecific single
chain antibody comprising a first binding domain that comprises a
variable heavy (VH) domain and a variable light (VL) domain of an
antibody that immunospecifically binds to the T-cell antigen CD3
and a second binding domain that comprises a VH domain and a VL
domain of an antibody that immunospecifically binds to an antigen
of interest (e.g., CD19 or EphA2 or CEA). The first and second
binding domains of a bispecific single chain antibody may be
derived from synthetic antibodies, monoclonal antibodies,
recombinantly produced antibodies, multispecific antibodies
(including bi-specific antibodies), human antibodies, humanized
antibodies, chimeric antibodies, intrabodies, single-chain Fvs
(scFvs) (e.g., including monospecific and bi-specific, etc.), Fab
fragments, F(ab') fragments, disulfide-linked Fvs (sdFv),
anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments
of any of the above. In a specific embodiment, an antibody of a
formulation of the invention is a bispecific single chain antibody
comprising a first binding domain that comprises a variable heavy
(VH) domain and a variable light (VL) domain of an antibody that
immunospecifically binds to the T-cell antigen CD3 and a second
binding domain that comprises a VH domain and a VL domain of an
antibody that immunospecifically binds to CD19, such as, but not
limited to, MT103. The antibody that immunospecifically binds to
the T-cell antigen CD3 and the antibody that immunospecifically
binds CD19 may be derived from synthetic antibodies, monoclonal
antibodies, recombinantly produced antibodies, multispecific
antibodies (including bi-specific antibodies), human antibodies,
humanized antibodies, chimeric antibodies, intrabodies,
single-chain Fvs (scFvs) (e.g., including monospecific and
bi-specific, etc.), Fab fragments, F(ab') fragments,
disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies,
and epitope-binding fragments of any of the above. In particular,
antibodies of the formulations of the present invention include
immunoglobulin molecules and immunologically active portions of
immunoglobulin molecules, i.e., molecules that contain an
antigen-binding domain that immunospecifically binds to an antigen
of interest (e.g., CD19 and/or CD3) (e.g., one or more
complementarity determining regions (CDRs) of an anti-CD19 or
anti-CD3 antibody).
[0019] As used herein, the term "binding domain" refers to a domain
comprising a three-dimensional structure capable of
immunospecifically binding to an epitope. Thus, said domain can
comprise the VH and/or VL domain of an antibody chain. In some
embodiments, said domain copmrises at least the VH domain. On the
other hand, said binding domains contained in the antibodies of a
formulation of the invention may comprise at least one
complementarity determining region (CDR) of an antibody chain
recognizing the CD19 and CD3 antigens, respectively. In this
respect, it is noted that the domains of the binding domains
present in the CD19-BiTE.RTM. (e.g., MT103) of the invention may
not only be derived from antibodies but also from other CD19 or CD3
binding proteins, such as naturally occurring surface receptors or
ligands. In accordance with the invention, said binding domain is
comprised in a domain.
[0020] As used herein, the term "single-chain Fv" or "scFv" refers
to antibody fragments comprising the VH and VL domains of an
antibody, wherein these domains are present in a single polypeptide
chain. Generally, the Fv polypeptide further comprises a
polypeptide linker between the VH and VL domains which enables the
scFv to form the desired structure for antigen binding. Methods for
producing scFvs are well known in the art. For a review of methods
for producing scFvs see Pluckthun in The Pharmacology of Monoclonal
Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New
York, pp. 269-315 (1994). In a specific embodiment, the
EphA2-BiTE.RTM.s of the invention are comprised of scFvs.
[0021] 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 except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. In addition to their specificity,
monoclonal antibodies are advantageous in that they can be
synthesized by hybridoma cells that are uncontaminated by other
immunoglobulin producing cells. Alternative production methods are
known to those trained in the art, for example, a monoclonal
antibody may be produced by cells stably or transiently transfected
with the heavy and light chain genes encoding the monoclonal
antibody.
[0022] 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
engineering of the antibody by any particular method. The term
"monoclonal" is used herein to refer to an antibody that is derived
from a clonal population of cells, including any eukaryotic,
prokaryotic, or phage clone, and not the method by which the
antibody was engineered. 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 any recombinant DNA method (see, e.g.,
U.S. Pat. No. 4,816,567), including isolation from phage antibody
libraries using the techniques described in Clackson et al.,
Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,
222:581-597 (1991), for example. These methods can be used to
produce monoclonal mammalian, chimeric, humanized, human, domain
antibodies, diabodies, vaccibodies, linear antibodies, and
bispecific antibodies, including BiTE.RTM. molecules.
[0023] All formulations of antibodies and/or antibody fragments
that specifically bind to an antigen of interest (e.g., CD19, or
EphA2, or CEA) are herein collectively referred to as "formulations
of the invention", "liquid formulations of the invention",
"lyophilized formulations of the invention", "reconstituted
formulations of the invention", "antibody liquid formulations of
the invention", "antibody lyophilized formulations of the
invention", "antibody reconstituted formulations of the invention"
or "antibody formulations of the invention".
[0024] The phrase "pharmaceutically acceptable" as used herein
means approved by a regulatory agency of the Federal or a state
government, or listed in the U.S. Pharmacopeia, European
Pharmacopia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans.
[0025] The terms "stability" and "stable" as used herein in the
context of a formulation comprising BiTE.RTM. molecules (e.g.,
MT103) refer to the resistance of the antibody (including antibody
fragment thereof) in the formulation to aggregation, degradation or
fragmentation under given manufacture, preparation, transportation
and storage conditions. The "stable" formulations of the invention
retain biological activity under given manufacture, preparation,
transportation and storage conditions. The stability of said
antibody (including antibody fragment thereof) can be assessed by
degrees of aggregation, degradation or fragmentation, as measured
by HPSEC, static light scattering (SLS), Fourier Transform Infrared
Spectroscopy (FTIR), circular dichroism (CD), urea unfolding
techniques, intrinsic tryptophan fluorescence, differential
scanning calorimetry, and/or ANS binding techniques, compared to a
reference formulation. For example, a reference formulation may be
a reference standard frozen at -70.degree. C. consisting of 10
mg/ml of BiTE.RTM. molecules (for example, but not limited to,
MT103) in PBS. The overall stability of a formulation comprising
BiTE.RTM. molecules can be assessed by various immunological assays
including, for example, ELISA and radioimmunoassay using isolated
antigen molecules.
[0026] The phrase "low to undetectable levels of aggregation" as
used herein refers to samples containing no more than about 5%, no
more than about 4%, no more than about 3%, no more than about 2%,
no more than about 1% and no more than about 0.5% aggregation by
weight of protein as measured by high performance size exclusion
chromatography (HPSEC) or static light scattering (SLS)
techniques.
[0027] The term "low to undetectable levels of fragmentation" as
used herein refers to samples containing equal to or more than
about 80%, about 85%, about 90%, about 95%, about 98% or about 99%
of the total protein, for example, in a single peak as determined
by HPSEC, or reduced Capillary Gel Electrophoresis (rCGE),
representing the non-degraded BiTE.RTM. molecule or a non-degraded
fragment thereof, and containing no other single peaks having more
than about 5%, more than about 4%, more than about 3%, more than
about 2%, more than about 1%, or more than about 0.5% of the total
protein in each. The term "reduced Capillary Gel Electrophoresis"
as used herein refers to capillary gel electrophoresis under
reducing conditions sufficient to reduce disulfide bonds in an
antibody.
[0028] "Isolated" when used to describe the various polypeptides
and antibodies disclosed herein, means a polypeptide or antibody
that has been identified, separated and/or recovered from a
component of its production environment. In some embodiments, the
isolated polypeptide is free of association with all other
components from its production environment. Contaminant components
of its production environment, such as that resulting from
recombinant transfected cells, are materials that would typically
interfere with diagnostic or therapeutic uses for the antibody, and
may include enzymes, hormones, and other proteinaceous or
non-proteinaceous solutes. In specific embodiments, the polypeptide
will be purified (1) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of a
spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under
non-reducing or reducing conditions using Coomassie blue or silver
stain. In some embodiments, an isolated antibody will be prepared
by at least one purification step.
[0029] The term "excipient" as used herein refers to an inert
substance which is commonly used as a diluent, vehicle,
preservative, binder or stabilizing agent for drugs which imparts a
beneficial physical property to a formulation, such as increased
protein stability, increased protein solubility, and decreased
viscosity. Examples of excipients include, but are not limited to,
proteins (for example, but not limited to, serum albumin), amino
acids (for example, but not limited to, aspartic acid, glutamic
acid, lysine, arginine, glycine), surfactants (for example, but not
limited to, SDS, Tween 20, Tween 80, polysorbate, polysorbate 80
and nonionic surfactants), saccharides (for example, but not
limited to, glucose, sucrose, maltose and trehalose), polyols (for
example, but not limited to, mannitol and sorbitol), fatty acids
and phospholipids (for example, but not limited to, alkyl
sulfonates and caprylate). For additional information regarding
excipients, see Remington's Pharmaceutical Sciences (by Joseph P.
Remington, 18.sup.th ed., Mack Publishing Co., Easton, Pa.), which
is incorporated herein in its entirety.
[0030] The term "lyophilized" or "freeze-dried" includes a state of
a substance that has been subjected to a drying procedure such as
lyophilization, where at least 50% of moisture has been
removed.
[0031] The phrase "bulking agent" includes a compound that is
pharmaceutically acceptable and that adds bulk to a lyo cake.
Bulking agents known to the art include, for example,
carbohydrates, including simple sugars such as dextrose, ribose,
fructose and the like, alcohol sugars such as mannitol, inositol
and sorbitol, disaccharides including trehalose, sucrose and
lactose, naturally occurring polymers such as starch, dextrans,
chitosan, hyaluronate, proteins (e.g., gelatin and serum albumin),
glycogen, and synthetic monomers and polymers.
[0032] A "lyoprotectant" is a molecule which, when combined with a
protein of interest, significantly prevents or reduces chemical
and/or physical instability of the protein upon lyophilization and
subsequent storage. Lyoprotectants include, but are not limited to,
sugars and their corresponding sugar alchohols; an amino acid such
as monosodium glutamate or histidine; a methylamine such as
betaine; a lyotropic salt such as magnesium sulfate; a polyol such
as trihydric or higher molecular weight sugar alcohols, e.g.
glycerin, dextran, erythritol, glycerol, arabitol, xylitol,
sorbitol, and mannitol; propylene glycol; polyethylene glycol;
Pluronics.RTM.; and combinations thereof. Additional examples of
lyoprotectants include, but are not limited to, glycerin and
gelatin, and the sugars mellibiose, melezitose, raffinose,
mannotriose and stachyose. Examples of reducing sugars include, but
are not limited to, glucose, maltose, lactose, maltulose,
iso-maltulose and lactulose. Examples of non-reducing sugars
include, but are not limited to, non-reducing glycosides of
polyhydroxy compounds selected from sugar alcohols and other
straight chain polyalcohols. Examples of sugar alcohols include,
but are not limited to, monoglycosides, compounds obtained by
reduction of disaccharides such as lactose, maltose, lactulose and
maltulose. The glycosidic side group can be either glucosidic or
galactosidic. Additional examples of sugar alcohols include, but
are not limited to, glucitol, maltitol, lactitol and iso-maltulose.
In specific embodiments, trehalose or sucrose is used as a
lyoprotectant.
[0033] The lyoprotectant is added to the pre-lyophilized
formulation in a "lyoprotecting amount" which means that, following
lyophilization of the protein in the presence of the lyoprotecting
amount of the lyoprotectant, the protein essentially retains its
physical and chemical stability and integrity upon lyophilization
and storage.
[0034] A "reconstituted" formulation is one which has been prepared
by dissolving a lyophilized antibody formulation in a diluent such
that the antibody is dispersed in the reconstituted formulation.
The reconstituted formulation is suitable for administration (e.g.
parenteral administration) to a patient to be treated with the
protein of interest and, in certain embodiments of the invention,
may be one which is suitable for intravenous administration.
[0035] The "diluent" of interest herein is one which is
pharmaceutically acceptable (safe and non-toxic for administration
to a human) and is useful for the preparation of a liquid
formulation, such as a formulation reconstituted after
lyophilization. In some embodiments, diluents include, but are not
limited to, sterile water, bacteriostatic water for injection
(BWFI), a pH buffered solution (e.g. phosphate-buffered saline),
sterile saline solution, Ringer's solution or dextrose solution. In
an alternative embodiment, diluents can include aqueous solutions
of salts and/or buffers.
[0036] An "isotonic" formulation is one which has essentially the
same osmotic pressure as human blood. Isotonic formulations will
generally have an osmotic pressure from about 250 to 350 mOsm. The
term "hypotonic" describes a formulation with an osmotic pressure
below that of human blood. Correspondingly, the term "hypertonic"
is used to describe a formulation with an osmotic pressure above
that of human blood. Isotonicity can be measured using a vapor
pressure or ice-freezing type osmometer.
[0037] As used herein, the term "subject" includes any human or
nonhuman animal. The term "nonhuman animal" includes all
vertebrates, for example, but not limited to, mammals and
non-mammals, such as nonhuman primates, sheep, dogs, cats, horses,
cows, chickens, amphibians, reptiles, etc.
[0038] By the terms "treat," "treating" or "treatment of" (or
grammatically equivalent terms) it is meant that the severity of
the subject's condition is reduced or at least partially improved
or ameliorated and/or that some alleviation, mitigation or decrease
in at least one clinical symptom is achieved and/or there is an
inhibition or delay in the progression of the condition and/or
prevention or delay of the onset of a disease or illness. Thus, the
terms "treat," "treating" or "treatment of" (or grammatically
equivalent terms) refer to both prophylactic and therapeutic
treatment regimes.
[0039] As used herein, a "sufficient amount" or "an amount
sufficient to" achieve a particular result refers to an amount of
an antibody or composition of the invention that is effective to
produce a desired effect, which is optionally a therapeutic effect
(i.e., by administration of a therapeutically effective amount).
For example, a "sufficient amount" or "an amount sufficient to" can
be an amount that is effective to deplete B cells.
[0040] A "therapeutically effective" amount as used herein is an
amount that provides some improvement or benefit to the subject.
Stated in another way, a "therapeutically effective" amount is an
amount that provides some alleviation, mitigation, and/or decrease
in at least one clinical symptom. Clinical symptoms associated with
the disorders that can be treated by the methods of the invention
are well-known to those skilled in the art. Further, those skilled
in the art will appreciate that the therapeutic effects need not be
complete or curative, as long as some benefit is provided to the
subject.
[0041] Concentrations, amounts, cell counts, percentages and other
numerical values may be presented herein in a range format. It is
also to be understood that such range format is used merely for
convenience and brevity and should be interpreted flexibly to
include not only the numerical values explicitly recited as the
limits of the range but also to include all the individual
numerical values or sub-ranges encompassed within that range as if
each numerical value and sub-range is explicitly recited.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1. Composition of formulations evaluated in the
excipient screen study.
[0043] FIG. 2. Post-reconstitution recovery from lyophilized
formulations.
[0044] FIG. 3. Bulk liquid stability: Change in % aggregate over
time at .about.50 ug/mL protein concentration.
[0045] FIG. 4. Bulk liquid stability: Change in % aggregate over
time at .about.300 ug/mL protein concentration.
[0046] FIG. 5. Percent protein recovery after 3 freeze/thaw cycles.
Data normalized to initial liquid control.
[0047] FIG. 6. Percent protein recovery after physical stress
(shaking). Data normalized to initial liquid control.
[0048] FIG. 7. Post-reconstitution stability at room temperature.
Results for each formulation are normalized to their respective
pre-lyophilization concentrations.
[0049] FIG. 8. Stability of lyophilized cakes at 40.degree. C. as
measured by percent recovery of MT103 from reconstituted cakes.
Results for each formulation are normalized to pre-lyophilization
concentrations.
[0050] FIG. 9. Composition of formulations evaluated in the pH
screen study.
[0051] FIG. 10. Effect of pH 6 vs 7 on recovery of MT103 (N=1).
[0052] FIG. 11. Composition of formulations evaluated in the lysine
HCl screen study.
[0053] FIG. 12. Percent recovery of MT103 after three freeze-thaw
cycles.
[0054] FIG. 13. Percent dimer content after three freeze-thaw
cycles.
[0055] FIG. 14. Percent MT103 recovery after two hours of
shaking.
[0056] FIG. 15. Percent dimer after two hours of shaking.
[0057] FIG. 16. Stability of MT103 in bulk liquid formulation at
4.degree. C.
[0058] FIG. 17. Stability of MT103 in bulk liquid formulation at
4.degree. C.
[0059] FIG. 18. Stability of lyophilized MT103 stored at 4.degree.
C.
[0060] FIG. 19. Stability of lyophilized MT103 stored at 40.degree.
C.
[0061] FIG. 20. Stability of lyophilized MT103 stored at 4.degree.
C.
[0062] FIG. 21. Stability of lyophilized MT103 stored at 40.degree.
C.
[0063] FIG. 22. Post-reconstitution stability of lyophilized MT103
at room temperature (-22.degree. C.) following reconstitution. 55
mg/mL MT103 in formulation #29 was lyophilized and subsequently
reconstituted.
[0064] FIG. 23. MT103 aggregation at 5.degree. C. as a function of
pH in the presence of basic amino acids.
[0065] FIG. 24. MT103 aggregation at 25.degree. C. as a function of
pH in the presence of basic amino acids.
[0066] FIG. 25. Determination of threshold levels of excipients.
Recovery tat 40.degree. C. in the presence of lysine.
[0067] FIGS. 26A and 26B show determination of threshold levels of
excipients. FIG. 26A shows recovery after three freeze/thaw cycles
or following shear stress. FIG. 26B show aggregate levels after
three freeze/thaw cycles or following shear stress.
[0068] FIGS. 27A, 27B and 27C show short term stability of MT103
formulations 25/25, 10/50, and 25/200. FIG. 27A shows composition
of formulations tested. FIG. 27B shows aggregate levels of
formulation 10/50 as a function of time at 5.degree. C., 25.degree.
C., and 40.degree. C. FIG. 27C shows aggregate levels of
formulation 25/25 as a function of time at 5.degree. C., 25.degree.
C., and 40.degree. C.
[0069] FIG. 28A shows stability of bulk liquid formulations 25/25,
10/50, and 25/200 at 5.degree. C. FIG. 28B shows stability of bulk
liquid formulations 25/25, 10/50, and 25/200 at 25.degree. C.
[0070] FIGS. 29A and 29B show mean MT103 serum concentrations in
CD-1 mice over a one day period following a single intravenous (IV)
or subcutaneous (SC) bolus injection of MT103 in different
formulations. FIG. 29A shows the mean MT103 concentration versus
time profile of serum samples collected from CD-1 mice after IV or
SC bolus infusion of 0.75 mg/kg of MT103 in one of four
formulations described in Table 12. FIG. 29B shows the mean MT103
concentration versus time profile of serum samples collected from
CD-1 mice after IV or SC bolus infusion of 2.5 mg/kg of MT103 in
one of four formulations described in Table 12. After bolus
injection of MT103 into CD-1 mice (n=4) serum samples were
collected at the indicated times shown in FIGS. 29A and 29B. The
serum concentrations of MT103 were measured by a specific ELISA
method and reported in FIGS. 29A and 29B as the arithmetic mean for
each sample at each time interval after infusion.
[0071] FIGS. 30A and 30B show mean MT103 serum concentrations in
CD-1 mice over a one day period following a single IV or SC bolus
infusion of MT103 in Formulation Z. FIG. 30A shows the mean MT103
concentration versus time profile of serum samples collected from
CD-1 mice after IV or SC bolus infusion of 5 mg/kg of MT103 in
Formulation Z. FIG. 30B shows the mean MT103 concentration versus
time profile of serum samples collected from CD-1 mice after IV or
SC bolus infusion of 1.6 mg/kg of MT103 in Formulation Z. After
bolus injection of MT103 into CD-1 mice (n=5) serum samples were
collected at the indicated times shown in FIGS. 30A and 30B. The
serum concentrations of MT103 were measured by a specific ELISA
method and reported in FIGS. 30A and 30B as the arithmetic mean for
each sample at each time interval after infusion.
[0072] FIGS. 31A and 31B show mean MT103 serum concentrations in
cynomolgus monkeys over a one week period following a single
intravenous (IV) or subcutaneous (SC) bolus injection of MT103.
FIGS. 31A and 31B show the mean MT103 concentration versus time
profile of serum samples collected from male cynomolgus monkeys
after IV or SC bolus infusion 0.5 mg/kg of MT103 in a cross-over
design with a one week washout period between the two phases (FIG.
31A shows Phase 1: IV then SC; FIG. 31B shows Phase 2: SC then IV).
After bolus injection of MT103 into cynomolgus monkeys (n=3) serum
samples were collected at the times indicated in FIGS. 31A and 31B.
The serum concentrations of MT103 were measured by a specific ELISA
method and reported in FIGS. 31A and 31B as the arithmetic mean for
each sample at each time interval after infusion.
[0073] FIG. 32. In CD-1 mice, SC administered MEDI-538 was 20-30%
bioavailable and exhibited serum concentrations that were
dose-proportional, reached a peak at 4 hrs, and had a similar
terminal half-life as IV administered drug. The mean MEDI-538
concentration versus time profile of serum samples collected from
CD-1 mice after IV and SC administration of 0.25 (triangles), 0.75
(circles), and 2.5 (squares) mg/kg of MEDI-538 is shown.
[0074] FIG. 33. Following SC administration in cynomolgus monkeys,
MEDI-538 was 22% bioavailable and exhibited serum concentrations
that reached a peak at 4 hrs, and had a similar terminal half-life
as IV administered drug. The mean MEDI-538 concentration versus
time profile of serum samples collected from cynomolgus monkeys
after IV and SC administration of 0.5 mg/kg of MEDI-538.
[0075] FIG. 34. In vivo mouse models for anti-tumor efficacy.
[0076] FIG. 35. SC treatment with MEDI-538 completely inhibits the
growth of SC engrafted CD19+ Namalwa B-lymphoma cells mixed with
human CD3+ T cells in NOD/SCID mice.
[0077] FIG. 36. SC treatment with MEDI-538 prolongs the survival of
NOD/SCID mice engrafted IV with CD19+Ramos B Lymphoma cells and
human CD3+ T cells.
[0078] FIGS. 37A and 37B show mediation of mouse T cell lysis of a
B cell lymphoma line (NALM-6) by HyS 103. FIG. 37A shows the %
lysis in Human Effector T cells. FIG. 37B shows the % lysis in
huCD19 Tg Mouse Effector T cells.
[0079] FIG. 38. Dose-dependent reduction of splenic B cells in
huCD19 transgenic mice following SC treatment with a single dose of
hys103.
[0080] FIG. 39. Dose-dependent reduction of bone marrow B cells in
huCD19 transgenic mice following SC treatment with a single dose of
hys103.
[0081] FIG. 40. Dose-dependent activation (CD69) of splenic T cells
in huCD19 transgenic mice following a single SC administration of
hys103.
[0082] FIG. 41. Dose-dependent activation (CD69) of bone marrow T
cells in huCD19 transgenic mice following a single SC
administration of hys103.
[0083] FIGS. 42A and 42B show PK analysis of hys103 in huCD19
transgenic mice. FIG. 42A shows hys103 serum concentration as a
function of time following i.v. administration of a single dose of
hys103. FIG. 42B shows hys103 serum concentration as a function of
time following s.c. administration of a single dose of hys103.
5. DETAILED DESCRIPTION
[0084] The present invention relates to stable formulations of
bispecific antibodies. In certain embodiments, the present
invention provides formulations of BiTE.RTM. molecules comprising a
first and a second binding site specific for the CD3 and CD19
antigens, respectively. In a specific embodiment, the present
invention provides formulations comprising the MT103.TM. BiTE.RTM.
molecule. In yet other embodiments, formulations of the invention
comprise a BiTE.RTM. molecule having at least a first and a second
binding site specific for CD3 and the EphA2 antigens, respectively.
In yet other embodiments, formulations of the invention comprise a
BiTE.RTM. molecule having at least a first and a second binding
site specific for CD3 and the carcinoembryonic antigen (CEA)
antigens, respectively.
[0085] In one embodiment, a formulation of the invention is for
parenteral administration. In one embodiment, a formulation of the
invention is an injectable formulation. In one embodiment, a
formulation of the invention is for intravenous, subcutaneous, or
intramuscular administration. In certain embodiments, formulations
of the invention comprise BiTE.RTM. molecules wherein said
formulations are for intravenous administration. In a specific
embodiment, a formulation of the invention comprise MT103 wherein
said formulation is for intravenous administration. In certain
embodiments, formulations of the invention comprise BiTE.RTM.
molecules wherein said formulations are for continuous intravenous
administration. In a specific embodiment, a formulation of the
invention comprise MT103 wherein said formulation is for continuous
intravenous administration. In certain embodiments, formulations of
the invention comprise BiTE.RTM. molecules wherein said
formulations are for subcutaneous administration. In a specific
embodiment, a formulation of the invention comprise MT103 wherein
said formulation is for subcutaneous administration.
[0086] In one embodiment, a formulation of the invention is for
continuous intravenous administration wherein said formulation
comprises between about 25 .mu.g/ml and about 250 .mu.g/ml of
BiTE.RTM. molecules or a fragment thereof. In a specific
embodiment, a formulation of the invention is for continuous
intravenous administration wherein said formulation comprises
between about 25 .mu.g/ml and about 250 .mu.g/ml of MT103. In one
embodiment, a formulation of the invention is for subcutaneous
administration wherein said formulation comprises between about 25
.mu.g/ml and about 250 .mu.g/ml of BiTE.RTM. molecules or a
fragment thereof. In a specific embodiment, a formulation of the
invention is for subcutaneous administration wherein said
formulation comprises between about 25 .mu.g/ml and about 250
.mu.g/ml of MT103.
[0087] In one embodiment, a formulation of the invention is
suitable for subcutaneous delivery. In one embodiment, the
bioavailability of a bispecific antibody formulated according to a
composition or method described herein is between about 10% and
about 100%.
[0088] In one embodiment, a formulation of the invention is for
aerosol administration.
[0089] The present invention also provides a pharmaceutical unit
dosage form suitable for parenteral administration to a human which
comprises BiTE.RTM. molecules formulation in a suitable container.
In one embodiment, a pharmaceutical unit dosage of the invention
comprises MT103. In one embodiment, a pharmaceutical unit dosage of
the invention comprises an intravenously, subcutaneously, or
intramuscularly delivered BiTE.RTM. molecule formulation. In
another embodiment, a pharmaceutical unit dosage of the invention
comprises aerosol delivered BiTE.RTM. molecule formulation. In a
specific embodiment, a pharmaceutical unit dosage of the invention
comprises a subcutaneously delivered MT103 formulation. In another
embodiment, a pharmaceutical unit dosage of the invention comprises
an intravenously delivered MT103 formulation. In another
embodiment, a pharmaceutical unit dosage of the invention comprises
an aerosol delivered BiTE.RTM. molecule formulation. In a further
embodiment, a pharmaceutical unit dosage of the invention comprises
an intranasally administered BiTE.RTM. molecule formulation.
[0090] In one embodiment, a formulation of the invention is
provided in a sealed container.
[0091] The present invention is further directed to methods for the
treatment of B cell disorders or diseases in human subjects,
including B cell malignancies, using the formulations of bispecific
antibodies or fragments thereof that comprise at least a first and
a second binding site specific for the CD3 and CD19 antigens,
respectively. The present invention is directed to methods for the
treatment and prevention of autoimmune disease as well as the
treatment and prevention of graft-versus-host disease (GVHD),
humoral rejection, and post-transplantation lymphoproliferative
disorder in human transplant recipients using formulations of
bispecific antibodies or fragments thereof that comprise at least a
first and a second binding site specific for the CD3 and CD19
antigens, respectively. In specific embodiments, the BiTE.RTM.
molecule of the formulations of the invention is MT103.
5.1. Liquid Formulations
[0092] In specific embodiments, the present invention encompasses
stable liquid formulations of bispecific antibodies which
formulations exhibit stability, low to undetectable levels of
antibody fragmentation, low to undetectable levels of aggregation,
very little to no loss of the biological activities of the
bispecific antibodies, and very little to no loss of antibody
through absorption to contact surfaces during manufacture,
preparation, transportation, and storage. The present invention
also encompasses stable liquid formulations of bispecific
antibodies that comprise at least a first and a second binding site
specific for the CD3 and CD19 antigens, respectively, said
formulations exhibiting low to undetectable levels of antibody
fragmentation, low to undetectable levels of aggregation, very
little to no loss of the biological activities, and very little to
no loss of antibody through absorption to contact surfaces during
manufacture, preparation, transportation, and storage. In specific
embodiments, the present invention provides stable liquid
formulations of MT103 which formulations exhibit stability, low to
undetectable levels of antibody fragmentation, low to undetectable
levels of aggregation, very little to no loss of the biological
activities of MT103, and very little to no loss of antibody through
absorption to contact surfaces during manufacture, preparation,
transportation, and storage.
[0093] In one embodiment, a liquid formulation of the invention is
an aqueous formulation. In a specific embodiment, a liquid
formulation of the invention is an aqueous formulation wherein the
aqueous carrier is distilled water.
[0094] In one embodiment, a formulation of the invention is
sterile.
[0095] In one embodiment, a formulation of the invention is
homogeneous.
[0096] In one embodiment, a formulation of the invention is
isotonic. In one embodiment, a formulation of the invention is
hypotonic. In one embodiment, a formulation of the invention is
hypertonic.
[0097] In certain embodiments, a liquid formulation of the
invention is suitable for lyophilization. In one embodiments, a
lyophilized formulation of the invention is stable from about one
month to about 2, 3, 4 or 5 years. In specific embodiments, a
lyophilized formulation of the invention is stable for more than 2
years.
[0098] The present invention provides stable liquid formulations of
MT103. The MT103 molecule was described in U.S. Pat. No.
7,112,324.
[0099] In one embodiment, a liquid formulation of the invention
comprises a BiTE.RTM. molecule. In one embodiment, a liquid
formulation of the invention comprises a BiTE.RTM. molecule that
comprises at least a first and a second binding domain specific for
the CD3 and CD19 antigens, respectively. In one embodiment, a
liquid formulation of the invention comprises a BiTE.RTM. molecule
that comprises at least a first and a second binding domain
specific for the CD3 and CD19 antigens, respectively, wherein the
first binding domain comprises the amino acid sequence of residues
283-525 of SEQ ID NO:5. In one embodiment, a liquid formulation of
the invention comprises a BiTE.RTM. molecule that comprises at
least a first and a second binding domain specific for the CD3 and
CD19 antigens, respectively, wherein the second binding domain
comprises the amino acid sequence of residues 28-277 of SEQ ID
NO:5. In another embodiment, a liquid formulation of the invention
comprises a BiTE.RTM. molecule that comprises at least a first and
a second binding domain specific for the CD3 and CD19 antigens,
respectively, wherein the first binding domain comprises the amino
acid sequence of residues 283-525 of SEQ ID NO:5 and the second
binding domain comprises the amino acid sequence of residues 28-277
of SEQ ID NO:5. In a further embodiment, a liquid formulation of
the invention comprises a BiTE.RTM. molecule wherein said BiTE.RTM.
molecule comprises an anti-CD19 VL domain having the amino acid
sequence of SEQ ID NO:1, an anti-CD19 VH domain having the amino
acid sequence of SEQ ID NO:2, an anti-CD3 VH domain having the
amino acid sequence of SEQ ID NO:3 and an anti-CD3 VL domain having
the amino acid sequence of SEQ ID NO:4. In one embodiment, a liquid
formulation of the invention comprises a BiTE.RTM. molecule, MT103,
wherein said a BiTE.RTM. molecule comprises the amino acid sequence
of residues 28-525 of SEQ ID NO:5. In a specific embodiment, a
liquid formulation of the invention comprises a BiTE.RTM. molecule,
MT103, wherein said a BiTE.RTM. molecule comprises the amino acid
sequence of SEQ ID NO:5.
[0100] The invention encompasses stable liquid formulations
comprising a single BiTE.RTM. molecule of interest, for example,
BiTE.RTM. molecules that comprises at least a first and a second
binding site specific for the CD3 and CD19 antigens, respectively.
The invention also encompasses stable liquid formulations
comprising two or more bispecific antibodies of interest, for
example, bispecific antibodies that comprise at least a first and a
second binding site specific for the CD3 and CD19 antigens,
respectively. In a specific embodiment, a stable liquid formulation
of the invention comprises MT103. In another embodiment, a stable
liquid formulation of the invention comprises two or more
bispecific antibodies, wherein one of the bispecific antibodies is
MT103.
[0101] In one embodiment, a formulation of the invention comprises
at least about 1 microgram/ml, at least about 5 micrograms/ml, at
least about 10 micrograms/ml, at least about 15 micrograms/ml, at
least about 20 micrograms/ml, at least about 25 micrograms/ml, at
least about 30 micrograms/ml, at least about 35 micrograms/ml, at
least about 40 micrograms/ml, at least about 45 micrograms/ml, at
least about 50 micrograms/ml, at least about 55 micrograms/ml, at
least about 60 micrograms/ml, at least about 70 micrograms/ml, at
least about 80 micrograms/ml, at least about 90 micrograms/ml, at
least about 100 micrograms/ml, at least about 110 micrograms/ml, at
least about 120 micrograms/ml, at least about 130 micrograms/ml, at
least about 140 micrograms/ml, at least about 150 micrograms/ml, at
least about 160 micrograms/ml, at least about 170 micrograms/ml, at
least about 180 micrograms/ml, at least about 190 micrograms/ml, at
least about 200 micrograms/ml, at least about 225 micrograms/ml, at
least about 250 micrograms/ml, at least about 275 micrograms/ml, at
least about 300 micrograms/ml at least about 325 micrograms/ml, at
least about 350 micrograms/ml, at least about 375 micrograms/ml, at
least about 400 micrograms/ml, at least about 500 micrograms/ml, at
least about 700 micrograms/ml, at least about 900 micrograms/ml, or
at least about 1000 micrograms/ml of BiTE.RTM. molecules or a
fragment thereof. In a further embodiment, a formulation of the
invention comprises at least about 1 mg/ml, at least about 2 mg/ml,
at least about 3 mg/ml, at least about 4 mg/ml, at least about 5
mg/ml, at least about 7 mg/ml, at least about 9 mg/ml, or at least
about 10 mg/ml of BiTE.RTM. molecules or a fragment thereof. In a
specific embodiment, a formulation of the invention comprises at
least about 55 micrograms/ml of BiTE.RTM. molecules or a fragment
thereof. In a specific embodiment, a formulation of the invention
comprises at least about 160 micrograms/ml of BiTE.RTM. molecules
or a fragment thereof. In a specific embodiment, a formulation of
the invention comprises at least about 150 micrograms/ml of
BiTE.RTM. molecules or a fragment thereof. In a specific
embodiment, a BiTE.RTM. molecule formulated as described herein is
specific for the CD19, EphA2 or CEA antigens.
[0102] In another embodiment, a formulation of the invention
comprises between about 1 microgram/ml and about 50 micrograms/ml,
between about 50 micrograms/ml and about 100 micrograms/ml, between
about 100 micrograms/ml and about 150 micrograms/ml, between about
150 micrograms/ml and about 200 micrograms/ml, between about 200
micrograms/ml and about 250 micrograms/ml, between about 250
micrograms/ml and about 300 micrograms/ml, between about 300
micrograms/ml and about 400 micrograms/ml, between about 400
micrograms/ml and about 500 micrograms/ml or between about 500
micrograms/ml and about 1000 micrograms/ml of BiTE.RTM. molecules
or a fragment thereof. In further embodiment, a formulation of the
invention comprises between about 1 mg/ml and about 2 mg/ml,
between about 2 mg/ml and about 3 mg/ml, between about 3 mg/ml and
about 5 mg/ml, or between about 5 mg/ml and about 10 mg/ml of
BiTE.RTM. molecules or a fragment thereof. In a specific
embodiment, a formulation of the invention comprises between about
25 micrograms/ml and about 75 micrograms/ml of BiTE.RTM. molecules
or a fragment thereof. In a specific embodiment, a formulation of
the invention comprises between about 150 micrograms/ml and about
200 micrograms/ml of BiTE.RTM. molecules or a fragment thereof. In
a specific embodiment, a formulation of the invention comprises
between about 140 micrograms/ml and about 170 micrograms/ml of
BiTE.RTM. molecules or a fragment thereof. In a specific
embodiment, a BiTE.RTM. molecule formulated as described herein is
specific for the CD19, EphA2 or CEA antigens.
[0103] In a further embodiment, a formulation described herein
comprises about 5 micrograms/ml, about 10 micrograms/ml, about 20
micrograms/ml, about 30 micrograms/ml, about 40 micrograms/ml,
about 50 micrograms/ml, about 60 micrograms/ml, about 70
micrograms/ml, about 80 micrograms/ml, about 90 micrograms/ml,
about 100 micrograms/ml, about 110 micrograms/ml, about 120
micrograms/ml, about 130 micrograms/ml, about 140 micrograms/ml,
about 150 micrograms/ml, about 160 micrograms/ml, about 170
micrograms/ml, about 180 micrograms/ml, about 190 micrograms/ml,
about 200 micrograms/ml, about 210 micrograms/ml, about 220
micrograms/ml, about 230 micrograms/ml, about 240 micrograms/ml,
about 250 micrograms/ml, about 260 micrograms/ml, about 270
micrograms/ml, about 280 micrograms/ml, about 290 micrograms/ml,
about 300 micrograms/ml, about 310 micrograms/ml, about 320
micrograms/ml, about 330 micrograms/ml, about 340 micrograms/ml,
about 350 micrograms/ml, about 500 micrograms/ml, about 700
micrograms/ml, about 900 micrograms/ml, or about 1000 micrograms/ml
of BiTE.RTM. molecules or a fragment thereof. In a further
embodiment, a formulation of the invention comprises about 1 mg/ml,
about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 7
mg/ml, about 9 mg/ml, or about 10 mg/ml of BiTE.RTM. molecules or a
fragment thereof. In a specific embodiment, a formulation of the
invention comprises about 55 micrograms/ml of BiTE.RTM. molecules
or a fragment thereof. In a specific embodiment, a formulation of
the invention comprises about 100 micrograms/ml of BiTE.RTM.
molecules or a fragment thereof. In a specific embodiment, a
formulation of the invention comprises about 160 micrograms/ml of
BiTE.RTM. molecules or a fragment thereof. In a specific
embodiment, a formulation of the invention comprises about 150
micrograms/ml of BiTE.RTM. molecules or a fragment thereof. In a
specific embodiment, a BiTE.RTM. molecule formulated as described
herein is specific for the CD19, EphA2 or CEA antigens.
[0104] In one embodiment, a formulation of the invention comprises
at least about 1 microgram/ml, at least about 5 micrograms/ml, at
least about 10 micrograms/ml, at least about 15 micrograms/ml, at
least about 20 micrograms/ml, at least about 25 micrograms/ml, at
least about 30 micrograms/ml, at least about 35 micrograms/ml, at
least about 40 micrograms/ml, at least about 45 micrograms/ml, at
least about 50 micrograms/ml, at least about 55 micrograms/ml, at
least about 60 micrograms/ml, at least about 70 micrograms/ml, at
least about 80 micrograms/ml, at least about 90 micrograms/ml, at
least about 100 micrograms/ml, at least about 110 micrograms/ml, at
least about 120 micrograms/ml, at least about 130 micrograms/ml, at
least about 140 micrograms/ml, at least about 150 micrograms/ml, at
least about 160 micrograms/ml, at least about 170 micrograms/ml, at
least about 180 micrograms/ml, at least about 190 micrograms/ml, at
least about 200 micrograms/ml, at least about 225 micrograms/ml, at
least about 250 micrograms/ml, at least about 275 micrograms/ml, at
least about 300 micrograms/ml at least about 325 micrograms/ml, at
least about 350 micrograms/ml, at least about 375 micrograms/ml, at
least about 400 micrograms/ml, at least about 500 micrograms/ml, at
least about 700 micrograms/ml, at least about 900 micrograms/ml, or
at least about 1000 micrograms/ml of MT103 or a fragment thereof.
In a further embodiment, a formulation of the invention comprises
at least about 1 mg/ml, at least about 2 mg/ml, at least about 3
mg/ml, at least about 4 mg/ml, at least about 5 mg/ml, at least
about 7 mg/ml, at least about 9 mg/ml, or at least about 10 mg/ml
of MT103 or a fragment thereof. In a specific embodiment, a
formulation of the invention comprises at least about 55
micrograms/ml of MT103 or a fragment thereof. In a specific
embodiment, a formulation of the invention comprises at least about
160 micrograms/ml of MT103 or a fragment thereof. In a specific
embodiment, a formulation of the invention comprises at least about
150 micrograms/ml of MT103 or a fragment thereof.
[0105] In another embodiment, a formulation of the invention
comprises between about 1 microgram/ml and about 50 micrograms/ml,
between about 50 micrograms/ml and about 100 micrograms/ml, between
about 100 micrograms/ml and about 150 micrograms/ml, between about
150 micrograms/ml and about 200 micrograms/ml, between about 200
micrograms/ml and about 250 micrograms/ml, between about 250
micrograms/ml and about 300 micrograms/ml, between about 300
micrograms/ml and about 400 micrograms/ml, or between about 400
micrograms/ml and about 500 micrograms/ml of BiTE.RTM. molecule or
a fragment thereof. In further embodiment, a formulation of the
invention comprises between about 1 mg/ml and about 2 mg/ml,
between about 2 mg/ml and about 3 mg/ml, between about 3 mg/ml and
about 5 mg/ml, or between about 5 mg/ml and about 10 mg/ml of MT103
or a fragment thereof. In a specific embodiment, a formulation of
the invention comprises between about 25 micrograms/ml and about 75
micrograms/ml of MT103 or a fragment thereof. In a further
embodiment, a formulation of the invention comprises between about
140 micrograms/ml and about 190 micrograms/ml of MT103 or a
fragment thereof. In a further embodiment, a formulation of the
invention comprises between about 140 micrograms/ml and about 160
micrograms/ml of MT103 or a fragment thereof.
[0106] In a further embodiment, a formulation described herein
comprises about 5 micrograms/ml, 10 micrograms/ml, 20
micrograms/ml, about 30 micrograms/ml, about 40 micrograms/ml,
about 50 micrograms/ml, about 60 micrograms/ml, about 70
micrograms/ml, about 80 micrograms/ml, about 90 micrograms/ml,
about 100 micrograms/ml, about 110 micrograms/ml, about 120
micrograms/ml, about 130 micrograms/ml, about 140 micrograms/ml,
about 150 micrograms/ml, about 160 micrograms/ml, about 170
micrograms/ml, about 180 micrograms/ml, about 190 micrograms/ml,
about 200 micrograms/ml, about 210 micrograms/ml, about 220
micrograms/ml, about 230 micrograms/ml, about 240 micrograms/ml,
about 250 micrograms/ml, about 260 micrograms/ml, about 270
micrograms/ml, about 280 micrograms/ml, about 290 micrograms/ml,
about 300 micrograms/ml, about 310 micrograms/ml, about 320
micrograms/ml, about 330 micrograms/ml, about 340 micrograms/ml,
about 350 micrograms/ml, about 500 micrograms/ml, about 700
micrograms/ml, about 900 micrograms/ml, or about 1000 micrograms/ml
of MT103 or a fragment thereof. In a further embodiment, a
formulation of the invention comprises about 1 mg/ml, about 2
mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 7 mg/ml,
about 9 mg/ml, or about 10 mg/ml of MT103 or a fragment thereof. In
a specific embodiment, a formulation of the invention comprises
about 55 micrograms/ml of MT103 or a fragment thereof. In a
specific embodiment, a formulation of the invention comprises about
100 micrograms/ml of MT103 or a fragment thereof. In a specific
embodiment, a formulation of the invention comprises about 160
micrograms/ml of MT103 or a fragment thereof. In a specific
embodiment, a formulation of the invention comprises about 150
micrograms/ml of MT103 or a fragment thereof.
[0107] Optionally, the formulations of the invention may further
comprise common excipients and/or additives such as buffering
agents, amino acids, saccharides, salts, surfactants, bulking
agents, and lyoprotectants. Additionally or alternatively, the
formulations of the invention may further comprise common
excipients and/or additives, such as, but not limited to,
solubilizers, diluents, binders, stabilizers, salts, lipophilic
solvents, chelators, preservatives, or the like.
[0108] In certain embodiments, the buffering agent is selected from
the group consisting of histidine, citrate, phosphate, glycine, and
acetate. In other embodiments the saccharide excipient is selected
from the group consisting of trehalose, sucrose, mannitol, maltose
and raffinose. In still other embodiments the surfactant is
selected from the group consisting of polysorbate 20, polysorbate
40, polysorbate 80, and Pluronic F68. In yet other embodiments the
salt is selected from the group consisting of NaCl, KCl,
MgCl.sub.2, and CaCl.sub.2. In further embodiments, the bulking
agent is selected from the group consisting of dextrose, ribose,
fructose, mannitol, inositol, sorbitol, trehalose, sucrose,
lactose, starch, dextrans, chitosan, hyaluronate, gelatin, serum
albumin, glycogen, synthetic monomers and synthetic polymers. In
additional embodiments, the lyoprotectant is selected from the
group consisting of monosodium glutamate, histidine; betaine;
magnesium sulfate; glycerin, dextran, erythritol, glycerol,
arabitol, xylitol, sorbitol, mannitol; propylene glycol;
polyethylene glycol; Pluronics.RTM. gelatin, mellibiose,
melezitose, raffinose, mannotriose, stachyose, glucose, maltose,
lactose, maltulose, iso-maltulose and lactulose, glucitol,
maltitol, lactitol, iso-maltulose, trehalose, sucrose and
combinations thereof. It is understood that a particular excipient
may serve more than one purpose in a formulation, for example,
trehalose may serve both as a bulking agent and a
lyoprotectant.
[0109] Optionally, the formulations of the invention may further
comprise other common auxiliary components, such as, but not
limited to, suitable excipients, polyols, solubilizers, diluents,
binders, stabilizers, lipophilic solvents, chelators,
preservatives, or the like.
[0110] The liquid formulations of the invention include a buffering
or pH adjusting agent to provide improved pH control. In one
embodiment, a formulation of the invention has a pH of between
about 4.0 and about 9.0, between about 5.0 and about 9.0, between
about 6.0 and about 8.0, between about 6.0 and about 7.5, between
about 6.5 and about 8.0, or between about 6.5 and about 7.5. In a
further embodiment, a formulation of the invention has a pH of
about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5,
about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5,
about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1,
about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7,
about 7.8, about 7.9, about 8.0, about 8.5, or about 9.0. In a
specific embodiment, a formulation of the invention has a pH of
about 7.0. In a further embodiment, a formulation of the invention
has a pH of about 6.0. The pH of a formulation generally should not
be equal to the isoelectric point of the particular BiTE.RTM.
molecule formulated.
[0111] Typically, the buffering agent is a salt prepared from an
organic or inorganic acid or base. Representative buffering agents
include, but are not limited to, organic acid salts such as salts
of citric acid, ascorbic acid, gluconic acid, carbonic acid,
tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,
tromethamine hydrochloride, or phosphate buffers. In addition,
amino acid components can also function in a buffering capacity.
Representative amino acid components which may be utilized in the
formulations of the invention as buffering agents include, but are
not limited to, glycine and histidine. In certain embodiments, the
buffering agent is selected from the group consisting of histidine,
citrate, phosphate, glycine, and acetate. In a specific embodiment,
the buffering agent is histidine. In another specific embodiment,
the buffering agent is citrate. The purity of the buffering agent
should be at least 98%, or at least 99%, or at least 99.5%. As used
herein, the term "purity" in the context of histidine refers to
chemical purity of histidine as understood in the art, e.g., as
described in The Merck Index, 13.sup.th ed., O'Neil et al. ed.
(Merck & Co., 2001).
[0112] Buffering agents are typically used at concentrations
between about 1 mM and about 200 mM or any range or value therein,
depending on the desired ionic strength and the buffering capacity
required. The usual concentrations of conventional buffering agents
employed in parenteral formulations can be found in: Pharmaceutical
Dosage Form: Parenteral Medications, Volume 1, 2.sup.nd Edition,
Chapter 5, p. 194, De Luca and Boylan, "Formulation of Small Volume
Parenterals", Table 5: Commonly used additives in Parenteral
Products. In one embodiment, the buffering agent is at a
concentration of about 1 mM, or of about 5 mM, or of about 10 mM,
or of about 15 mM, or of about 20 mM, or of about 25 mM, or of
about 30 mM, or of about 35 mM, or of about 40 mM, or of about 45
mM, or of about 50 mM, or of about 60 mM, or of about 70 mM, or of
about 80 mM, or of about 90 mM, or of about 100 mM. In one
embodiment, the buffering agent is at a concentration of 1 mM, or
of 5 mM, or of 10 mM, or of 15 mM, or of 20 mM, or of 25 mM, or of
30 mM, or of 35 mM, or of 40 mM, or of 45 mM, or of 50 mM, or of 60
mM, or of 70 mM, or of 80 mM, or of 90 mM, or of 100 mM. In a
specific embodiment, the buffering agent is at a concentration of
between about 10 mM and about 50 mM. In another specific
embodiment, the buffering agent is at a concentration of between 10
mM and 50 mM.
[0113] In certain embodiments, a formulation of the invention
comprises a buffering agent. In one embodiment, said buffering
agent is selected from the group consisting of histidine, citrate,
phosphate, Tris, glycine, and acetate. In a specific embodiment, a
formulation of the invention comprises citrate as a buffering
agent. In a further embodiment, a formulation of the invention
comprises a citrate buffer.
[0114] In one embodiment, a formulation of the invention comprises
at least about 1 mM, at least about 5 mM, at least about 10 mM, at
least about 20 mM, at least about 30 mM, at least about 40 mM, at
least about 50 mM, at least about 75 mM, at least about 100 mM, at
least about 150 mM, or at least about 200 mM citrate. In another
embodiment, a formulation of the invention comprises between about
1 mM and about 5 mM, between about 5 mM and about 10 mM, between
about 10 mM and about 15 mM, between about 15 mM and about 20 mM,
between about 20 mM and about 30 mM, between about 30 mM and about
50 mM, between about 50 mM and about 70 mM, between about 70 mM and
about 100 mM, between about 100 mM and about 150 mM, between about
150 mM and about 200 mM, or between about 200 mM and about 300 mM
citrate. In a further embodiment of the invention comprises about 1
mM, about 5 mM, about 10 mM, about 20 mM, about 25 mM, about 30 mM,
about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM,
about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM,
or about 200 mM citrate. In a specific embodiment, a formulation of
the invention comprises about 25 mM citrate. In a specific
embodiment, a formulation of the invention comprises about 30 mM
citrate.
[0115] In one embodiment, a formulation of the invention comprises
at least about 1 mM, at least about 5 mM, at least about 10 mM, at
least about 20 mM, at least about 30 mM, at least about 40 mM, at
least about 50 mM, at least about 75 mM, at least about 100 mM, at
least about 150 mM, or at least about 200 mM citrate buffer. In
another embodiment, a formulation of the invention comprises
between about 1 mM and about 5 mM, between about 5 mM and about 10
mM, between about 10 mM and about 15 mM, between about 15 mM and
about 20 mM, between about 20 mM and about 30 mM, between about 30
mM and about 50 mM, between about 50 mM and about 70 mM, between
about 70 mM and about 100 mM, between about 100 mM and about 150
mM, between about 150 mM and about 200 mM, or between about 200 mM
and about 300 mM citrate buffer. In a further embodiment of the
invention comprises about 1 mM, about 5 mM, about 10 mM, about 20
mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45
mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90
mM, about 100 mM, about 150 mM, or about 200 mM citrate buffer. In
a specific embodiment, a formulation of the invention comprises
about 25 mM citrate buffer. In a specific embodiment, a formulation
of the invention comprises about 30 mM citrate buffer.
[0116] In certain embodiments, the formulations of the invention
comprise a carbohydrate excipient. Carbohydrate excipients can act,
e.g., as viscosity enhancing agents, stabilizers, bulking agents,
solubilizing agents, lyoprotectants and/or the like. Carbohydrate
excipients are generally present at between about 1% to about 99%
by weight/volume. In one embodiment, the carbohydrate excipient is
present at between about 0.1% to about 50%. In another embodiment,
the carbohydrate excipient is present at between about 0.5% to
about 40%. In a specific embodiment, the carbohydrate excipient is
present at between about 1% to about 2%, or between about 2% to
about 5%, or between about 5% to about 10%, or between about 10% to
about 15%, between about 15% and about 20%, between about 20% and
about 25%, between about 25% and about 30%, or between about 30%
and about 40%. In other specific embodiments, the carbohydrate
excipient is present at 1%, or at 2%, or at 3%, or at 5%, or at 6%,
or at 6.5%, or at 7%, or at 8%, or at 9%, or at 10%, or at 11%, or
at 12%, or at 13%, or at 14%, or at 15%, or at 16%, or at 17%, or
at 18%, or at 20%, or at 25%, or at 30%.
[0117] Carbohydrate excipients suitable for use in the formulations
of the invention include, for example, monosaccharides such as
fructose, maltose, galactose, glucose, D-mannose, sorbose, and the
like; disaccharides, such as lactose, sucrose, trehalose,
cellobiose, and the like; polysaccharides, such as raffinose,
melezitose, maltodextrins, dextrans, starches, and the like; and
alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol
sorbitol (glucitol) and the like. In one embodiment, the
carbohydrate excipients for use in the present invention are
selected from the group consisting of, sucrose, trehalose, lactose,
mannitol, and raffinose. In a specific embodiment, the carbohydrate
excipient is trehalose. In another specific embodiment, the
carbohydrate excipient is mannitol. In yet another specific
embodiment, the carbohydrate excipient is sucrose. In still another
specific embodiment, the carbohydrate excipient is raffinose. The
purity of the carbohydrate excipient should be at least 98%, or at
least 99%, or at least 99.5%.
[0118] In one embodiment, a formulation of the invention comprises
at least about 1%, at least about 2.5%, at least about 5%, at least
about 6%, at least about 7%, at least about 8%, at least about 9%,
at least about 10%, at least about 11%, at least about 12%, at
least about 13%, at least about 14%, at least about 15%, at least
about 16%, at least about 17%, at least about 18%, at least about
19%, at least about 20%, at least about 30%, or at least about 40%
trehalose dihydrate. In another embodiment, a formulation of the
invention comprises between about 1% and about 40%, between about
1% and about 5%, between about 5% and about 10%, between about 10%
and about 15%, between about 15% and about 20%, between about 20%
and about 25%, between about 25% and about 30%, or between about
30% and about 40% trehalose dihydrate. In a further embodiment, a
formulation of the invention comprises about 1%, about 2%, about
4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,
about 11%, about 12%, about 13%, about 14%, about 15%, about 16%,
about 17%, about 18%, about 19%, about 20%, about 30%, or about 40%
trehalose dihydrate. In a specific embodiment, a formulation of the
invention comprises about 15% trehalose dihydrate. In a specific
embodiment, a formulation of the invention comprises about 5%
trehalose dihydrate. In a specific embodiment, a formulation of the
invention comprises about 6% trehalose dihydrate. In a specific
embodiment, a formulation of the invention comprises about 6.5%
trehalose dihydrate.
[0119] In one embodiment, the molar ratio of a lyoprotectant (e.g.,
trehalose) and BiTE.RTM. molecules of a formulation of the
invention is at least about 10,000, at least about 50,000, at least
about 100,000, at least about 200,000, at least about 300,000, at
least about 400,000, at least about 500,000, at least about
600,000, at least about 700,000, at least about 800,000, at least
about 900,000, or at least about 1,000,000. In another embodiment,
the molar ratio of a lyoprotectant (e.g., trehalose) and BiTE.RTM.
molecules of a formulation of the invention is about 10,000, about
50,000, about 100,000, about 200,000, about 300,000, about 400,000,
about 500,000, about 600,000, about 700,000, about 800,000, about
900,000, or about 1,000,000. In a further embodiment, the molar
ratio of a lyoprotectant (e.g., trehalose) and BiTE.RTM. molecules
of a formulation of the invention is between about 10,000 and about
1,000,000, between about 100,000 and about 900,000, or between
about 500,000 and about 900,000.
[0120] In one embodiment, a formulation of the invention comprises
an excipient. In a specific embodiment, a formulation of the
invention comprises at least one excipient selected from the group
consisting of: sugar, salt, surfactant, amino acid, polyol,
chelating agent, emulsifier and preservative. In one embodiment, a
formulation of the invention comprises a salt. In one embodiment, a
formulation of the invention comprises a salt selected from the
group consisting of: NaCl, KCl, CaCl.sub.2, and MgCl.sub.2. In a
specific embodiment, a formulation of the invention comprises
NaCl.
[0121] The formulations of the invention may further comprise a
surfactant. The term "surfactant" as used herein refers to organic
substances having amphipathic structures; namely, they are composed
of groups of opposing solubility tendencies, typically an
oil-soluble hydrocarbon chain and a water-soluble ionic group.
Surfactants can be classified, depending on the charge of the
surface-active moiety, into anionic, cationic, and nonionic
surfactants. Surfactants are often used as wetting, emulsifying,
solubilizing, and dispersing agents for various pharmaceutical
compositions and preparations of biological materials.
Pharmaceutically acceptable surfactants like polysorbates (e.g.
polysorbates 20 or 80); polyoxamers (e.g. poloxamer 188); Triton;
sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or
stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or
stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;
lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,
myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine
(e.g. lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUA.TM. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc), can optionally be added to the formulations
of the invention to reduce aggregation. Surfactants are
particularly useful if a pump or plastic container is used to
administer the formulation. The presence of a pharmaceutically
acceptable surfactant mitigates the propensity for the protein to
aggregate. In a specific embodiment, the formulations of the
invention comprise a polysorbate which is at a concentration
ranging from between about 0.001% to about 1%, or about 0.001% to
about 0.5%, or about 0.01% to about 0.2%. In other specific
embodiments, the formulations of the invention comprise a
polysorbate which is at a concentration of 0.01%, or 0.02%, or
0.03%, or 0.04%, or 0.05%, or 0.06%, or 0.07%, or 0.08%, or 0.09%,
or 0.1%, or 0.15%, or 0.2%. In another specific embodiment, the
polysorbate is polysorbate-80.
[0122] In one embodiment, a formulation of the invention comprises
a surfactant. In one embodiment, a formulation of the invention
comprises Polysorbate 20, Polysorbate 40, Polysorbate 60, or
Polysorbate 80. In a specific embodiment, a formulation of the
invention comprises Polysorbate 80.
[0123] In one embodiment, a formulation of the invention comprises
at least about 0.01%, at least about 0.02%, at least about 0.05%,
at least about 0.1%, at least about 0.2%, at least about 0.3%, at
least about 0.4%, or at least about 0.5% Polysorbate 80. In another
embodiment, a formulation of the invention comprises between about
0.01% and about 0.5%, between about 0.01% and about 0.3%, between
about 0.001% and about 0.2%, between about 0.02% and about 0.5%,
between about 0.02% and about 0.3%, between about 0.02% and about
0.2%, between about 0.05% and about 0.5%, between about 0.05% and
about 0.3%, between about 0.05% and about 0.2%, between about
0.075% and about 0.5%, between about 0.075% and about 0.3%, or
between about 0.075% and about 0.2% Polysorbate 80. In a further
embodiment, a formulation of the invention comprises about 0.01%,
about 0.02%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about
0.4%, or about 0.5% Polysorbate 80. In a specific embodiment, a
formulation of the invention comprises about 0.1% Polysorbate 80.
In a specific embodiment, a formulation of the invention comprises
about 0.01% Polysorbate 80. In a specific embodiment, a formulation
of the invention comprises about 0.05% Polysorbate 80. In a
specific embodiment, a formulation of the invention comprises about
0.1% Polysorbate 80. In a specific embodiment, a formulation of the
invention comprises about 0.02% Polysorbate 80.
[0124] In one embodiment, a formulation of the invention comprises
an amino acid. In one embodiment, a formulation of the invention
comprises an amino acid salt. In one embodiment, a formulation of
the invention comprises an amino acid selected from the group
consisting of lysine, arginine, and histidine. In one embodiment, a
formulation of the invention comprises an amino acid salt selected
from the group comprising lysine HCl and arginine HCl. In a
specific embodiment, a formulation of the invention comprises
lysine HCl.
[0125] In one embodiment, a formulation of the invention comprises
at least about 25 mM lysine HCl, at least about 50 mM lysine HCl,
at least about 100 mM lysine HCl, at least about 150 mM lysine HCl,
at least about 200 mM lysine HCl, at least about 250 mM lysine HCl,
at least about 300 mM lysine HCl, at least about 350 mM lysine HCl,
or at least about 400 mM lysine HCl. In another embodiment, a
formulation of the invention comprises between about 25 mM and
about 250 mM, between about 25 mM and about 300 mM, between about
25 mM and about 350 mM, between about 25 mM and about 400 mM,
between about 50 mM and about 250 mM, between about 50 mM and about
300 mM, between about 50 mM and about 350 mM, between about 50 mM
and about 400 mM, between about 100 mM and about 250 mM, between
about 100 mM and about 300 mM, between about 100 mM and about 400
mM, between about 150 mM and about 250 mM, between about 150 mM and
about 300 mM, or between about 150 mM and about 400 mM lysine HCl.
In a further embodiment, a formulation of the invention comprises
about 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM,
about 250 mM, about 300 mM, about 350 mM, or about 400 mM lysine
HCl. In a specific embodiment, a formulation of the invention
comprises about 25 mM lysine HCl. In a specific embodiment, a
formulation of the invention comprises about 50 mM lysine HCl. In a
specific embodiment, a formulation of the invention comprises about
75 mM lysine HCl. In a specific embodiment, a formulation of the
invention comprises about 100 mM lysine HCl. In a specific
embodiment, a formulation of the invention comprises about 200 mM
lysine HCl.
[0126] In one embodiment, a formulation of the invention comprises
trehalose and lysine HCl. In one embodiment, a formulation of the
invention comprises trehalose and lysine HCl at a molar ratio of
about 0.1, about 0.5, about 0.75, about 1, about 5, about 10, about
20, about 30, about 40, about 50, about 60, about 70, about 80,
about 90, about 100, about 200, or about 300, In one embodiment, a
formulation of the invention comprises trehalose and lysine HCl at
a molar ratio of about 1.5, about 1.7, about 1.8, about 1.9, about
2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about
2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about
3.2, about 3.3, about 3.4, about 3.5, or about 4. In a specific
embodiment, a formulation of the invention comprises trehalose and
lysine HCl at a molar ratio of about 2.1. In a specific embodiment,
a formulation of the invention comprises trehalose and lysine HCl
at a molar ratio of about 2.2. In a specific embodiment, a
formulation of the invention comprises trehalose and lysine HCl at
a molar ratio of about 2.4. In a specific embodiment, a formulation
of the invention comprises trehalose and lysine HCl at a molar
ratio of about 2.5. In a specific embodiment, a formulation of the
invention comprises trehalose and lysine HCl at a molar ratio of
about 2.6. In a specific embodiment, a formulation of the invention
comprises trehalose and lysine HCl at a molar ratio of about
2.7.
[0127] Optionally, the formulations of the invention may further
comprise other common excipients and/or additives including, but
not limited to, diluents, binders, stabilizers, lipophilic
solvents, preservatives, adjuvants, or the like. Pharmaceutically
acceptable excipients and/or additives may be used in the
formulations of the invention. Commonly used excipients/additives,
such as pharmaceutically acceptable chelators (for example, but not
limited to, EDTA, DTPA or EGTA) can optionally be added to the
formulations of the invention to reduce aggregation. These
additives are particularly useful if a pump or plastic container is
used to administer the formulation.
[0128] Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,
chlorocresol, benzyl alcohol, phenylmercuric nitrite,
phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride
(for example, but not limited to, hexahydrate), alkylparaben
(methyl, ethyl, propyl, butyl and the like), benzalkonium chloride,
benzethonium chloride, sodium dehydroacetate and thimerosal, or
mixtures thereof can optionally be added to the formulations of the
invention at any suitable concentration such as between about
0.001% to about 5%, or any range or value therein. The
concentration of preservative used in the formulations of the
invention is a concentration sufficient to yield an microbial
effect. Such concentrations are dependent on the preservative
selected and are readily determined by the skilled artisan.
[0129] Other contemplated excipients/additives, which may be
utilized in the formulations of the invention include, for example,
flavoring agents, antimicrobial agents, sweeteners, antioxidants,
antistatic agents, lipids such as phospholipids or fatty acids,
steroids such as cholesterol, protein excipients such as serum
albumin (human serum albumin (HSA), recombinant human albumin
(rHA)), gelatin, casein, salt-forming counterions such as sodium
and the like. These and additional known pharmaceutical excipients
and/or additives suitable for use in the formulations of the
invention are known in the art, e.g., as listed in "Remington: The
Science & Practice of Pharmacy", 21.sup.st ed., Lippincott
Williams & Wilkins, (2005), and in the "Physician's Desk
Reference", 60.sup.th ed., Medical Economics, Montvale, N.J.
(2005).
[0130] It will be understood by one skilled in the art that the
formulations of the invention may be isotonic with human blood,
that is the formulations of the invention have essentially the same
osmotic pressure as human blood. Such isotonic formulations will
generally have an osmotic pressure from about 250 mOSm to about 350
mOSm. Isotonicity can be measured by, for example, using a vapor
pressure or ice-freezing type osmometer. Tonicity of a formulation
is adjusted by the use of tonicity modifiers. "Tonicity modifiers"
are those pharmaceutically acceptable inert substances that can be
added to the formulation to provide an isotonity of the
formulation. Tonicity modifiers suitable for this invention
include, but are not limited to, saccharides, salts and amino
acids.
[0131] In one embodiment, a formulation of the invention comprises
citrate, lysine HCl, trehalose, and Polysorbate 80. In one
embodiment, a formulation of the invention comprises lysine HCl,
trehalose, and Polysorbate 80. In one embodiment, a formulation of
the invention comprises citrate, trehalose, and Polysorbate 80. In
one embodiment, a formulation of the invention comprises citrate,
lysine HCl, and Polysorbate 80. In one embodiment, a formulation of
the invention comprises citrate, lysine HCl, and trehalose. In one
embodiment, a formulation of the invention comprises citrate and
lysine HCl. In one embodiment, a formulation of the invention
comprises citrate and trehalose. In one embodiment, a formulation
of the invention comprises citrate and Polysorbate 80. In one
embodiment, a formulation of the invention comprises lysine HCl and
trehalose. In one embodiment, a formulation of the invention
comprises lysine HCl and Polysorbate 80. In one embodiment, a
formulation of the invention comprises trehalose, and Polysorbate
80. In one embodiment, a formulation of the invention comprises
lysine HCl.
[0132] In one embodiment, a formulation of the invention comprises
citrate, lysine HCl, trehalose and Polysorbate 80. In one
embodiment, a formulation of the invention comprises between about
5 mM and about 125 mM citrate, between about 25 mM and about 400 mM
lysine HCl, between about 3% and about 50% trehalose, and between
about 0.001% and about 1% Polysorbate 80, wherein said formulation
has a pH of between about 4.0 and about 8.0. In another embodiment,
a formulation of the invention comprises between about 10 mM and
about 50 mM citrate, between about 100 mM and about 300 mM lysine
HCl, between about 10% and about 20% trehalose, and between about
0.01% and about 0.2% Polysorbate 80, wherein said formulation has a
pH of between about 5.0 and about 8.0. In a further embodiment, a
formulation of the invention comprises about 25 mM citrate, about
200 mM lysine HCl, about 15% trehalose and about 0.1% Polysorbate
80, wherein said formulation has a pH of about 7.0.
[0133] In one embodiment, a formulation of the invention comprises
between about 10 micrograms/ml and about 100 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In another embodiment, a formulation of the
invention comprises between about 50 micrograms/ml and about 60
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises between about 110
micrograms/ml and about 210 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In another embodiment, a formulation of the invention
comprises between about 150 micrograms/ml and about 170
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In a further embodiment, a
formulation of the invention comprises about 55 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In another embodiment, a formulation of the
invention comprises about 160 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In another embodiment, a formulation of the invention
comprises about 150 micrograms/ml BiTE.RTM., about 30 mM citrate,
about 75 mM lysine HCl, about 6.5% trehalose and about 0.02%
Polysorbate 80 and has a pH of about 7.0. In another embodiment, a
formulation of the invention comprises about 150 micrograms/ml
BiTE.RTM., about 30 mM citrate, about 75 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80 and has a pH of about
7.0.
[0134] In one embodiment, a formulation of the invention comprises
between about 10 micrograms/ml and about 100 micrograms/ml MT103,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In another embodiment, a formulation of the invention
comprises between about 50 micrograms/ml and about 60 micrograms/ml
MT103, about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises between about 110 micrograms/ml and about 210
micrograms/ml MT103, about 25 mM citrate, about 200 mM lysine HCl,
about 15% trehalose and about 0.1% Polysorbate 80, wherein said
formulation has a pH of about 7.0. In another embodiment, a
formulation of the invention comprises between about 150
micrograms/ml and about 170 micrograms/ml MT103, about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In a further embodiment, a formulation of the invention
comprises about 55 micrograms/ml MT103, about 25 mM citrate, about
200 mM lysine HCl, about 15% trehalose and about 0.1% Polysorbate
80, wherein said formulation has a pH of about 7.0. In another
embodiment, a formulation of the invention comprises about 160
micrograms/ml MT103, about 25 mM citrate, about 200 mM lysine HCl,
about 15% trehalose and about 0.1% Polysorbate 80, wherein said
formulation has a pH of about 7.0. In another embodiment, a
formulation of the invention comprises about 150 micrograms/ml
MT103, about 30 mM citrate, about 75 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80 and has a pH of about 7.0.
In another embodiment, a formulation of the invention comprises
about 150 micrograms/ml MT103, about 30 mM citrate, about 75 mM
lysine HCl, about 6% trehalose and about 0.02% Polysorbate 80 and
has a pH of about 7.0.
[0135] In one embodiment, a formulation of the invention consists
of between about 10 micrograms/ml and about 100 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In another embodiment, a formulation of the
invention consists of between about 50 micrograms/ml and about 60
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention consists of between about 110
micrograms/ml and about 210 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In another embodiment, a formulation of the invention consists
of between about 150 micrograms/ml and about 170 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In a further embodiment, a formulation of
the invention consists of about 55 micrograms/ml BiTE.RTM., about
25 mM citrate, about 200 mM lysine HCl, about 15% trehalose and
about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In another embodiment, a formulation of the invention
consists of about 160 micrograms/ml BiTE.RTM., about 25 mM citrate,
about 200 mM lysine HCl, about 15% trehalose and about 0.1%
Polysorbate 80, wherein said formulation has a pH of about 7.0. In
another embodiment, a formulation of the invention consists of
about 150 micrograms/ml BiTE.RTM., about 30 mM citrate, about 75 mM
lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate 80 and
has a pH of about 7.0. In another embodiment, a formulation of the
invention consists of about 150 micrograms/ml BiTE.RTM., about 30
mM citrate, about 75 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80 and has a pH of about 7.0.
[0136] In one embodiment, a formulation of the invention consists
of between about 10 micrograms/ml and about 100 micrograms/ml
MT103, about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In another embodiment, a formulation of the
invention consists of between about 50 micrograms/ml and about 60
micrograms/ml MT103, about 25 mM citrate, about 200 mM lysine HCl,
about 15% trehalose and about 0.1% Polysorbate 80, wherein said
formulation has a pH of about 7.0. In one embodiment, a formulation
of the invention consists of between about 110 micrograms/ml and
about 210 micrograms/ml MT103, about 25 mM citrate, about 200 mM
lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In another
embodiment, a formulation of the invention consists of between
about 150 micrograms/ml and about 170 micrograms/ml MT103, about 25
mM citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In a further embodiment, a formulation of the invention
consists of about 55 micrograms/ml MT103, about 25 mM citrate,
about 200 mM lysine HCl, about 15% trehalose and about 0.1%
Polysorbate 80, wherein said formulation has a pH of about 7.0. In
another embodiment, a formulation of the invention consists of
about 160 micrograms/ml MT103, about 25 mM citrate, about 200 mM
lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In another
embodiment, a formulation of the invention consists of about 150
micrograms/ml MT103, about 30 mM citrate, about 75 mM lysine HCl,
about 6.5% trehalose and about 0.02% Polysorbate 80 and has a pH of
about 7.0. In another embodiment, a formulation of the invention
consists of about 150 micrograms/ml MT103, about 30 mM citrate,
about 75 mM lysine HCl, about 6% trehalose and about 0.02%
Polysorbate 80 and has a pH of about 7.0.
[0137] In one embodiment, a formulation of the invention comprises
at least about 10 micrograms/ml BiTE.RTM., about 25 mM citrate,
about 200 mM lysine HCl, about 15% trehalose and about 0.1%
Polysorbate 80, wherein said formulation has a pH of about 7.0. In
one embodiment, a formulation of the invention comprises at least
about 20 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises at least about 30
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 40
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 50
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 55
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 60
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 70
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 80
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 90
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 100
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 110
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 120
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 130
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 140
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 150
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 160
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 170
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 180
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 190
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 200
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0.
[0138] In one embodiment, a formulation of the invention comprises
about 10 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 20 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 30 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 40 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 50 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 55 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 60 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 70 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 80 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 90 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 100 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 110 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 120 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200
mM lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 130 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 140 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 150 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200
mM lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 160 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 170 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 180 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200
mM lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 190 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 200 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0.
[0139] In one embodiment, a formulation of the invention comprises
at least about 10 micrograms/ml BiTE.RTM., about 25 mM citrate,
about 200 mM lysine HCl, about 6.5% trehalose and about 0.02%
Polysorbate 80, wherein said formulation has a pH of about 7.0. In
one embodiment, a formulation of the invention comprises at least
about 20 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises at least about 30
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 40
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 50
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 55
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 60
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 70
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 80
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 90
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 100
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 110
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 120
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 130
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 140
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 150
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 160
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 170
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 180
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 190
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 200
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0.
[0140] In one embodiment, a formulation of the invention comprises
about 10 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 20 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 30 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 40 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 50 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 55 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 60 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 70 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 80 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 90 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 100 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 110 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 120 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200
mM lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 130 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 140 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 150 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200
mM lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 160 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 170 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 180 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200
mM lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 190 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 200 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0.
[0141] In one embodiment, a formulation of the invention comprises
at least about 10 micrograms/ml BiTE.RTM., about 25 mM citrate,
about 200 mM lysine HCl, about 6% trehalose and about 0.02%
Polysorbate 80, wherein said formulation has a pH of about 7.0. In
one embodiment, a formulation of the invention comprises at least
about 20 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 6% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises at least about 30
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 40
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 50
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 55
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 60
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 70
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 80
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 90
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 100
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 110
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 120
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 130
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 140
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 150
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 160
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 170
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 180
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 190
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
formulation of the invention comprises at least about 200
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0.
[0142] In one embodiment, a formulation of the invention comprises
about 10 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 6% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 20 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 30 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 40 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 6% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 50 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 55 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 60 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 6% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 70 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 80 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 90 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 6% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 100 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 110 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 120 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200
mM lysine HCl, about 6% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 130 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 140 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 150 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200
mM lysine HCl, about 6% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 160 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 170 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a formulation of the invention comprises
about 180 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200
mM lysine HCl, about 6% trehalose and about 0.02% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In one embodiment,
a formulation of the invention comprises about 190 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a formulation of the
invention comprises about 200 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0.
[0143] In one embodiment the formulations of the invention are
pyrogen-free formulations which are substantially free of
endotoxins and/or related pyrogenic substances. Endotoxins include
toxins that are confined inside a microorganism and are released
only when the microorganisms are broken down or die. Pyrogenic
substances also include fever-inducing, thermostable substances
(glycoproteins) from the outer membrane of bacteria and other
microorganisms. Both of these substances can cause fever,
hypotension and shock if administered to humans. Due to the
potential harmful effects, even low amounts of endotoxins must be
removed from intravenously administered pharmaceutical drug
solutions. The Food & Drug Administration ("FDA") has set an
upper limit of 5 endotoxin units (EU) per dose per kilogram body
weight in a single one hour period for intravenous drug
applications (The United States Pharmacopeial Convention,
Pharmacopeial Forum 26 (1):223 (2000)). When therapeutic proteins
are administered in amounts of several hundred or thousand
milligrams per kilogram body weight, as can be the case with
antibodies, even trace amounts of harmful and dangerous endotoxin
must be removed. In certain specific embodiments, the endotoxin and
pyrogen levels in the composition are less then 10 EU/mg, or less
then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less
then 0.01 EU/mg, or less then 0.001 EU/mg.
[0144] In one embodiment, the formulations of the invention are
sterile. The formulations of the invention may be sterilized by
various sterilization methods, including sterile filtration,
radiation, etc. In one embodiment, the antibody formulation is
filter-sterilized with a presterilized 0.22-micron filter. Sterile
compositions for injection can be formulated according to
conventional pharmaceutical practice as described in "Remington:
The Science & Practice of Pharmacy", 21.sup.st ed., Lippincott
Williams & Wilkins, (2005). Formulations comprising antibodies,
such as those disclosed herein, ordinarily will be stored in
lyophilized form or in solution. It is contemplated that sterile
compositions comprising antibodies are placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having an adapter that allows retrieval of the
formulation, such as a stopper pierceable by a hypodermic injection
needle.
5.1.1. Stability of Liquid Formulations
[0145] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules that is susceptible to aggregation,
fragmentation and/or loss due to absorption to contact
surfaces.
[0146] In one embodiment, a formulation of the invention stabilizes
BiTE.RTM. molecules. In one embodiment, a formulation of the
invention prevents aggregation of BiTE.RTM. molecules. In another
embodiment, a formulation of the invention prevents fragmentation
of BiTE.RTM. molecules. In a further embodiment, a formulation of
the invention prevents loss of BiTE.RTM. molecules due to
absorption to contact surfaces.
[0147] In one embodiment, a formulation of the invention stabilizes
MT103. In one embodiment, a formulation of the invention prevents
aggregation of MT103. In another embodiment, a formulation of the
invention prevents fragmentation of MT103. In a further embodiment,
a formulation of the invention prevents loss of MT103 due to
absorption to contact surfaces.
5.1.1.1. Stability Over Time
[0148] In one embodiment, a formulation of the invention is stable
upon storage at about 40.degree. C. for at least about 1 day, at
least about 2 days, at least about 3 days, at least about 4 days,
at least about 5 days, at least about 6 days, at least about 7
days, at least about 8 days, at least about 9 days, at least about
10 days, at least about 15 days, at least about 20 days, at least
about 25 days, or at least about 30 days. In one embodiment, a
formulation of the invention is stable upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, or at least about 4 weeks. In one embodiment,
a formulation of the invention is stable upon storage at about
40.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, or at least about 6 months.
[0149] In one embodiment, a formulation of the invention is stable
upon storage at about 4.degree. C. for at least about 1 day, at
least about 2 days, at least about 3 days, at least about 4 days,
at least about 5 days, at least about 6 days, at least about 7
days, at least about 8 days, at least about 9 days, at least about
10 days. In one embodiment, a formulation of the invention is
stable upon storage at about 4.degree. C. for at least about 1
week, at least about 2 weeks, at least about 3 weeks, or at least
about 4 weeks. In one embodiment, a formulation of the invention is
stable upon storage at about 5.degree. C. for at least about 1
month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, at least about 6 months,
at least about 7 months, at least about 8 months, at least about 9
months, at least about 10 months, at least about 11 months, or at
least about 12 months. In one embodiment, a formulation of the
invention is stable upon storage at about 5.degree. C. for at least
about 1 year, at least about 2 years, at least about 3 years, at
least about 4 years, at least about 5 years, at least about 6
years, at least about 7 years, at least about 8 years, at least
about 9 years, at least about 10 years, at least about 11 years, or
at least about 12 years.
[0150] In one embodiment, a formulation of the invention is stable
upon storage at about 40.degree. C. for about 1 day, about 2 days,
about 3 days, about 4 days, about 5 days, about 6 days, about 7
days, about 8 days, about 9 days, about 10 days, about 15 days,
about 20 days, about 25 days, or about 30 days. In one embodiment,
a formulation of the invention is stable upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, or
about 4 weeks. In one embodiment, a formulation of the invention is
stable upon storage at about 40.degree. C. for about 1 month, about
2 months, about 3 months, about 4 months, about 5 months, or about
6 months.
[0151] In one embodiment, a formulation of the invention is stable
upon storage at about 4.degree. C. for about 1 day, about 2 days,
about 3 days, about 4 days, about 5 days, about 6 days, about 7
days, about 8 days, about 9 days, about 10 days. In one embodiment,
a formulation of the invention is stable upon storage at about
4.degree. C. for about 1 week, about 2 weeks, about 3 weeks, or
about 4 weeks. In one embodiment, a formulation of the invention is
stable upon storage at about 5.degree. C. for about 1 month, about
2 months, about 3 months, about 4 months, about 5 months, about 6
months, about 7 months, about 8 months, about 9 months, about 10
months, about 11 months, or about 12 months. In one embodiment, a
formulation of the invention is stable upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, about 5 years, about 6 years, about 7 years, about 8
years, about 9 years, about 10 years, about 11 years, or about 12
years.
5.1.1.2. Binding Activity
[0152] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains at least 90% of
binding ability to its target polypeptides compared to a reference
antibody representing the antibody prior to storage at about
40.degree. C. for at least about 1 day, at least about 2 days, at
least about 3 days, at least about 4 days, at least about 5 days,
at least about 6 days, or at least about 7 days. In one embodiment,
a formulation of the invention comprises BiTE.RTM. molecules,
wherein said antibody retains at least 90% of binding ability to
its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, or at least
about 6 weeks.
[0153] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains at least 90% of
binding ability to its target polypeptides compared to a reference
antibody representing the antibody prior to storage at about
5.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, at least about 6 weeks, at least about 7 weeks, at least
about 8 weeks, at least about 9 weeks, at least about 10 weeks, at
least about 11 weeks, or at least about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein said antibody retains at least 90% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for at least about 1 month, at least about 2 months, at least about
3 months, at least about 4 months, at least about 5 months, at
least about 6 months, at least about 7 months, at least about 8
months, at least about 9 months, at least about 10 months, at least
about 11 months, or at least about 12 months.
[0154] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains 90% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for about 1 day, about 2 days, about 3 days, about 4 days, about 5
days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
said antibody retains 90% of binding ability to its target
polypeptides compared to a reference antibody representing the
antibody prior to storage at about 40.degree. C. for about 1 week,
about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or
about 6 weeks.
[0155] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains 90% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein said antibody retains 90% of binding ability to
its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for about 1 month, about 2 months, about 3 months, about 4 months,
about 5 months, about 6 months, about 7 months, about 8 months,
about 9 months, about 10 months, about 11 months, or about 12
months.
[0156] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains at least 90% of binding
ability to CD19 and CD3 antigens compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for at least about 1 day, at least about 2 days, at least about 3
days, at least about 4 days, at least about 5 days, at least about
6 days, or at least about 7 days. In one embodiment, a formulation
of the invention comprises MT103, wherein said antibody retains at
least 90% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to storage at
about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks.
[0157] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains at least 90% of binding
ability to CD19 and CD3 antigens compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises MT103, wherein said antibody retains at
least 90% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to storage at
about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months.
[0158] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains 90% of binding ability to CD19
and CD3 antigens compared to a reference antibody representing the
antibody prior to storage at about 40.degree. C. for about 1 day,
about 2 days, about 3 days, about 4 days, about 5 days, about 6
days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein said antibody retains 90% of
binding ability to CD19 and CD3 antigens compared to a reference
antibody representing the antibody prior to storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0159] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains 90% of binding ability to CD19
and CD3 antigens compared to a reference antibody representing the
antibody prior to storage at about 5.degree. C. for about 1 week,
about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6
weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks,
about 11 weeks, or about 12 weeks. In one embodiment, a formulation
of the invention comprises MT103, wherein said antibody retains 90%
of binding ability to CD19 and CD3 antigens compared to a reference
antibody representing the antibody prior to storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months.
[0160] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains at least 95% of
binding ability to its target polypeptides compared to a reference
antibody representing the antibody prior to storage at about
40.degree. C. for at least about 1 day, at least about 2 days, at
least about 3 days, at least about 4 days, at least about 5 days,
at least about 6 days, or at least about 7 days. In one embodiment,
a formulation of the invention comprises BiTE.RTM. molecules,
wherein said antibody retains at least 95% of binding ability to
its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, or at least
about 6 weeks.
[0161] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains at least 95% of
binding ability to its target polypeptides compared to a reference
antibody representing the antibody prior to storage at about
5.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, at least about 6 weeks, at least about 7 weeks, at least
about 8 weeks, at least about 9 weeks, at least about 10 weeks, at
least about 11 weeks, or at least about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein said antibody retains at least 95% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for at least about 1 month, at least about 2 months, at least about
3 months, at least about 4 months, at least about 5 months, at
least about 6 months, at least about 7 months, at least about 8
months, at least about 9 months, at least about 10 months, at least
about 11 months, or at least about 12 months.
[0162] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains 95% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for about 1 day, about 2 days, about 3 days, about 4 days, about 5
days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
said antibody retains 95% of binding ability to its target
polypeptides compared to a reference antibody representing the
antibody prior to storage at about 40.degree. C. for about 1 week,
about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or
about 6 weeks.
[0163] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains 95% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein said antibody retains 95% of binding ability to
its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for about 1 month, about 2 months, about 3 months, about 4 months,
about 5 months, about 6 months, about 7 months, about 8 months,
about 9 months, about 10 months, about 11 months, or about 12
months.
[0164] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains at least 95% of binding
ability to CD19 and CD3 antigens compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for at least about 1 day, at least about 2 days, at least about 3
days, at least about 4 days, at least about 5 days, at least about
6 days, or at least about 7 days. In one embodiment, a formulation
of the invention comprises MT103, wherein said antibody retains at
least 95% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to storage at
about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks.
[0165] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains at least 95% of binding
ability to CD19 and CD3 antigens compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises MT103, wherein said antibody retains at
least 95% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to storage at
about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months.
[0166] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains 95% of binding ability to CD19
and CD3 antigens compared to a reference antibody representing the
antibody prior to storage at about 40.degree. C. for about 1 day,
about 2 days, about 3 days, about 4 days, about 5 days, about 6
days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein said antibody retains 95% of
binding ability to CD19 and CD3 antigens compared to a reference
antibody representing the antibody prior to storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0167] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains 95% of binding ability to CD19
and CD3 antigens compared to a reference antibody representing the
antibody prior to storage at about 5.degree. C. for about 1 week,
about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6
weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks,
about 11 weeks, or about 12 weeks. In one embodiment, a formulation
of the invention comprises MT103, wherein said antibody retains 95%
of binding ability to CD19 and CD3 antigens compared to a reference
antibody representing the antibody prior to storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months.
[0168] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains at least 99% of
binding ability to its target polypeptides compared to a reference
antibody representing the antibody prior to storage at about
40.degree. C. for at least about 1 day, at least about 2 days, at
least about 3 days, at least about 4 days, at least about 5 days,
at least about 6 days, or at least about 7 days. In one embodiment,
a formulation of the invention comprises BiTE.RTM. molecules,
wherein said antibody retains at least 99% of binding ability to
its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, or at least
about 6 weeks.
[0169] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains at least 99% of
binding ability to its target polypeptides compared to a reference
antibody representing the antibody prior to storage at about
5.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, at least about 6 weeks, at least about 7 weeks, at least
about 8 weeks, at least about 9 weeks, at least about 10 weeks, at
least about 11 weeks, or at least about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein said antibody retains at least 99% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for at least about 1 month, at least about 2 months, at least about
3 months, at least about 4 months, at least about 5 months, at
least about 6 months, at least about 7 months, at least about 8
months, at least about 9 months, at least about 10 months, at least
about 11 months, or at least about 12 months.
[0170] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains 99% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for about 1 day, about 2 days, about 3 days, about 4 days, about 5
days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
said antibody retains 99% of binding ability to its target
polypeptides compared to a reference antibody representing the
antibody prior to storage at about 40.degree. C. for about 1 week,
about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or
about 6 weeks.
[0171] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains 99% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein said antibody retains 99% of binding ability to
its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for about 1 month, about 2 months, about 3 months, about 4 months,
about 5 months, about 6 months, about 7 months, about 8 months,
about 9 months, about 10 months, about 11 months, or about 12
months.
[0172] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains at least 99% of binding
ability to CD19 and CD3 antigens compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for at least about 1 day, at least about 2 days, at least about 3
days, at least about 4 days, at least about 5 days, at least about
6 days, or at least about 7 days. In one embodiment, a formulation
of the invention comprises MT103, wherein said antibody retains at
least 99% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to storage at
about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks.
[0173] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains at least 99% of binding
ability to CD19 and CD3 antigens compared to a reference antibody
representing the antibody prior to storage at about 5.degree. C.
for at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises MT103, wherein said antibody retains at
least 99% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to storage at
about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months.
[0174] In one embodiment, a formulation of the invention comprises
MT103, wherein said antibody retains 99% of binding ability to CD19
and CD3 antigens compared to a reference antibody representing the
antibody prior to storage at about 40.degree. C. for about 1 day,
about 2 days, about 3 days, about 4 days, about 5 days, about 6
days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein said antibody retains 99% of
binding ability to CD19 and CD3 antigens compared to a reference
antibody representing the antibody prior to storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
5.1.1.3. Aggregate Formation
[0175] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 1% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 40.degree.
C. for at least about 1 day, at least about 2 days, at least about
3 days, at least about 4 days, at least about 5 days, at least
about 6 days, or at least about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 1% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for at least about 1
week, at least about 2 weeks, at least about 3 weeks, at least
about 4 weeks, at least about 5 weeks, or at least about 6
weeks.
[0176] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 1% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 5.degree. C.
for at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
1% of said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months.
[0177] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 1% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 40.degree.
C. for about 1 day, about 2 days, about 3 days, about 4 days, about
5 days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 1% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6
weeks.
[0178] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 1% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 5.degree. C.
for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 1% of said antibody forms an aggregate
as determined by HPSEC upon storage at about 5.degree. C. for about
1 month, about 2 months, about 3 months, about 4 months, about 5
months, about 6 months, about 7 months, about 8 months, about 9
months, about 10 months, about 11 months, or about 12 months.
[0179] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 1% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
or at least about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, or at least about 6 weeks.
[0180] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 1% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 5.degree. C. for at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, or at least about 12 months.
[0181] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 1% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0182] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 1% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 5.degree. C. for about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 1%
of said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for about 1 month, about 2 months,
about 3 months, about 4 months, about 5 months, about 6 months,
about 7 months, about 8 months, about 9 months, about 10 months,
about 11 months, or about 12 months.
[0183] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 2% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 40.degree.
C. for at least about 1 day, at least about 2 days, at least about
3 days, at least about 4 days, at least about 5 days, at least
about 6 days, or at least about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 2% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for at least about 1
week, at least about 2 weeks, at least about 3 weeks, at least
about 4 weeks, at least about 5 weeks, or at least about 6
weeks.
[0184] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 2% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 5.degree. C.
for at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
2% of said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months.
[0185] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 2% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 40.degree.
C. for about 1 day, about 2 days, about 3 days, about 4 days, about
5 days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 2% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6
weeks.
[0186] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 2% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 5.degree. C.
for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 2% of said antibody forms an aggregate
as determined by HPSEC upon storage at about 5.degree. C. for about
1 month, about 2 months, about 3 months, about 4 months, about 5
months, about 6 months, about 7 months, about 8 months, about 9
months, about 10 months, about 11 months, or about 12 months.
[0187] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 2% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
or at least about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, or at least about 6 weeks.
[0188] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 2% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 5.degree. C. for at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, or at least about 12 months.
[0189] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 2% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0190] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 2% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 5.degree. C. for about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 2%
of said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for about 1 month, about 2 months,
about 3 months, about 4 months, about 5 months, about 6 months,
about 7 months, about 8 months, about 9 months, about 10 months,
about 11 months, or about 12 months.
[0191] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 3% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 40.degree.
C. for at least about 1 day, at least about 2 days, at least about
3 days, at least about 4 days, at least about 5 days, at least
about 6 days, or at least about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 3% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for at least about 1
week, at least about 2 weeks, at least about 3 weeks, at least
about 4 weeks, at least about 5 weeks, or at least about 6
weeks.
[0192] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 3% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 5.degree. C.
for at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
3% of said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months.
[0193] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 3% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 40.degree.
C. for about 1 day, about 2 days, about 3 days, about 4 days, about
5 days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 3% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6
weeks.
[0194] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 3% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 5.degree. C.
for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 3% of said antibody forms an aggregate
as determined by HPSEC upon storage at about 5.degree. C. for about
1 month, about 2 months, about 3 months, about 4 months, about 5
months, about 6 months, about 7 months, about 8 months, about 9
months, about 10 months, about 11 months, or about 12 months.
[0195] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 3% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
or at least about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, or at least about 6 weeks.
[0196] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 3% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 5.degree. C. for at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, or at least about 12 months.
[0197] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 3% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0198] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 3% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 5.degree. C. for about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 3%
of said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for about 1 month, about 2 months,
about 3 months, about 4 months, about 5 months, about 6 months,
about 7 months, about 8 months, about 9 months, about 10 months,
about 11 months, or about 12 months.
[0199] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 4% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 40.degree.
C. for at least about 1 day, at least about 2 days, at least about
3 days, at least about 4 days, at least about 5 days, at least
about 6 days, or at least about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 4% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for at least about 1
week, at least about 2 weeks, at least about 3 weeks, at least
about 4 weeks, at least about 5 weeks, or at least about 6
weeks.
[0200] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 4% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 5.degree. C.
for at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
4% of said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months.
[0201] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 4% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 40.degree.
C. for about 1 day, about 2 days, about 3 days, about 4 days, about
5 days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 4% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6
weeks.
[0202] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 4% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 5.degree. C.
for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules BiTE.RTM. molecule, wherein less than 4% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months.
[0203] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 4% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
or at least about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, or at least about 6 weeks.
[0204] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 4% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 5.degree. C. for at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, or at least about 12 months.
[0205] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 4% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0206] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 4% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 5.degree. C. for about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 4%
of said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for about 1 month, about 2 months,
about 3 months, about 4 months, about 5 months, about 6 months,
about 7 months, about 8 months, about 9 months, about 10 months,
about 11 months, or about 12 months.
[0207] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules BiTE.RTM. molecule, wherein less than 5% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 day, at least
about 2 days, at least about 3 days, at least about 4 days, at
least about 5 days, at least about 6 days, or at least about 7
days. In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules BiTE.RTM. molecule, wherein less than 5% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks.
[0208] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules BiTE.RTM. molecule, wherein less than 5% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, at least about 6 weeks, at least about 7
weeks, at least about 8 weeks, at least about 9 weeks, at least
about 10 weeks, at least about 11 weeks, or at least about 12
weeks. In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules BiTE.RTM. molecule, wherein less than 5% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months.
[0209] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 40.degree.
C. for about 1 day, about 2 days, about 3 days, about 4 days, about
5 days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 5% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6
weeks.
[0210] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 5.degree. C.
for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 5% of said antibody forms an aggregate
as determined by HPSEC upon storage at about 5.degree. C. for about
1 month, about 2 months, about 3 months, about 4 months, about 5
months, about 6 months, about 7 months, about 8 months, about 9
months, about 10 months, about 11 months, or about 12 months.
[0211] In one embodiment, a formulation of the invention comprises
MT103MT103, wherein less than 5% of said antibody forms an
aggregate as determined by HPSEC upon storage at about 40.degree.
C. for at least about 1 day, at least about 2 days, at least about
3 days, at least about 4 days, at least about 5 days, at least
about 6 days, or at least about 7 days. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 5%
of said antibody forms an aggregate as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks.
[0212] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 5.degree. C. for at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, or at least about 12 months.
[0213] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0214] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody forms an aggregate as
determined by HPSEC upon storage at about 5.degree. C. for about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 5%
of said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for about 1 month, about 2 months,
about 3 months, about 4 months, about 5 months, about 6 months,
about 7 months, about 8 months, about 9 months, about 10 months,
about 11 months, or about 12 months.
5.1.1.4. Dimer formation
[0215] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 1% of said antibody forms a
dimer as determined by HPSEC upon storage at about 40.degree. C.
for at least about 1 day, at least about 2 days, at least about 3
days, at least about 4 days, at least about 5 days, at least about
6 days, or at least about 7 days. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
1% of said antibody forms a dimer as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks.
[0216] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 1% of said antibody forms a
dimer as determined by HPSEC upon storage at about 5.degree. C. for
at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
1% of said antibody forms a dimer as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months.
[0217] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 1% of said antibody forms a
dimer as determined by HPSEC upon storage at about 40.degree. C.
for about 1 day, about 2 days, about 3 days, about 4 days, about 5
days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 1% of said antibody forms a dimer as determined by HPSEC
upon storage at about 40.degree. C. for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6
weeks.
[0218] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 1% of said antibody forms a
dimer as determined by HPSEC upon storage at about 5.degree. C. for
about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5
weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks,
about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 1% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for about 1
month, about 2 months, about 3 months, about 4 months, about 5
months, about 6 months, about 7 months, about 8 months, about 9
months, about 10 months, about 11 months, or about 12 months.
[0219] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 1% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
or at least about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, or at least about 6 weeks.
[0220] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 1% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, or at least about 12 months.
[0221] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 1% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0222] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 1% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 1%
of said antibody forms a dimer as determined by HPSEC upon storage
at about 5.degree. C. for about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 7
months, about 8 months, about 9 months, about 10 months, about 11
months, or about 12 months.
[0223] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 2% of said antibody forms a
dimer as determined by HPSEC upon storage at about 40.degree. C.
for at least about 1 day, at least about 2 days, at least about 3
days, at least about 4 days, at least about 5 days, at least about
6 days, or at least about 7 days. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
2% of said antibody forms a dimer as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks.
[0224] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 2% of said antibody forms a
dimer as determined by HPSEC upon storage at about 5.degree. C. for
at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
2% of said antibody forms a dimer as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months.
[0225] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 2% of said antibody forms a
dimer as determined by HPSEC upon storage at about 40.degree. C.
for about 1 day, about 2 days, about 3 days, about 4 days, about 5
days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 2% of said antibody forms a dimer as determined by HPSEC
upon storage at about 40.degree. C. for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6
weeks.
[0226] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 2% of said antibody forms a
dimer as determined by HPSEC upon storage at about 5.degree. C. for
about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5
weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks,
about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 2% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for about 1
month, about 2 months, about 3 months, about 4 months, about 5
months, about 6 months, about 7 months, about 8 months, about 9
months, about 10 months, about 11 months, or about 12 months.
[0227] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 2% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
or at least about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, or at least about 6 weeks.
[0228] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 2% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, or at least about 12 months.
[0229] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 2% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0230] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 2% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecule MT103,
wherein less than 2% of said antibody forms a dimer as determined
by HPSEC upon storage at about 5.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months,
about 6 months, about 7 months, about 8 months, about 9 months,
about 10 months, about 11 months, or about 12 months.
[0231] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 3% of said antibody forms a
dimer as determined by HPSEC upon storage at about 40.degree. C.
for at least about 1 day, at least about 2 days, at least about 3
days, at least about 4 days, at least about 5 days, at least about
6 days, or at least about 7 days. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
3% of said antibody forms a dimer as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks.
[0232] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 3% of said antibody forms a
dimer as determined by HPSEC upon storage at about 5.degree. C. for
at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
3% of said antibody forms a dimer as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months.
[0233] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 3% of said antibody forms a
dimer as determined by HPSEC upon storage at about 40.degree. C.
for about 1 day, about 2 days, about 3 days, about 4 days, about 5
days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 3% of said antibody forms a dimer as determined by HPSEC
upon storage at about 40.degree. C. for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6
weeks.
[0234] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 3% of said antibody forms a
dimer as determined by HPSEC upon storage at about 5.degree. C. for
about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5
weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks,
about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 3% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for about 1
month, about 2 months, about 3 months, about 4 months, about 5
months, about 6 months, about 7 months, about 8 months, about 9
months, about 10 months, about 11 months, or about 12 months.
[0235] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 3% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
or at least about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, or at least about 6 weeks.
[0236] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 3% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, or at least about 12 months.
[0237] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 3% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0238] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 3% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 3%
of said antibody forms a dimer as determined by HPSEC upon storage
at about 5.degree. C. for about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 7
months, about 8 months, about 9 months, about 10 months, about 11
months, or about 12 months.
[0239] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 4% of said antibody forms a
dimer as determined by HPSEC upon storage at about 40.degree. C.
for at least about 1 day, at least about 2 days, at least about 3
days, at least about 4 days, at least about 5 days, at least about
6 days, or at least about 7 days. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
4% of said antibody forms a dimer as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks.
[0240] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 4% of said antibody forms a
dimer as determined by HPSEC upon storage at about 5.degree. C. for
at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
4% of said antibody forms a dimer as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months.
[0241] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 4% of said antibody forms a
dimer as determined by HPSEC upon storage at about 40.degree. C.
for about 1 day, about 2 days, about 3 days, about 4 days, about 5
days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 4% of said antibody forms a dimer as determined by HPSEC
upon storage at about 40.degree. C. for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6
weeks.
[0242] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 4% of said antibody forms a
dimer as determined by HPSEC upon storage at about 5.degree. C. for
about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5
weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks,
about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 4% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for about 1
month, about 2 months, about 3 months, about 4 months, about 5
months, about 6 months, about 7 months, about 8 months, about 9
months, about 10 months, about 11 months, or about 12 months.
[0243] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 4% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
or at least about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, or at least about 6 weeks.
[0244] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 4% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, or at least about 12 months.
[0245] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 4% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0246] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 4% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 4%
of said antibody forms a dimer as determined by HPSEC upon storage
at about 5.degree. C. for about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 7
months, about 8 months, about 9 months, about 10 months, about 11
months, or about 12 months.
[0247] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody forms a
dimer as determined by HPSEC upon storage at about 40.degree. C.
for at least about 1 day, at least about 2 days, at least about 3
days, at least about 4 days, at least about 5 days, at least about
6 days, or at least about 7 days. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
5% of said antibody forms a dimer as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks.
[0248] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody forms a
dimer as determined by HPSEC upon storage at about 5.degree. C. for
at least about 1 week, at least about 2 weeks, at least about 3
weeks, at least about 4 weeks, at least about 5 weeks, at least
about 6 weeks, at least about 7 weeks, at least about 8 weeks, at
least about 9 weeks, at least about 10 weeks, at least about 11
weeks, or at least about 12 weeks. In one embodiment, a formulation
of the invention comprises BiTE.RTM. molecules, wherein less than
5% of said antibody forms a dimer as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months.
[0249] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody forms a
dimer as determined by HPSEC upon storage at about 40.degree. C.
for about 1 day, about 2 days, about 3 days, about 4 days, about 5
days, about 6 days, or about 7 days. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 5% of said antibody forms a dimer as determined by HPSEC
upon storage at about 40.degree. C. for about 1 week, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6
weeks.
[0250] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody forms a
dimer as determined by HPSEC upon storage at about 5.degree. C. for
about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5
weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks,
about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 5% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for about 1
month, about 2 months, about 3 months, about 4 months, about 5
months, about 6 months, about 7 months, about 8 months, about 9
months, about 10 months, about 11 months, or about 12 months.
[0251] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
or at least about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, or at least about 6 weeks.
[0252] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for at least
about 1 week, at least about 2 weeks, at least about 3 weeks, at
least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 month, at least about 2 months,
at least about 3 months, at least about 4 months, at least about 5
months, at least about 6 months, at least about 7 months, at least
about 8 months, at least about 9 months, at least about 10 months,
at least about 11 months, or at least about 12 months.
[0253] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, or about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks.
[0254] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody forms a dimer as
determined by HPSEC upon storage at about 5.degree. C. for about 1
week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 5%
of said antibody forms a dimer as determined by HPSEC upon storage
at about 5.degree. C. for about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 7
months, about 8 months, about 9 months, about 10 months, about 11
months, or about 12 months.
5.1.1.5. Protein Recovery Following Shaking or Freeze/Thaw
Cycles
[0255] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 1% of said antibody forms an
aggregate as determined by HPSEC upon shaking a vial filled to half
of its volume with a sample of said formulation for 4 hours at a
speed of 400 shakes per minute. In one embodiment, a formulation of
the invention comprises BiTE.RTM. molecules, wherein less than 1%
of said antibody forms an aggregate as determined by HPSEC upon
subjecting the formulation to three freeze/thaw cycles.
[0256] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 1% of said antibody forms an aggregate as
determined by HPSEC upon shaking a vial filled to half of its
volume with a sample of said formulation for 4 hours at a speed of
400 shakes per minute. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms an aggregate as determined by HPSEC upon subjecting the
formulation to three freeze/thaw cycles.
[0257] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 2% of said antibody forms an
aggregate as determined by HPSEC upon shaking a vial filled to half
of its volume with a sample of said formulation for 4 hours at a
speed of 400 shakes per minute. In one embodiment, a formulation of
the invention comprises BiTE.RTM. molecules, wherein less than 2%
of said antibody forms an aggregate as determined by HPSEC upon
subjecting the formulation to three freeze/thaw cycles.
[0258] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 2% of said antibody forms an aggregate as
determined by HPSEC upon shaking a vial filled to half of its
volume with a sample of said formulation for 4 hours at a speed of
400 shakes per minute. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms an aggregate as determined by HPSEC upon subjecting the
formulation to three freeze/thaw cycles.
[0259] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 3% of said antibody forms an
aggregate as determined by HPSEC upon shaking a vial filled to half
of its volume with a sample of said formulation for 4 hours at a
speed of 400 shakes per minute. In one embodiment, a formulation of
the invention comprises BiTE.RTM. molecules, wherein less than 3%
of said antibody forms an aggregate as determined by HPSEC upon
subjecting the formulation to three freeze/thaw cycles.
[0260] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 3% of said antibody forms an aggregate as
determined by HPSEC upon shaking a vial filled to half of its
volume with a sample of said formulation for 4 hours at a speed of
400 shakes per minute. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms an aggregate as determined by HPSEC upon subjecting the
formulation to three freeze/thaw cycles.
[0261] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 4% of said antibody forms an
aggregate as determined by HPSEC upon shaking a vial filled to half
of its volume with a sample of said formulation for 4 hours at a
speed of 400 shakes per minute. In one embodiment, a formulation of
the invention comprises BiTE.RTM. molecules, wherein less than 4%
of said antibody forms an aggregate as determined by HPSEC upon
subjecting the formulation to three freeze/thaw cycles.
[0262] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 4% of said antibody forms an aggregate as
determined by HPSEC upon shaking a vial filled to half of its
volume with a sample of said formulation for 4 hours at a speed of
400 shakes per minute. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms an aggregate as determined by HPSEC upon subjecting the
formulation to three freeze/thaw cycles.
[0263] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody forms an
aggregate as determined by HPSEC upon shaking a vial filled to half
of its volume with a sample of said formulation for 4 hours at a
speed of 400 shakes per minute. In one embodiment, a formulation of
the invention comprises BiTE.RTM. molecules, wherein less than 5%
of said antibody forms an aggregate as determined by HPSEC upon
subjecting the formulation to three freeze/thaw cycles.
[0264] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody forms an aggregate as
determined by HPSEC upon shaking a vial filled to half of its
volume with a sample of said formulation for 4 hours at a speed of
400 shakes per minute. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms an aggregate as determined by HPSEC upon subjecting the
formulation to three freeze/thaw cycles.
5.1.1.6. Dimer Formation Upon Shaking or Freeze/Thaw Cycles
[0265] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 1% of said antibody forms a
dimer as determined by HPSEC upon shaking a vial filled to half of
its volume with a sample of said formulation for 4 hours at a speed
of 400 shakes per minute. In one embodiment, a formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody forms a dimer as determined by HPSEC upon subjecting
the formulation to three freeze/thaw cycles.
[0266] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 1% of said antibody forms a dimer as
determined by HPSEC upon shaking a vial filled to half of its
volume with a sample of said formulation for 4 hours at a speed of
400 shakes per minute. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms a dimer as determined by HPSEC upon subjecting the
formulation to three freeze/thaw cycles.
[0267] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 2% of said antibody forms a
dimer as determined by HPSEC upon shaking a vial filled to half of
its volume with a sample of said formulation for 4 hours at a speed
of 400 shakes per minute. In one embodiment, a formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody forms a dimer as determined by HPSEC upon subjecting
the formulation to three freeze/thaw cycles.
[0268] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 2% of said antibody forms a dimer as
determined by HPSEC upon shaking a vial filled to half of its
volume with a sample of said formulation for 4 hours at a speed of
400 shakes per minute. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms a dimer as determined by HPSEC upon subjecting the
formulation to three freeze/thaw cycles.
[0269] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 3% of said antibody forms a
dimer as determined by HPSEC upon shaking a vial filled to half of
its volume with a sample of said formulation for 4 hours at a speed
of 400 shakes per minute. In one embodiment, a formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody forms a dimer as determined by HPSEC upon subjecting
the formulation to three freeze/thaw cycles.
[0270] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 3% of said antibody forms a dimer as
determined by HPSEC upon shaking a vial filled to half of its
volume with a sample of said formulation for 4 hours at a speed of
400 shakes per minute. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms a dimer as determined by HPSEC upon subjecting the
formulation to three freeze/thaw cycles.
[0271] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 4% of said antibody forms a
dimer as determined by HPSEC upon shaking a vial filled to half of
its volume with a sample of said formulation for 4 hours at a speed
of 400 shakes per minute. In one embodiment, a formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody forms a dimer as determined by HPSEC upon subjecting
the formulation to three freeze/thaw cycles.
[0272] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 4% of said antibody forms a dimer as
determined by HPSEC upon shaking a vial filled to half of its
volume with a sample of said formulation for 4 hours at a speed of
400 shakes per minute. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms a dimer as determined by HPSEC upon subjecting the
formulation to three freeze/thaw cycles.
[0273] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody forms a
dimer as determined by HPSEC upon shaking a vial filled to half of
its volume with a sample of said formulation for 4 hours at a speed
of 400 shakes per minute. In one embodiment, a formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody forms a dimer as determined by HPSEC upon subjecting
the formulation to three freeze/thaw cycles.
[0274] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody forms a dimer as
determined by HPSEC upon shaking a vial filled to half of its
volume with a sample of said formulation for 4 hours at a speed of
400 shakes per minute. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms a dimer as determined by HPSEC upon subjecting the
formulation to three freeze/thaw cycles.
5.1.1.7. Recovery Following Lyophilization
[0275] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 90%, at least about
95%, at least about 97%, at least about 98%, or at least about 99%
of said antibody may be recovered from a vial upon shaking said
vial for 4 hours at a speed of 400 shakes per minute wherein said
vial is filled to half of its volume with said formulation. In
another embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 90%, at least about
95%, at least about 97%, at least about 98%, or at least about 99%
of said antibody may be recovered from a vial upon subjecting the
formulation to three freeze/thaw cycles wherein said vial is filled
to half of its volume with said formulation. In a further
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein at least about 90%, at least about 95%, at least
about 97%, at least about 98%, or at least about 99% of said
antibody may be recovered by reconstituting a lyophilized cake
generated from said formulation.
[0276] In one embodiment, a formulation of the invention comprises
MT103, wherein at least about 90%, at least about 95%, at least
about 97%, at least about 98%, or at least about 99% of said
antibody may be recovered from a vial upon shaking said vial for 4
hours at a speed of 400 shakes per minute wherein said vial is
filled to half of its volume with said formulation. In another
embodiment, a formulation of the invention comprises MT103, wherein
at least about 90%, at least about 95%, at least about 97%, at
least about 98%, or at least about 99% of said antibody may be
recovered from a vial upon subjecting the formulation to three
freeze/thaw cycles wherein said vial is filled to half of its
volume with said formulation. In a further embodiment, a
formulation of the invention comprises MT103, wherein at least
about 90%, at least about 95%, at least about 97%, at least about
98%, or at least about 99% of said antibody may be recovered by
reconstituting a lyophilized cake generated from said
formulation.
[0277] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 90% of said antibody
may be recovered from a vial upon shaking said vial for 4 hours at
a speed of 400 shakes per minute wherein said vial is filled to
half of its volume with said formulation. In another embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
at least about 90% of said antibody may be recovered from a vial
upon subjecting the formulation to three freeze/thaw cycles wherein
said vial is filled to half of its volume with said formulation. In
a further embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 90% of said antibody
may be recovered by reconstituting a lyophilized cake generated
from said formulation.
[0278] In one embodiment, a formulation of the invention comprises
MT103, wherein at least about 90% of said antibody may be recovered
from a vial upon shaking said vial for 4 hours at a speed of 400
shakes per minute wherein said vial is filled to half of its volume
with said formulation. In another embodiment, a formulation of the
invention comprises MT103, wherein at least about 90% of said
antibody may be recovered from a vial upon subjecting the
formulation to three freeze/thaw cycles wherein said vial is filled
to half of its volume with said formulation. In a further
embodiment, a formulation of the invention comprises MT103, wherein
at least about 90% of said antibody may be recovered by
reconstituting a lyophilized cake generated from said
formulation.
[0279] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 95% of said antibody
may be recovered from a vial upon shaking said vial for 4 hours at
a speed of 400 shakes per minute wherein said vial is filled to
half of its volume with said formulation. In another embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
at least about 95% of said antibody may be recovered from a vial
upon subjecting the formulation to three freeze/thaw cycles wherein
said vial is filled to half of its volume with said formulation. In
a further embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 95% of said antibody
may be recovered by reconstituting a lyophilized cake generated
from said formulation.
[0280] In one embodiment, a formulation of the invention comprises
MT103, wherein at least about 95% of said antibody may be recovered
from a vial upon shaking said vial for 4 hours at a speed of 400
shakes per minute wherein said vial is filled to half of its volume
with said formulation. In another embodiment, a formulation of the
invention comprises MT103, wherein at least about 95% of said
antibody may be recovered from a vial upon subjecting the
formulation to three freeze/thaw cycles wherein said vial is filled
to half of its volume with said formulation. In a further
embodiment, a formulation of the invention comprises MT103, wherein
at least about 95% of said antibody may be recovered by
reconstituting a lyophilized cake generated from said
formulation.
[0281] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 97% of said antibody
may be recovered from a vial upon shaking said vial for 4 hours at
a speed of 400 shakes per minute wherein said vial is filled to
half of its volume with said formulation. In another embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
at least about 97% of said antibody may be recovered from a vial
upon subjecting the formulation to three freeze/thaw cycles wherein
said vial is filled to half of its volume with said formulation. In
a further embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 97% of said antibody
may be recovered by reconstituting a lyophilized cake generated
from said formulation.
[0282] In one embodiment, a formulation of the invention comprises
MT103, wherein at least about 97% of said antibody may be recovered
from a vial upon shaking said vial for 4 hours at a speed of 400
shakes per minute wherein said vial is filled to half of its volume
with said formulation. In another embodiment, a formulation of the
invention comprises MT103, wherein at least about 97% of said
antibody may be recovered from a vial upon subjecting the
formulation to three freeze/thaw cycles wherein said vial is filled
to half of its volume with said formulation. In a further
embodiment, a formulation of the invention comprises MT103, wherein
at least about 97% of said antibody may be recovered by
reconstituting a lyophilized cake generated from said
formulation.
[0283] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 98% of said antibody
may be recovered from a vial upon shaking said vial for 4 hours at
a speed of 400 shakes per minute wherein said vial is filled to
half of its volume with said formulation. In another embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
at least about 98% of said antibody may be recovered from a vial
upon subjecting the formulation to three freeze/thaw cycles wherein
said vial is filled to half of its volume with said formulation. In
a further embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 98% of said antibody
may be recovered by reconstituting a lyophilized cake generated
from said formulation.
[0284] In one embodiment, a formulation of the invention comprises
MT103, wherein at least about 98% of said antibody may be recovered
from a vial upon shaking said vial for 4 hours at a speed of 400
shakes per minute wherein said vial is filled to half of its volume
with said formulation. In another embodiment, a formulation of the
invention comprises MT103, wherein at least about 98% of said
antibody may be recovered from a vial upon subjecting the
formulation to three freeze/thaw cycles wherein said vial is filled
to half of its volume with said formulation. In a further
embodiment, a formulation of the invention comprises MT103, wherein
at least about 98% of said antibody may be recovered by
reconstituting a lyophilized cake generated from said
formulation.
[0285] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 99% of said antibody
may be recovered from a vial upon shaking said vial for 4 hours at
a speed of 400 shakes per minute wherein said vial is filled to
half of its volume with said formulation. In another embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
at least about 99% of said antibody may be recovered from a vial
upon subjecting the formulation to three freeze/thaw cycles wherein
said vial is filled to half of its volume with said formulation. In
a further embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein at least about 99% of said antibody
may be recovered by reconstituting a lyophilized cake generated
from said formulation.
[0286] In one embodiment, a formulation of the invention comprises
MT103, wherein at least about 99% of said antibody may be recovered
from a vial upon shaking said vial for 4 hours at a speed of 400
shakes per minute wherein said vial is filled to half of its volume
with said formulation. In another embodiment, a formulation of the
invention comprises MT103, wherein at least about 99% of said
antibody may be recovered from a vial upon subjecting the
formulation to three freeze/thaw cycles wherein said vial is filled
to half of its volume with said formulation. In a further
embodiment, a formulation of the invention comprises MT103, wherein
at least about 99% of said antibody may be recovered by
reconstituting a lyophilized cake generated from said
formulation.
5.1.1.8. Fragmentation
[0287] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at about
40.degree. C. for at least about 1 day, at least about 2 days, at
least about 3 days, at least about 4 days, at least about 5 days,
at least about 6 days, or at least about 7 days. In one embodiment,
a formulation of the invention comprises BiTE.RTM. molecules,
wherein less than 5% of said antibody is fragmented as determined
by RP-HPLC upon storage at about 40.degree. C. for at least about 1
week, at least about 2 weeks, at least about 3 weeks, at least
about 4 weeks, at least about 5 weeks, or at least about 6
weeks.
[0288] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for at least about 1 week, at least about 2 weeks, at least
about 3 weeks, at least about 4 weeks, at least about 5 weeks, at
least about 6 weeks, at least about 7 weeks, at least about 8
weeks, at least about 9 weeks, at least about 10 weeks, at least
about 11 weeks, or at least about 12 weeks. In one embodiment, a
formulation of the invention comprises BiTE.RTM. molecules, wherein
less than 5% of said antibody is fragmented as determined by
RP-HPLC upon storage at about 5.degree. C. for at least about 1
month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, at least about 6 months,
at least about 7 months, at least about 8 months, at least about 9
months, at least about 10 months, at least about 11 months, or at
least about 12 months.
[0289] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at about
40.degree. C. for about 1 day, about 2 days, about 3 days, about 4
days, about 5 days, about 6 days, or about 7 days. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 5% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 40.degree. C. for about
1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
or about 6 weeks.
[0290] In one embodiment, a formulation of the invention comprises
BiTE.RTM. molecules, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9
weeks, about 10 weeks, about 11 weeks, or about 12 weeks. In one
embodiment, a formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 5% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 5.degree. C. for about
1 month, about 2 months, about 3 months, about 4 months, about 5
months, about 6 months, about 7 months, about 8 months, about 9
months, about 10 months, about 11 months, or about 12 months.
[0291] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
or at least about 7 days. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at about
40.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, at least about 4 weeks, at least about 5
weeks, or at least about 6 weeks.
[0292] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 5.degree. C. for at
least about 1 week, at least about 2 weeks, at least about 3 weeks,
at least about 4 weeks, at least about 5 weeks, at least about 6
weeks, at least about 7 weeks, at least about 8 weeks, at least
about 9 weeks, at least about 10 weeks, at least about 11 weeks, or
at least about 12 weeks. In one embodiment, a formulation of the
invention comprises MT103, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for at least about 1 month, at least about 2 months, at least
about 3 months, at least about 4 months, at least about 5 months,
at least about 6 months, at least about 7 months, at least about 8
months, at least about 9 months, at least about 10 months, at least
about 11 months, or at least about 12 months.
[0293] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 40.degree. C. for about
1 day, about 2 days, about 3 days, about 4 days, about 5 days,
about 6 days, or about 7 days. In one embodiment, a formulation of
the invention comprises MT103, wherein less than 5% of said
antibody is fragmented as determined by RP-HPLC upon storage at
about 40.degree. C. for about 1 week, about 2 weeks, about 3 weeks,
about 4 weeks, about 5 weeks, or about 6 weeks.
[0294] In one embodiment, a formulation of the invention comprises
MT103, wherein less than 5% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 5.degree. C. for about
1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about
10 weeks, about 11 weeks, or about 12 weeks. In one embodiment, a
formulation of the invention comprises MT103, wherein less than 5%
of said antibody is fragmented as determined by RP-HPLC upon
storage at about 5.degree. C. for about 1 month, about 2 months,
about 3 months, about 4 months, about 5 months, about 6 months,
about 7 months, about 8 months, about 9 months, about 10 months,
about 11 months, or about 12 months.
5.1.1.9. Visual Characteristics
[0295] In one embodiment, a formulation of the invention is clear
and colorless as determined by visual inspection upon storage at
about 40.degree. C. f for at least about 1 day, at least about 2
days, at least about 3 days, at least about 4 days, at least about
5 days, at least about 6 days, or at least about 7 days. In one
embodiment, a formulation of the invention is clear and colorless
as determined by visual inspection upon storage at about 40.degree.
C. for at least about 1 week, at least about 2 weeks, at least
about 3 weeks, at least about 4 weeks, at least about 5 weeks, or
at least about 6 weeks.
[0296] In one embodiment, a formulation of the invention is clear
and colorless as determined by visual inspection upon storage at
about 5.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, at least about 6 weeks, at least about 7 weeks, at
least about 8 weeks, at least about 9 weeks, at least about 10
weeks, at least about 11 weeks, or at least about 12 weeks. In one
embodiment, a formulation of the invention is clear and colorless
as determined by visual inspection upon storage at about 5.degree.
C. for at least about 1 month, at least about 2 months, at least
about 3 months, at least about 4 months, at least about 5 months,
at least about 6 months, at least about 7 months, at least about 8
months, at least about 9 months, at least about 10 months, at least
about 11 months, or at least about 12 months.
[0297] In one embodiment, a formulation of the invention is clear
and colorless as determined by visual inspection upon storage at
about 40.degree. C. f for about 1 day, about 2 days, about 3 days,
about 4 days, about 5 days, about 6 days, or about 7 days. In one
embodiment, a formulation of the invention is clear and colorless
as determined by visual inspection upon storage at about 40.degree.
C. for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,
about 5 weeks, or about 6 weeks.
[0298] In one embodiment, a formulation of the invention is clear
and colorless as determined by visual inspection upon storage at
about 5.degree. C. for about 1 week, about 2 weeks, about 3 weeks,
about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8
weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12
weeks. In one embodiment, a formulation of the invention is clear
and colorless as determined by visual inspection upon storage at
about 5.degree. C. for about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 7
months, about 8 months, about 9 months, about 10 months, about 11
months, or about 12 months.
5.2. Lyophilized Formulations
[0299] The present invention also encompasses lyophilized
formulations comprising bispecific antibodies which formulations
exhibit stability, low to undetectable levels of antibody
fragmentation, low to undetectable levels of aggregation, and very
little to no loss of the biological activities of the bispecific
antibodies during manufacture, preparation, transportation, and
storage. In certain embodiments, a lyophilized formulation of the
invention comprises bispecific antibodies comprising at least a
first and a second binding site specific for the CD3 and CD19
antigens, respectively. In specific embodiments, the present
invention provides lyophilized formulations comprising MT103 (see,
U.S. Pat. No. 7,112,324).
[0300] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules that comprises at least a first and a second binding site
specific for the CD3 and CD19 antigens, respectively. In a further
embodiment, a lyophilized formulation of the invention comprises
BiTE.RTM. molecules wherein said antibody comprises an anti-CD19 VL
domain having the amino acid sequence of SEQ ID NO:1, an anti-CD19
VH domain having the amino acid sequence of SEQ ID NO:2, an
anti-CD3 VH domain having the amino acid sequence of SEQ ID NO:3
and an anti-CD3 VL domain having the amino acid sequence of SEQ ID
NO:4. In a specific embodiment, a lyophilized formulation of the
invention comprises MT103, wherein said antibody comprises the
amino acid sequence of SEQ ID NO:5.
[0301] The invention encompasses stable lyophilized formulations
comprising a single BiTE.RTM. molecule of interest, for example,
BiTE.RTM. molecules that comprises at least a first and a second
binding site specific for the CD3 and CD19 antigens, respectively.
The invention also encompasses stable lyophilized formulations
comprising two or more bispecific antibodies of interest, for
example, bispecific antibodies that comprise at least a first and a
second binding site specific for the CD3 and CD19 antigens,
respectively. In a specific embodiment, a stable lyophilized
formulation of the invention comprises MT103. In another
embodiment, a stable lyophilized formulation of the invention
comprises two or more bispecific antibodies, wherein one of the
bispecific antibodies is MT103.
[0302] In one embodiment, a lyophilized formulation of the
invention comprises citrate, lysine HCl, trehalose, and Polysorbate
80. In one embodiment, a lyophilized formulation of the invention
comprises lysine HCl, trehalose, and Polysorbate 80. In one
embodiment, a lyophilized formulation of the invention comprises
citrate, trehalose, and Polysorbate 80. In one embodiment, a
lyophilized formulation of the invention comprises citrate, lysine
HCl, and Polysorbate 80. In one embodiment, a lyophilized
formulation of the invention comprises citrate, lysine HCl, and
trehalose. In one embodiment, a lyophilized formulation of the
invention comprises citrate and lysine HCl. In one embodiment, a
lyophilized formulation of the invention comprises citrate and
trehalose. In one embodiment, a lyophilized formulation of the
invention comprises citrate and Polysorbate 80. In one embodiment,
a lyophilized formulation of the invention comprises lysine HCl and
trehalose. In one embodiment, a lyophilized formulation of the
invention comprises lysine HCl and Polysorbate 80. In one
embodiment, a lyophilized formulation of the invention comprises
trehalose, and Polysorbate 80. In one embodiment, a lyophilized
formulation of the invention comprises lysine HCl.
[0303] In one embodiment, a lyophilized formulation of the
invention comprises citrate, lysine HCl, trehalose and Polysorbate
80. In one embodiment, a lyophilized formulation of the invention
comprises citrate, lysine HCl, and trehalose at a molar ratio of
between about 1 and about 5 mole citrate to between about 4 and
about 40 mole trehalose to between about 5 and about 20 mole lysine
HCl. In a specific embodiment, a lyophilized formulation of the
invention comprises citrate, lysine HCl, and trehalose at a molar
ratio of about 1 mole citrate to about 16 mole trehalose to about 8
mole lysine HCl.
[0304] In a specific embodiment, a lyophilized formulation of the
invention comprises MT103, citrate, lysine HCl, trehalose and
Polysorbate 80.
[0305] In a specific embodiment, a lyophilized formulation of the
invention consists of MT103, citrate, lysine HCl, trehalose and
Polysorbate 80.
[0306] In one embodiment, a lyophilized formulation of the
invention has a residual moisture content that is less than about
10%, less than about 5%, less than about 3%, less than about 2%,
less than about 1%. In a further embodiment, a lyophilized
formulation of the invention has a residual moisture content of
about 10%, about 5%, about 4%, about 3%, about 2%, about 1%.
[0307] In certain embodiments, a lyophilized formulation of the
invention is derived from a liquid formulation of the
invention.
5.2.1. Stability of Lyophilized Formulations
[0308] In one embodiment, a lyophilized lyophilized formulation of
the invention comprises BiTE.RTM. molecules that is susceptible to
aggregation, fragmentation and/or loss due to absorption to contact
surfaces.
[0309] In one embodiment, a lyophilized formulation of the
invention stabilizes BiTE.RTM. molecules. In one embodiment, a
lyophilized formulation of the invention prevents aggregation of
BiTE.RTM. molecules. In another embodiment, a lyophilized
formulation of the invention prevents fragmentation of BiTE.RTM.
molecules. In a further embodiment, a lyophilized formulation of
the invention prevents loss of BiTE.RTM. molecules due to
absorption to contact surfaces.
[0310] In one embodiment, a lyophilized formulation of the
invention stabilizes MT103. In one embodiment, a lyophilized
formulation of the invention prevents aggregation of MT103. In
another embodiment, a lyophilized formulation of the invention
prevents fragmentation of MT103. In a further embodiment, a
lyophilized formulation of the invention prevents loss of MT103 due
to absorption to contact surfaces.
[0311] In one embodiment, a lyophilized formulation of the
invention stabilizes BiTE.RTM. molecules. In one embodiment, a
lyophilized formulation of the invention reduces aggregation of
BiTE.RTM. molecules. In another embodiment, a lyophilized
formulation of the invention reduces fragmentation of BiTE.RTM.
molecules. In a further embodiment, a lyophilized formulation of
the invention reduces loss of BiTE.RTM. molecules due to absorption
to contact surfaces.
[0312] In one embodiment, a lyophilized formulation of the
invention stabilizes MT103. In one embodiment, a lyophilized
formulation of the invention reduces aggregation of MT103. In
another embodiment, a lyophilized formulation of the invention
reduces fragmentation of MT103. In a further embodiment, a
lyophilized formulation of the invention reduces loss of MT103 due
to absorption to contact surfaces.
5.2.2. Stability Over Time
[0313] In one embodiment, a lyophilized formulation of the
invention is stable upon storage at about 40.degree. C. for at
least about 1 day, at least about 2 days, at least about 3 days, at
least about 4 days, at least about 5 days, at least about 6 days,
at least about 7 days, at least about 8 days, at least about 9
days, at least about 10 days, at least about 15 days, at least
about 20 days, at least about 25 days, or at least about 30 days.
In one embodiment, a lyophilized formulation of the invention is
stable upon storage at about 40.degree. C. for at least about 1
week, at least about 2 weeks, at least about 3 weeks, or at least
about 4 weeks. In one embodiment, a lyophilized formulation of the
invention is stable upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0314] In one embodiment, a lyophilized formulation of the
invention is stable upon storage at about 4.degree. C. for at least
about 1 day, at least about 2 days, at least about 3 days, at least
about 4 days, at least about 5 days, at least about 6 days, at
least about 7 days, at least about 8 days, at least about 9 days,
at least about 10 days. In one embodiment, a lyophilized
formulation of the invention is stable upon storage at about
4.degree. C. for at least about 1 week, at least about 2 weeks, at
least about 3 weeks, or at least about 4 weeks. In one embodiment,
a lyophilized formulation of the invention is stable upon storage
at about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention is
stable upon storage at about 5.degree. C. for at least about 1
year, at least about 2 years, at least about 3 years, at least
about 4 years, at least about 5 years, at least about 6 years, at
least about 7 years, at least about 8 years, at least about 9
years, at least about 10 years, at least about 11 years, or at
least about 12 years.
[0315] In one embodiment, a lyophilized formulation of the
invention is stable upon storage at about 40.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, about 7 days, about 8 days, about 9 days, about 10 days,
about 15 days, about 20 days, about 25 days, or about 30 days. In
one embodiment, a lyophilized formulation of the invention is
stable upon storage at about 40.degree. C. for about 1 week, about
2 weeks, about 3 weeks, or about 4 weeks. In one embodiment, a
lyophilized formulation of the invention is stable upon storage at
about 40.degree. C. for about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, or about 6 months.
[0316] In one embodiment, a lyophilized formulation of the
invention is stable upon storage at about 4.degree. C. for about 1
day, about 2 days, about 3 days, about 4 days, about 5 days, about
6 days, about 7 days, about 8 days, about 9 days, about 10 days. In
one embodiment, a lyophilized formulation of the invention is
stable upon storage at about 4.degree. C. for about 1 week, about 2
weeks, about 3 weeks, or about 4 weeks. In one embodiment, a
lyophilized formulation of the invention is stable upon storage at
about 5.degree. C. for about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 7
months, about 8 months, about 9 months, about 10 months, about 11
months, or about 12 months. In one embodiment, a lyophilized
formulation of the invention is stable upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, about 5 years, about 6 years, about 7 years, about 8
years, about 9 years, about 10 years, about 11 years, or about 12
years.
5.2.2.1. Protein Recovery
[0317] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein at least about 90%
of said antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for at least about
1 week, at least about 2 weeks, at least about 3 weeks, at least
about 4 weeks, at least about 5 weeks, or at least about 6 weeks.
In one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein at least about 90% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for at least about
1 month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, or at least about 6
months.
[0318] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein at least about 90%
of said antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 5.degree. C. for at least about 1
month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, at least about 6 months,
at least about 7 months, at least about 8 months, at least about 9
months, at least about 10 months, at least about 11 months, or at
least about 12 months. In one embodiment, a lyophilized formulation
of the invention comprises BiTE.RTM. molecules, wherein at least
about 90% of said antibody is recovered by reconstituting said
lyophilized formulation upon storage at about 5.degree. C. for at
least about 1 year, at least about 2 years, at least about 3 years,
at least about 4 years, or at least about 5 years.
[0319] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 90% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for about 1 week,
about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or
about 6 weeks. In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 90% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months, or
about 6 months.
[0320] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 90% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 5.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months,
about 6 months, about 7 months, about 8 months, about 9 months,
about 10 months, about 11 months, or about 12 months. In one
embodiment, a lyophilized formulation of the invention comprises
MT103, wherein at least about 90% of said antibody is recovered by
reconstituting said lyophilized formulation upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
[0321] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein at least about 95%
of said antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for at least about
1 week, at least about 2 weeks, at least about 3 weeks, at least
about 4 weeks, at least about 5 weeks, or at least about 6 weeks.
In one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein at least about 95% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for at least about
1 month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, or at least about 6
months.
[0322] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein at least about 95%
of said antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 5.degree. C. for at least about 1
month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, at least about 6 months,
at least about 7 months, at least about 8 months, at least about 9
months, at least about 10 months, at least about 11 months, or at
least about 12 months. In one embodiment, a lyophilized formulation
of the invention comprises BiTE.RTM. molecules, wherein at least
about 95% of said antibody is recovered by reconstituting said
lyophilized formulation upon storage at about 5.degree. C. for at
least about 1 year, at least about 2 years, at least about 3 years,
at least about 4 years, or at least about 5 years.
[0323] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 95% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for about 1 week,
about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or
about 6 weeks. In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 95% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months, or
about 6 months.
[0324] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 95% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 5.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months,
about 6 months, about 7 months, about 8 months, about 9 months,
about 10 months, about 11 months, or about 12 months. In one
embodiment, a lyophilized formulation of the invention comprises
MT103, wherein at least about 95% of said antibody is recovered by
reconstituting said lyophilized formulation upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
[0325] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein at least about 97%
of said antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for at least about
1 week, at least about 2 weeks, at least about 3 weeks, at least
about 4 weeks, at least about 5 weeks, or at least about 6 weeks.
In one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein at least about 97% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for at least about
1 month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, or at least about 6
months.
[0326] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein at least about 97%
of said antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 5.degree. C. for at least about 1
month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, at least about 6 months,
at least about 7 months, at least about 8 months, at least about 9
months, at least about 10 months, at least about 11 months, or at
least about 12 months. In one embodiment, a lyophilized formulation
of the invention comprises BiTE.RTM. molecules, wherein at least
about 97% of said antibody is recovered by reconstituting said
lyophilized formulation upon storage at about 5.degree. C. for at
least about 1 year, at least about 2 years, at least about 3 years,
at least about 4 years, or at least about 5 years.
[0327] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 97% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for about 1 week,
about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or
about 6 weeks. In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 97% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months, or
about 6 months.
[0328] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 97% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 5.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months,
about 6 months, about 7 months, about 8 months, about 9 months,
about 10 months, about 11 months, or about 12 months. In one
embodiment, a lyophilized formulation of the invention comprises
MT103, wherein at least about 97% of said antibody is recovered by
reconstituting said lyophilized formulation upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
[0329] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein at least about 98%
of said antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for at least about
1 week, at least about 2 weeks, at least about 3 weeks, at least
about 4 weeks, at least about 5 weeks, or at least about 6 weeks.
In one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein at least about 98% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for at least about
1 month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, or at least about 6
months.
[0330] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein at least about 98%
of said antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 5.degree. C. for at least about 1
month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, at least about 6 months,
at least about 7 months, at least about 8 months, at least about 9
months, at least about 10 months, at least about 11 months, or at
least about 12 months. In one embodiment, a lyophilized formulation
of the invention comprises BiTE.RTM. molecules, wherein at least
about 98% of said antibody is recovered by reconstituting said
lyophilized formulation upon storage at about 5.degree. C. for at
least about 1 year, at least about 2 years, at least about 3 years,
at least about 4 years, or at least about 5 years.
[0331] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 98% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for about 1 week,
about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or
about 6 weeks. In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 98% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months, or
about 6 months.
[0332] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 98% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 5.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months,
about 6 months, about 7 months, about 8 months, about 9 months,
about 10 months, about 11 months, or about 12 months. In one
embodiment, a lyophilized formulation of the invention comprises
MT103, wherein at least about 98% of said antibody is recovered by
reconstituting said lyophilized formulation upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
[0333] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein at least about 99%
of said antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for at least about
1 week, at least about 2 weeks, at least about 3 weeks, at least
about 4 weeks, at least about 5 weeks, or at least about 6 weeks.
In one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein at least about 99% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for at least about
1 month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, or at least about 6
months.
[0334] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein at least about 99%
of said antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 5.degree. C. for at least about 1
month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, at least about 6 months,
at least about 7 months, at least about 8 months, at least about 9
months, at least about 10 months, at least about 11 months, or at
least about 12 months. In one embodiment, a lyophilized formulation
of the invention comprises BiTE.RTM. molecules, wherein at least
about 99% of said antibody is recovered by reconstituting said
lyophilized formulation upon storage at about 5.degree. C. for at
least about 1 year, at least about 2 years, at least about 3 years,
at least about 4 years, or at least about 5 years.
[0335] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 99% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for about 1 week,
about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or
about 6 weeks. In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 99% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 40.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months, or
about 6 months.
[0336] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein at least about 99% of said
antibody is recovered by reconstituting said lyophilized
formulation upon storage at about 5.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months,
about 6 months, about 7 months, about 8 months, about 9 months,
about 10 months, about 11 months, or about 12 months. In one
embodiment, a lyophilized formulation of the invention comprises
MT103, wherein at least about 99% of said antibody is recovered by
reconstituting said lyophilized formulation upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
5.2.2.2. Binding Activity
[0337] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein said antibody
retains at least 90% of binding ability to its target polypeptides
compared to a reference antibody representing the antibody prior to
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks. In one embodiment,
a lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein said antibody retains at least 90% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for at least about 1 month, at least about 2 months, at least about
3 months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0338] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein said antibody
retains at least 90% of binding ability to its target polypeptides
compared to a reference antibody representing the antibody prior to
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months. In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein said antibody
retains at least 90% of binding ability to its target polypeptides
compared to a reference antibody representing the antibody prior to
storage at about 5.degree. C. for at least about 1 year, at least
about 2 years, at least about 3 years, at least about 4 years, or
at least about 5 years.
[0339] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein said antibody
retains 90% of binding ability to its target polypeptides compared
to a reference antibody representing the antibody prior to storage
at about 40.degree. C. for about 1 week, about 2 weeks, about 3
weeks, about 4 weeks, about 5 weeks, or about 6 weeks. In one
embodiment, a lyophilized formulation of the invention comprises
BiTE.RTM. molecules, wherein said antibody retains 90% of binding
ability to its target polypeptides compared to a reference antibody
representing the antibody prior to storage at about 40.degree. C.
for about 1 month, about 2 months, about 3 months, about 4 months,
about 5 months, or about 6 months.
[0340] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein said antibody
retains 90% of binding ability to its target polypeptides compared
to a reference antibody representing the antibody prior to storage
at about 5.degree. C. for about 1 month, about 2 months, about 3
months, about 4 months, about 5 months, about 6 months, about 7
months, about 8 months, about 9 months, about 10 months, about 11
months, or about 12 months. In one embodiment, a lyophilized
formulation of the invention comprises BiTE.RTM. molecules, wherein
said antibody retains 90% of binding ability to its target
polypeptides compared to a reference antibody representing the
antibody prior to storage at about 5.degree. C. for about 1 year,
about 2 years, about 3 years, about 4 years, or about 5 years.
[0341] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein said antibody retains at least
90% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to storage at
about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
said antibody retains at least 90% of binding ability to CD19 and
CD3 antigens compared to a reference antibody representing the
antibody prior to storage at about 40.degree. C. for at least about
1 month, at least about 2 months, at least about 3 months, at least
about 4 months, at least about 5 months, or at least about 6
months.
[0342] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein said antibody retains at least
90% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to storage at
about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises MT103, wherein said antibody retains at least 90% of
binding ability to CD19 and CD3 antigens compared to a reference
antibody representing the antibody prior to storage at about
5.degree. C. for at least about 1 year, at least about 2 years, at
least about 3 years, at least about 4 years, or at least about 5
years.
[0343] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein said antibody retains 90% of
binding ability to CD19 and CD3 antigens compared to a reference
antibody representing the antibody prior to storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
said antibody retains 90% of binding ability to CD19 and CD3
antigens compared to a reference antibody representing the antibody
prior to storage at about 40.degree. C. for about 1 month, about 2
months, about 3 months, about 4 months, about 5 months, or about 6
months.
[0344] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein said antibody retains 90% of
binding ability to CD19 and CD3 antigens compared to a reference
antibody representing the antibody prior to storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein said antibody retains 90% of
binding ability to CD19 and CD3 antigens compared to a reference
antibody representing the antibody prior to storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
5.2.2.3. Aggregate Formation
[0345] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks. In one embodiment,
a lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 1% of said antibody forms an aggregate
as determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0346] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months. In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 year, at least
about 2 years, at least about 3 years, at least about 4 years, or
at least about 5 years.
[0347] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 1% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for about 1 month, about
2 months, about 3 months, about 4 months, about 5 months, or about
6 months.
[0348] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein less than 1% of said
antibody forms an aggregate as determined by HPSEC upon storage at
about 5.degree. C. for about 1 year, about 2 years, about 3 years,
about 4 years, or about 5 years.
[0349] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks. In one embodiment,
a lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 2% of said antibody forms an aggregate
as determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0350] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months. In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 year, at least
about 2 years, at least about 3 years, at least about 4 years, or
at least about 5 years.
[0351] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 2% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for about 1 month, about
2 months, about 3 months, about 4 months, about 5 months, or about
6 months.
[0352] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein less than 2% of said
antibody forms an aggregate as determined by HPSEC upon storage at
about 5.degree. C. for about 1 year, about 2 years, about 3 years,
about 4 years, or about 5 years.
[0353] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks. In one embodiment,
a lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 3% of said antibody forms an aggregate
as determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0354] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months. In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 year, at least
about 2 years, at least about 3 years, at least about 4 years, or
at least about 5 years.
[0355] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 3% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for about 1 month, about
2 months, about 3 months, about 4 months, about 5 months, or about
6 months.
[0356] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein less than 3% of said
antibody forms an aggregate as determined by HPSEC upon storage at
about 5.degree. C. for about 1 year, about 2 years, about 3 years,
about 4 years, or about 5 years.
[0357] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks. In one embodiment,
a lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 4% of said antibody forms an aggregate
as determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0358] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months. In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 year, at least
about 2 years, at least about 3 years, at least about 4 years, or
at least about 5 years.
[0359] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 4% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for about 1 month, about
2 months, about 3 months, about 4 months, about 5 months, or about
6 months.
[0360] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein less than 4% of said
antibody forms an aggregate as determined by HPSEC upon storage at
about 5.degree. C. for about 1 year, about 2 years, about 3 years,
about 4 years, or about 5 years.
[0361] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 40.degree. C. for at least about 1 week, at least
about 2 weeks, at least about 3 weeks, at least about 4 weeks, at
least about 5 weeks, or at least about 6 weeks. In one embodiment,
a lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 5% of said antibody forms an aggregate
as determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0362] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 month, at least
about 2 months, at least about 3 months, at least about 4 months,
at least about 5 months, at least about 6 months, at least about 7
months, at least about 8 months, at least about 9 months, at least
about 10 months, at least about 11 months, or at least about 12
months. In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for at least about 1 year, at least
about 2 years, at least about 3 years, at least about 4 years, or
at least about 5 years.
[0363] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 5% of said antibody forms an aggregate as determined by
HPSEC upon storage at about 40.degree. C. for about 1 month, about
2 months, about 3 months, about 4 months, about 5 months, or about
6 months.
[0364] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein less than 5% of said
antibody forms an aggregate as determined by HPSEC upon storage at
about 5.degree. C. for about 1 year, about 2 years, about 3 years,
about 4 years, or about 5 years.
5.2.2.4. Dimer Formation
[0365] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 1% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0366] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein less than 1% of said
antibody forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 year, at least about 2 years, at
least about 3 years, at least about 4 years, or at least about 5
years.
[0367] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 1% of said antibody forms a dimer as determined by HPSEC
upon storage at about 40.degree. C. for about 1 month, about 2
months, about 3 months, about 4 months, about 5 months, or about 6
months.
[0368] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein less than 1% of said
antibody forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
[0369] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 2% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0370] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein less than 2% of said
antibody forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 year, at least about 2 years, at
least about 3 years, at least about 4 years, or at least about 5
years.
[0371] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 2% of said antibody forms a dimer as determined by HPSEC
upon storage at about 40.degree. C. for about 1 month, about 2
months, about 3 months, about 4 months, about 5 months, or about 6
months.
[0372] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein less than 2% of said
antibody forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
[0373] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 3% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0374] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein less than 3% of said
antibody forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 year, at least about 2 years, at
least about 3 years, at least about 4 years, or at least about 5
years.
[0375] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 3% of said antibody forms a dimer as determined by HPSEC
upon storage at about 40.degree. C. for about 1 month, about 2
months, about 3 months, about 4 months, about 5 months, or about 6
months.
[0376] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein less than 3% of said
antibody forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
[0377] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 4% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0378] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein less than 4% of said
antibody forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 year, at least about 2 years, at
least about 3 years, at least about 4 years, or at least about 5
years.
[0379] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 4% of said antibody forms a dimer as determined by HPSEC
upon storage at about 40.degree. C. for about 1 month, about 2
months, about 3 months, about 4 months, about 5 months, or about 6
months.
[0380] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein less than 4% of said
antibody forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
[0381] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 5% of said antibody forms a dimer as
determined by HPSEC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0382] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein less than 5% of said
antibody forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for at least about 1 year, at least about 2 years, at
least about 3 years, at least about 4 years, or at least about 5
years.
[0383] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms a dimer as determined by HPSEC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 5% of said antibody forms a dimer as determined by HPSEC
upon storage at about 40.degree. C. for about 1 month, about 2
months, about 3 months, about 4 months, about 5 months, or about 6
months.
[0384] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 month, about 2 months, about 3 months,
about 4 months, about 5 months, about 6 months, about 7 months,
about 8 months, about 9 months, about 10 months, about 11 months,
or about 12 months. In one embodiment, a lyophilized formulation of
the invention comprises MT103, wherein less than 5% of said
antibody forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 year, about 2 years, about 3 years, about
4 years, or about 5 years.
5.2.2.5. Fragmentation
[0385] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 1% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0386] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein less than 1% of said
antibody is fragmented as determined by RP-HPLC upon storage at
about 5.degree. C. for at least about 1 year, at least about 2
years, at least about 3 years, at least about 4 years, or at least
about 5 years.
[0387] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 1% of said antibody is
fragmented as determined by RP-HPLC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 1% of said antibody is fragmented as determined by
RP-HPLC upon storage at about 40.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months, or
about 6 months.
[0388] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 1% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 month, about 2 months, about 3 months, about 4
months, about 5 months, about 6 months, about 7 months, about 8
months, about 9 months, about 10 months, about 11 months, or about
12 months. In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 1% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 year, about 2 years, about 3 years, about 4 years,
or about 5 years.
[0389] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 2% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0390] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein less than 2% of said
antibody is fragmented as determined by RP-HPLC upon storage at
about 5.degree. C. for at least about 1 year, at least about 2
years, at least about 3 years, at least about 4 years, or at least
about 5 years.
[0391] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 2% of said antibody is
fragmented as determined by RP-HPLC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 2% of said antibody is fragmented as determined by
RP-HPLC upon storage at about 40.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months, or
about 6 months.
[0392] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 2% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 month, about 2 months, about 3 months, about 4
months, about 5 months, about 6 months, about 7 months, about 8
months, about 9 months, about 10 months, about 11 months, or about
12 months. In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 2% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 year, about 2 years, about 3 years, about 4 years,
or about 5 years.
[0393] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 3% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0394] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein less than 3% of said
antibody is fragmented as determined by RP-HPLC upon storage at
about 5.degree. C. for at least about 1 year, at least about 2
years, at least about 3 years, at least about 4 years, or at least
about 5 years.
[0395] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 3% of said antibody is
fragmented as determined by RP-HPLC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 3% of said antibody is fragmented as determined by
RP-HPLC upon storage at about 40.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months, or
about 6 months.
[0396] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 3% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 month, about 2 months, about 3 months, about 4
months, about 5 months, about 6 months, about 7 months, about 8
months, about 9 months, about 10 months, about 11 months, or about
12 months. In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 3% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 year, about 2 years, about 3 years, about 4 years,
or about 5 years.
[0397] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 4% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0398] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein less than 4% of said
antibody is fragmented as determined by RP-HPLC upon storage at
about 5.degree. C. for at least about 1 year, at least about 2
years, at least about 3 years, at least about 4 years, or at least
about 5 years.
[0399] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 4% of said antibody is
fragmented as determined by RP-HPLC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 4% of said antibody is fragmented as determined by
RP-HPLC upon storage at about 40.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months, or
about 6 months.
[0400] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 4% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 month, about 2 months, about 3 months, about 4
months, about 5 months, about 6 months, about 7 months, about 8
months, about 9 months, about 10 months, about 11 months, or about
12 months. In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 4% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 year, about 2 years, about 3 years, about 4 years,
or about 5 years.
[0401] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 40.degree. C. for at least about 1 week, at least about 2
weeks, at least about 3 weeks, at least about 4 weeks, at least
about 5 weeks, or at least about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises BiTE.RTM.
molecules, wherein less than 5% of said antibody is fragmented as
determined by RP-HPLC upon storage at about 40.degree. C. for at
least about 1 month, at least about 2 months, at least about 3
months, at least about 4 months, at least about 5 months, or at
least about 6 months.
[0402] In one embodiment, a lyophilized formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 5.degree. C. for at least about 1 month, at least about 2
months, at least about 3 months, at least about 4 months, at least
about 5 months, at least about 6 months, at least about 7 months,
at least about 8 months, at least about 9 months, at least about 10
months, at least about 11 months, or at least about 12 months. In
one embodiment, a lyophilized formulation of the invention
comprises BiTE.RTM. molecules, wherein less than 5% of said
antibody is fragmented as determined by RP-HPLC upon storage at
about 5.degree. C. for at least about 1 year, at least about 2
years, at least about 3 years, at least about 4 years, or at least
about 5 years.
[0403] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at about
40.degree. C. for about 1 week, about 2 weeks, about 3 weeks, about
4 weeks, about 5 weeks, or about 6 weeks. In one embodiment, a
lyophilized formulation of the invention comprises MT103, wherein
less than 5% of said antibody is fragmented as determined by
RP-HPLC upon storage at about 40.degree. C. for about 1 month,
about 2 months, about 3 months, about 4 months, about 5 months, or
about 6 months.
[0404] In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 month, about 2 months, about 3 months, about 4
months, about 5 months, about 6 months, about 7 months, about 8
months, about 9 months, about 10 months, about 11 months, or about
12 months. In one embodiment, a lyophilized formulation of the
invention comprises MT103, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 year, about 2 years, about 3 years, about 4 years,
or about 5 years.
[0405] 5.3. RECONSTITUTED FORMULATIONS
[0406] The present invention further provides reconstituted liquid
formulations of bispecific antibodies which formulations exhibit
stability, low to undetectable levels of antibody fragmentation,
low to undetectable levels of aggregation, very little to no loss
of the biological activities of the bispecific antibodies, and very
little to no loss of antibody through absorption to contact
surfaces during preparation, storage and use. In certain
embodiments, a reconstituted liquid formulation of the invention
comprises BiTE.RTM. molecules comprising at least a first and a
second binding site specific for the CD3 and CD19 antigens,
respectively. In specific embodiments, a reconstituted liquid
formulation of the invention comprises MT103 (see, U.S. Pat. No.
7,112,324).
[0407] In certain embodiments, reconstituted liquid formulation of
the invention is prepared from a lyophilized formulation described
herein.
[0408] In one embodiment, a reconstituted liquid formulation of the
invention comprises BiTE.RTM. molecules at the same concentration
as the pre-lyophilized liquid formulation.
[0409] In one embodiment, a reconstituted liquid formulation of the
invention comprises BiTE.RTM. molecules at a higher concentration
than the pre-lyophilized liquid formulation. In specific
embodiments, a reconstituted liquid formulation of the invention
comprises about 2 fold, about 3 fold, about 4 fold, about 5 fold,
about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10
fold, about 15 fold, about 20 fold, about 30 fold, about 40 fold
higher concentration of BiTE.RTM. molecules than the
pre-lyophilized liquid formulation.
[0410] In one embodiment, a reconstituted liquid formulation of the
invention comprises BiTE.RTM. molecules at a lower concentration
than the pre-lyophilized liquid formulation. In specific
embodiments, a reconstituted liquid formulation of the invention
comprises about 2 fold, about 3 fold, about 4 fold, about 5 fold,
about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10
fold, about 15 fold, about 20 fold, about 30 fold, about 40 fold
lower concentration of BiTE.RTM. molecules than the pre-lyophilized
liquid formulation.
[0411] In one embodiment, a reconstituted liquid formulation of the
invention comprises MT103 at the same concentration as the
pre-lyophilized liquid formulation.
[0412] In one embodiment, a reconstituted liquid formulation of the
invention comprises MT103 at a higher concentration than the
pre-lyophilized liquid formulation. In specific embodiments, a
reconstituted liquid formulation of the invention comprises about 2
fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about
7 fold, about 8 fold, about 9 fold, about 10 fold, about 15 fold,
about 20 fold, about 30 fold, about 40 fold higher concentration of
MT103 than the pre-lyophilized liquid formulation.
[0413] In one embodiment, a reconstituted liquid formulation of the
invention comprises MT103 at a lower concentration than the
pre-lyophilized liquid formulation. In specific embodiments, a
reconstituted liquid formulation of the invention comprises about 2
fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about
7 fold, about 8 fold, about 9 fold, about 10 fold, about 15 fold,
about 20 fold, about 30 fold, about 40 fold lower concentration of
MT103 than the pre-lyophilized liquid formulation.
[0414] In one embodiment, a reconstituted liquid formulation of the
invention is an aqueous formulation. In a specific embodiment, a
reconstituted liquid formulation of the invention is an aqueous
formulation wherein the aqueous carrier is distilled water.
[0415] In one embodiment, a reconstituted formulation of the
invention is sterile.
[0416] In one embodiment, a reconstituted formulation of the
invention is homogeneous.
[0417] In one embodiment, a reconstituted formulation of the
invention is isotonic. In one embodiment, a reconstituted
formulation of the invention is hypotonic. In one embodiment, a
reconstituted formulation of the invention is hypertonic.
[0418] In one embodiment, a reconstituted liquid formulation of the
invention comprises BiTE.RTM. molecules. In one embodiment, a
reconstituted liquid formulation of the invention comprises
BiTE.RTM. molecules that comprises at least a first and a second
binding site specific for the CD3 and CD19 antigens, respectively.
In a further embodiment, a reconstituted liquid formulation of the
invention comprises BiTE.RTM. molecules wherein said antibody
comprises an anti-CD19 VL domain having the amino acid sequence of
SEQ ID NO:1, an anti-CD19 VH domain having the amino acid sequence
of SEQ ID NO:2, an anti-CD3 VH domain having the amino acid
sequence of SEQ ID NO:3 and an anti-CD3 VL domain having the amino
acid sequence of SEQ ID NO:4. In a specific embodiment, a
reconstituted liquid formulation of the invention comprises MT103,
wherein said antibody comprises the amino acid sequence of SEQ ID
NO:5.
[0419] The invention encompasses reconstituted liquid formulations
comprising a single BiTE.RTM. molecule of interest, for example,
BiTE.RTM. molecules that comprises at least a first and a second
binding site specific for the CD3 and CD19 antigens, respectively.
The invention also encompasses reconstituted liquid formulations
comprising two or more bispecific antibodies of interest, for
example, bispecific antibodies that comprise at least a first and a
second binding site specific for the CD3 and CD19 antigens,
respectively. In a specific embodiment, a reconstituted liquid
formulation of the invention comprises MT103. In another
embodiment, a reconstituted liquid formulation of the invention
comprises two or more bispecific antibodies, wherein one of the
bispecific antibodies is MT103.
[0420] In one embodiment, a reconstituted liquid formulation of the
invention comprises at least one excipient selected from the group
consisting of citrate, lysine HCl, trehalose, and Polysorbate
80.
[0421] In one embodiment, a lyophilized formulation of the
invention comprises citrate, lysine HCl, trehalose and Polysorbate
80. In one embodiment, a lyophilized formulation of the invention
comprises citrate, lysine HCl, and trehalose at a molar ratio of
between about 1 and about 5 mole citrate to between about 4 and
about 40 mole trehalose to between about 5 and about 20 mole lysine
HCl. In a specific embodiment, a lyophilized formulation of the
invention comprises citrate, lysine HCl, and trehalose at a molar
ratio of about 1 mole citrate to about 16 mole trehalose to about 8
mole lysine HCl.
[0422] In a specific embodiment, a lyophilized formulation of the
invention comprises MT103, citrate, lysine HCl, trehalose and
Polysorbate 80.
[0423] In a specific embodiment, a lyophilized formulation of the
invention consists of MT103, citrate, lysine HCl, trehalose and
Polysorbate 80.
[0424] In one embodiment, a reconstituted liquid formulation of the
invention comprises citrate, lysine HCl, trehalose and Polysorbate
80. In one embodiment, a reconstituted liquid formulation of the
invention comprises between about 5 mM and about 125 mM citrate,
between about 25 mM and about 400 mM lysine HCl, between about 3%
and about 50% trehalose, and between about 0.001% and about 1%
Polysorbate 80, wherein said formulation has a pH of between about
4.0 and about 8.0. In another embodiment, a reconstituted liquid
formulation of the invention comprises between about 10 mM and
about 50 mM citrate, between about 100 mM and about 300 mM lysine
HCl, between about 10% and about 20% trehalose, and between about
0.01% and about 0.2% Polysorbate 80, wherein said formulation has a
pH of between about 5.0 and about 8.0. In a further embodiment, a
reconstituted liquid formulation of the invention comprises about
25 mM citrate, about 200 mM lysine HCl, about 15% trehalose and
about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0.
[0425] In one embodiment, a reconstituted liquid formulation of the
invention comprises between about 10 micrograms/ml and about 100
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In another embodiment, a
reconstituted liquid formulation of the invention comprises between
about 50 micrograms/ml and about 60 micrograms/ml BiTE.RTM., about
25 mM citrate, about 200 mM lysine HCl, about 15% trehalose and
about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises between about 110 micrograms/ml and about
210 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In another
embodiment, a reconstituted liquid formulation of the invention
comprises between about 150 micrograms/ml and about 170
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In a further embodiment, a
reconstituted liquid formulation of the invention comprises about
55 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In another
embodiment, a reconstituted liquid formulation of the invention
comprises about 160 micrograms/ml BiTE.RTM., about 25 mM citrate,
about 200 mM lysine HCl, about 15% trehalose and about 0.1%
Polysorbate 80, wherein said formulation has a pH of about 7.0. In
another embodiment, a reconstituted liquid formulation of the
invention comprises about 150 micrograms/ml BiTE.RTM., about 30 mM
citrate, about 75 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80 and has a pH of about 7.0. In another
embodiment, a reconstituted liquid formulation of the invention
comprises about 150 micrograms/ml BiTE.RTM., about 30 mM citrate,
about 75 mM lysine HCl, about 6% trehalose and about 0.02%
Polysorbate 80 and has a pH of about 7.0.
[0426] In one embodiment, a reconstituted liquid formulation of the
invention comprises between about 10 micrograms/ml and about 100
micrograms/ml MT103, about 25 mM citrate, about 200 mM lysine HCl,
about 15% trehalose and about 0.1% Polysorbate 80, wherein said
formulation has a pH of about 7.0. In another embodiment, a
reconstituted liquid formulation of the invention comprises between
about 50 micrograms/ml and about 60 micrograms/ml MT103, about 25
mM citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises between about 110 micrograms/ml and about 210
micrograms/ml MT103, about 25 mM citrate, about 200 mM lysine HCl,
about 15% trehalose and about 0.1% Polysorbate 80, wherein said
formulation has a pH of about 7.0. In another embodiment, a
reconstituted liquid formulation of the invention comprises between
about 150 micrograms/ml and about 170 micrograms/ml MT103, about 25
mM citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In a further embodiment, a reconstituted liquid formulation of
the invention comprises about 55 micrograms/ml MT103, about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In another embodiment, a reconstituted liquid formulation of
the invention comprises about 160 micrograms/ml MT103, about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In another embodiment, a reconstituted liquid formulation of
the invention comprises about 150 micrograms/ml MT103, about 30 mM
citrate, about 75 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80 and has a pH of about 7.0. In another
embodiment, a reconstituted liquid formulation of the invention
comprises about 150 micrograms/ml MT103, about 30 mM citrate, about
75 mM lysine HCl, about 6% trehalose and about 0.02% Polysorbate 80
and has a pH of about 7.0.
[0427] In one embodiment, a reconstituted liquid formulation of the
invention consists of between about 10 micrograms/ml and about 100
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In another embodiment, a
reconstituted liquid formulation of the invention consists of
between about 50 micrograms/ml and about 60 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a reconstituted liquid
formulation of the invention consists of between about 110
micrograms/ml and about 210 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In another embodiment, a reconstituted liquid formulation of
the invention consists of between about 150 micrograms/ml and about
170 micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM
lysine HCl, about 15% trehalose and about 0.1% Polysorbate 80,
wherein said formulation has a pH of about 7.0. In a further
embodiment, a reconstituted liquid formulation of the invention
consists of about 55 micrograms/ml BiTE.RTM., about 25 mM citrate,
about 200 mM lysine HCl, about 15% trehalose and about 0.1%
Polysorbate 80, wherein said formulation has a pH of about 7.0. In
another embodiment, a reconstituted liquid formulation of the
invention consists of about 160 micrograms/ml BiTE.RTM., about 25
mM citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In another embodiment, a reconstituted liquid formulation of
the invention consists of about 150 micrograms/ml BiTE.RTM., about
30 mM citrate, about 75 mM lysine HCl, about 6.5% trehalose and
about 0.02% Polysorbate 80 and has a pH of about 7.0. In another
embodiment, a reconstituted liquid formulation of the invention
consisits of about 150 micrograms/ml BiTE.RTM., about 30 mM
citrate, about 75 mM lysine HCl, about 6% trehalose and about 0.02%
Polysorbate 80 and has a pH of about 7.0.
[0428] In one embodiment, a reconstituted liquid formulation of the
invention consists of between about 10 micrograms/ml and about 100
micrograms/ml MT103, about 25 mM citrate, about 200 mM lysine HCl,
about 15% trehalose and about 0.1% Polysorbate 80, wherein said
formulation has a pH of about 7.0. In another embodiment, a
reconstituted liquid formulation of the invention consists of
between about 50 micrograms/ml and about 60 micrograms/ml MT103,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention consists of between about 110 micrograms/ml and about
210 micrograms/ml MT103, about 25 mM citrate, about 200 mM lysine
HCl, about 15% trehalose and about 0.1% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In another embodiment, a
reconstituted liquid formulation of the invention consists of
between about 150 micrograms/ml and about 170 micrograms/ml MT103,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In a further embodiment, a reconstituted liquid
formulation of the invention consists of about 55 micrograms/ml
MT103, about 25 mM citrate, about 200 mM lysine HCl, about 15%
trehalose and about 0.1% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In another embodiment, a reconstituted
liquid formulation of the invention consists of about 160
micrograms/ml MT103, about 25 mM citrate, about 200 mM lysine HCl,
about 15% trehalose and about 0.1% Polysorbate 80, wherein said
formulation has a pH of about 7.0. In another embodiment, a
reconstituted liquid formulation of the invention consists of about
150 micrograms/ml MT103, about 30 mM citrate, about 75 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80 and has a
pH of about 7.0. In another embodiment, a reconstituted liquid
formulation of the invention consisits of about 150 micrograms/ml
MT103, about 30 mM citrate, about 75 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80 and has a pH of about
7.0.
[0429] In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 10 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 20 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 30 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 40 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 50 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 55 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 60 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 70 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 80 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 90 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 100 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 110 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 120 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 130 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 140 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 150 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 160 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 170 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 180 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 190 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0. In one embodiment, a reconstituted liquid formulation of
the invention comprises at least about 200 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 15% trehalose
and about 0.1% Polysorbate 80, wherein said formulation has a pH of
about 7.0.
[0430] In one embodiment, a reconstituted liquid formulation of the
invention comprises about 10 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 20 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 30 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 40 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 50 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 55 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 60 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 70 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 80 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 90 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 100 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 110 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 120 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 130 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 140 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 150 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 160 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 170 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 180 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 190 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 200 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 15% trehalose and about
0.1% Polysorbate 80, wherein said formulation has a pH of about
7.0.
[0431] In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 10 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 6.5% trehalose
and about 0.02% Polysorbate 80, wherein said formulation has a pH
of about 7.0. In one embodiment, a reconstituted liquid formulation
of the invention comprises at least about 20 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a reconstituted liquid
formulation of the invention comprises at least about 30
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
reconstituted liquid formulation of the invention comprises at
least about 40 micrograms/ml BiTE.RTM., about 25 mM citrate, about
200 mM lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate
80, wherein said formulation has a pH of about 7.0. In one
embodiment, a reconstituted liquid formulation of the invention
comprises at least about 50 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 55 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 6.5% trehalose
and about 0.02% Polysorbate 80, wherein said formulation has a pH
of about 7.0. In one embodiment, a reconstituted liquid formulation
of the invention comprises at least about 60 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a reconstituted liquid
formulation of the invention comprises at least about 70
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
reconstituted liquid formulation of the invention comprises at
least about 80 micrograms/ml BiTE.RTM., about 25 mM citrate, about
200 mM lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate
80, wherein said formulation has a pH of about 7.0. In one
embodiment, a reconstituted liquid formulation of the invention
comprises at least about 90 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 100 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 6.5% trehalose
and about 0.02% Polysorbate 80, wherein said formulation has a pH
of about 7.0. In one embodiment, a reconstituted liquid formulation
of the invention comprises at least about 110 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a reconstituted liquid
formulation of the invention comprises at least about 120
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
reconstituted liquid formulation of the invention comprises at
least about 130 micrograms/ml BiTE.RTM., about 25 mM citrate, about
200 mM lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate
80, wherein said formulation has a pH of about 7.0. In one
embodiment, a reconstituted liquid formulation of the invention
comprises at least about 140 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 150 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 6.5% trehalose
and about 0.02% Polysorbate 80, wherein said formulation has a pH
of about 7.0. In one embodiment, a reconstituted liquid formulation
of the invention comprises at least about 160 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6.5%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a reconstituted liquid
formulation of the invention comprises at least about 170
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6.5% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
reconstituted liquid formulation of the invention comprises at
least about 180 micrograms/ml BiTE.RTM., about 25 mM citrate, about
200 mM lysine HCl, about 6.5% trehalose and about 0.02% Polysorbate
80, wherein said formulation has a pH of about 7.0. In one
embodiment, a reconstituted liquid formulation of the invention
comprises at least about 190 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 200 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 6.5% trehalose
and about 0.02% Polysorbate 80, wherein said formulation has a pH
of about 7.0.
[0432] In one embodiment, a reconstituted liquid formulation of the
invention comprises about 10 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 20 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 30 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 40 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 50 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 55 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 60 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 70 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 80 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 90 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 100 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 110 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 120 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 130 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 140 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 150 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 160 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 170 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 180 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 190 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 200 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6.5% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0.
[0433] In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 10 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 6% trehalose
and about 0.02% Polysorbate 80, wherein said formulation has a pH
of about 7.0. In one embodiment, a reconstituted liquid formulation
of the invention comprises at least about 20 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a reconstituted liquid
formulation of the invention comprises at least about 30
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
reconstituted liquid formulation of the invention comprises at
least about 40 micrograms/ml BiTE.RTM., about 25 mM citrate, about
200 mM lysine HCl, about 6% trehalose and about 0.02% Polysorbate
80, wherein said formulation has a pH of about 7.0. In one
embodiment, a reconstituted liquid formulation of the invention
comprises at least about 50 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 55 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 6% trehalose
and about 0.02% Polysorbate 80, wherein said formulation has a pH
of about 7.0. In one embodiment, a reconstituted liquid formulation
of the invention comprises at least about 60 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a reconstituted liquid
formulation of the invention comprises at least about 70
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
reconstituted liquid formulation of the invention comprises at
least about 80 micrograms/ml BiTE.RTM., about 25 mM citrate, about
200 mM lysine HCl, about 6% trehalose and about 0.02% Polysorbate
80, wherein said formulation has a pH of about 7.0. In one
embodiment, a reconstituted liquid formulation of the invention
comprises at least about 90 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 100 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 6% trehalose
and about 0.02% Polysorbate 80, wherein said formulation has a pH
of about 7.0. In one embodiment, a reconstituted liquid formulation
of the invention comprises at least about 110 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a reconstituted liquid
formulation of the invention comprises at least about 120
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
reconstituted liquid formulation of the invention comprises at
least about 130 micrograms/ml BiTE.RTM., about 25 mM citrate, about
200 mM lysine HCl, about 6% trehalose and about 0.02% Polysorbate
80, wherein said formulation has a pH of about 7.0. In one
embodiment, a reconstituted liquid formulation of the invention
comprises at least about 140 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 150 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 6% trehalose
and about 0.02% Polysorbate 80, wherein said formulation has a pH
of about 7.0. In one embodiment, a reconstituted liquid formulation
of the invention comprises at least about 160 micrograms/ml
BiTE.RTM., about 25 mM citrate, about 200 mM lysine HCl, about 6%
trehalose and about 0.02% Polysorbate 80, wherein said formulation
has a pH of about 7.0. In one embodiment, a reconstituted liquid
formulation of the invention comprises at least about 170
micrograms/ml BiTE.RTM., about 25 mM citrate, about 200 mM lysine
HCl, about 6% trehalose and about 0.02% Polysorbate 80, wherein
said formulation has a pH of about 7.0. In one embodiment, a
reconstituted liquid formulation of the invention comprises at
least about 180 micrograms/ml BiTE.RTM., about 25 mM citrate, about
200 mM lysine HCl, about 6% trehalose and about 0.02% Polysorbate
80, wherein said formulation has a pH of about 7.0. In one
embodiment, a reconstituted liquid formulation of the invention
comprises at least about 190 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises at least about 200 micrograms/ml BiTE.RTM.,
about 25 mM citrate, about 200 mM lysine HCl, about 6% trehalose
and about 0.02% Polysorbate 80, wherein said formulation has a pH
of about 7.0.
[0434] In one embodiment, a reconstituted liquid formulation of the
invention comprises about 10 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 20 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 30 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 40 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 50 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 55 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 60 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 70 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 80 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 90 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 100 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 110 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 120 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 130 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 140 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 150 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 160 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 170 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 180 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 190 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0. In one embodiment, a reconstituted liquid formulation of the
invention comprises about 200 micrograms/ml BiTE.RTM., about 25 mM
citrate, about 200 mM lysine HCl, about 6% trehalose and about
0.02% Polysorbate 80, wherein said formulation has a pH of about
7.0.
5.3.1. Stability of Reconstituted Formulations
[0435] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules that is susceptible to
aggregation, fragmentation and/or loss due to absorption to contact
surfaces.
[0436] In one embodiment, a reconstituted formulation of the
invention stabilizes BiTE.RTM. molecules in that the formulation of
the invention prevents aggregation of BiTE.RTM. molecules as
measured by HPSEC. In another embodiment, a reconstituted
formulation of the invention prevents fragmentation of BiTE.RTM.
molecules as measured by RP-HPLC. In a further embodiment, a
reconstituted formulation of the invention prevents loss of
BiTE.RTM. molecules due to absorption to contact surfaces.
[0437] In one embodiment, a reconstituted formulation of the
invention stabilizes MT103 in that the formulation of the invention
prevents aggregation of MT103 as measured by HPSEC. In another
embodiment, a reconstituted formulation of the invention prevents
fragmentation of MT103 as measured by RP=HPLC. In a further
embodiment, a reconstituted formulation of the invention prevents
loss of MT103 due to absorption to contact surfaces.
[0438] In one embodiment, a reconstituted formulation of the
invention is stable upon storage at room temperature for at least
about 1 hour, at least about 2 hours, at least about 3 hours, at
least about 4 hours, at least about 5 hours, at least about 6
hours, at least about 7 hours, at least about 8 hours, at least
about 9 hours, at least about 12 hours, at least about 18 hours, or
at least about 24 hours as measured by HPSEC.
[0439] In one embodiment, a reconstituted formulation of the
invention is stable upon storage at about 4.degree. C. for at least
about 1 hour, at least about 2 hours, at least about 3 hours, at
least about 4 hours, at least about 5 hours, at least about 6
hours, at least about 7 hours, at least about 8 hours, at least
about 9 hours, at least about 12 hours, at least about 15 hours, at
least about 18 hours, or at least about 24 hours as measured by
HPSEC.
[0440] In one embodiment, a reconstituted formulation of the
invention is stable upon storage at room temperature for about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours,
about 6 hours, about 7 hours, about 8 hours, about 9 hours, about
12 hours, about 18 hours, or about 24 hours as measured by
HPSEC.
[0441] In one embodiment, a reconstituted formulation of the
invention is stable upon storage at about 4.degree. C. for about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours,
about 6 hours, about 7 hours, about 8 hours, about 9 hours, about
12 hours, about 15 hours, about 18 hours, or about 24 hours as
measured by HPSEC.
5.3.1.1. Binding Activity
[0442] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein said antibody
retains at least 90% of binding ability to its target polypeptides
compared to a reference antibody representing the antibody prior to
lyophilization. Binding activity can be measured in a number of
ways known to one of skill in the art, for example, by using a
BIAcore or ELISA assay.
[0443] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein said antibody
retains at least 90% of binding ability to its target polypeptides
compared to a reference antibody representing the antibody prior to
storage at room temperature for about 1 hour, about 2 hours, about
3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 18
hours, or about 24 hours.
[0444] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein said antibody
retains at least 90% of binding ability to its target polypeptides
compared to a reference antibody representing the antibody prior to
storage at about 5.degree. C. for about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 15
hours, about 18 hours, or about 24 hours.
[0445] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein said antibody retains at least
90% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to
lyophilization.
[0446] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein said antibody retains at least
90% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to storage at
room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0447] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein said antibody retains at least
90% of binding ability to CD19 and CD3 antigens compared to a
reference antibody representing the antibody prior to storage at
about 5.degree. C. for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
5.3.1.2. Aggregate Formation
[0448] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1%, less
than 2%, less than 3%, less than 4%, or less than 5% of said
antibody forms an aggregate as determined by HPSEC.
[0449] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 1%, less than 2%, less
than 3%, less than 4%, or less than 5% of said antibody forms an
aggregate as determined by HPSEC.
[0450] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody forms an aggregate as determined by HPSEC upon
storage at room temperature for about 1 hour, about 2 hours, about
3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 18
hours, or about 24 hours.
[0451] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 15
hours, about 18 hours, or about 24 hours.
[0452] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms an aggregate as determined by HPSEC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0453] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 hour, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0454] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody forms an aggregate as determined by HPSEC upon
storage at room temperature for about 1 hour, about 2 hours, about
3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 18
hours, or about 24 hours.
[0455] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 15
hours, about 18 hours, or about 24 hours.
[0456] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms an aggregate as determined by HPSEC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0457] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 hour, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0458] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody forms an aggregate as determined by HPSEC upon
storage at room temperature for about 1 hour, about 2 hours, about
3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 18
hours, or about 24 hours.
[0459] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 15
hours, about 18 hours, or about 24 hours.
[0460] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms an aggregate as determined by HPSEC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0461] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 hour, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0462] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody forms an aggregate as determined by HPSEC upon
storage at room temperature for about 1 hour, about 2 hours, about
3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 18
hours, or about 24 hours.
[0463] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 15
hours, about 18 hours, or about 24 hours.
[0464] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms an aggregate as determined by HPSEC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0465] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 hour, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0466] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody forms an aggregate as determined by HPSEC upon
storage at room temperature for about 1 hour, about 2 hours, about
3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 18
hours, or about 24 hours.
[0467] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody forms an aggregate as determined by HPSEC upon
storage at about 5.degree. C. for about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 15
hours, about 18 hours, or about 24 hours.
[0468] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms an aggregate as determined by HPSEC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0469] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms an aggregate as determined by HPSEC upon storage at about
5.degree. C. for about 1 hour, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
5.3.1.3. Dimer Formation
[0470] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1%, less
than 2%, less than 3%, less than 4%, or less than 5% of said
antibody forms a dimer as determined by HPSEC.
[0471] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 1%, less than 2%, less
than 3%, less than 4%, or less than 5% of said antibody forms a
dimer as determined by HPSEC.
[0472] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody forms a dimer as determined by HPSEC upon storage at
room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0473] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 5.degree. C. for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0474] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms a dimer as determined by HPSEC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0475] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 1% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 hour, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0476] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody forms a dimer as determined by HPSEC upon storage at
room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0477] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 5.degree. C. for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0478] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms a dimer as determined by HPSEC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0479] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 2% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 hour, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0480] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody forms a dimer as determined by HPSEC upon storage at
room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0481] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 5.degree. C. for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0482] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms a dimer as determined by HPSEC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0483] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 3% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 hour, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0484] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody forms a dimer as determined by HPSEC upon storage at
room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0485] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 5.degree. C. for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0486] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms a dimer as determined by HPSEC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0487] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 4% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 hour, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0488] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody forms a dimer as determined by HPSEC upon storage at
room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0489] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody forms a dimer as determined by HPSEC upon storage at
about 5.degree. C. for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
[0490] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms a dimer as determined by HPSEC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0491] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 5% of said antibody
forms a dimer as determined by HPSEC upon storage at about
5.degree. C. for about 1 hour, about 2 hours, about 3 hours, about
4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 15 hours, about 18
hours, or about 24 hours.
5.3.1.4. Fragmentation
[0492] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1%, less
than 2%, less than 3%, less than 4%, or less than 5% of said
antibody is fragmented as determined by RP-HPLC.
[0493] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 1%, less than 2%, less
than 3%, less than 4%, or less than 5% of said antibody is
fragmented as determined by RP-HPLC.
[0494] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody is fragmented as determined by RP-HPLC upon storage
at room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0495] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 1% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 5.degree. C. for about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 12 hours, about 15 hours, about
18 hours, or about 24 hours.
[0496] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 1% of said antibody is
fragmented as determined by RP-HPLC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0497] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 1% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9
hours, about 12 hours, about 15 hours, about 18 hours, or about 24
hours.
[0498] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody is fragmented as determined by RP-HPLC upon storage
at room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0499] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 2% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 5.degree. C. for about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 12 hours, about 15 hours, about
18 hours, or about 24 hours.
[0500] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 2% of said antibody is
fragmented as determined by RP-HPLC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0501] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 2% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9
hours, about 12 hours, about 15 hours, about 18 hours, or about 24
hours.
[0502] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody is fragmented as determined by RP-HPLC upon storage
at room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0503] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 3% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 5.degree. C. for about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 12 hours, about 15 hours, about
18 hours, or about 24 hours.
[0504] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 3% of said antibody is
fragmented as determined by RP-HPLC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0505] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 3% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9
hours, about 12 hours, about 15 hours, about 18 hours, or about 24
hours.
[0506] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody is fragmented as determined by RP-HPLC upon storage
at room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0507] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 4% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 5.degree. C. for about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 12 hours, about 15 hours, about
18 hours, or about 24 hours.
[0508] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 4% of said antibody is
fragmented as determined by RP-HPLC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0509] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 4% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9
hours, about 12 hours, about 15 hours, about 18 hours, or about 24
hours.
[0510] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody is fragmented as determined by RP-HPLC upon storage
at room temperature for about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0511] In one embodiment, a reconstituted formulation of the
invention comprises BiTE.RTM. molecules, wherein less than 5% of
said antibody is fragmented as determined by RP-HPLC upon storage
at about 5.degree. C. for about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 12 hours, about 15 hours, about
18 hours, or about 24 hours.
[0512] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at room
temperature for about 1 hour, about 2 hours, about 3 hours, about 4
hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours,
about 9 hours, about 12 hours, about 18 hours, or about 24
hours.
[0513] In one embodiment, a reconstituted formulation of the
invention comprises MT103, wherein less than 5% of said antibody is
fragmented as determined by RP-HPLC upon storage at about 5.degree.
C. for about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9
hours, about 12 hours, about 15 hours, about 18 hours, or about 24
hours.
5.3.1.5. Visual Characteristics
[0514] In one embodiment, a reconstituted formulation of the
invention is clear and colorless as determined by visual
inspection.
[0515] In one embodiment, a reconstituted formulation of the
invention is clear and colorless as determined by visual inspection
upon storage at room temperature for about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 18
hours, or about 24 hours.
[0516] In one embodiment, a reconstituted formulation of the
invention is clear and colorless as determined by visual inspection
upon storage at about 5.degree. C. for about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 12 hours, about 15
hours, about 18 hours, or about 24 hours.
[0517] In one embodiment, a reconstituted formulation of the
invention is clear and colorless as determined by visual inspection
upon storage at room temperature for at least about 1 hour, at
least about 2 hours, at least about 3 hours, at least about 4
hours, at least about 5 hours, at least about 6 hours, at least
about 7 hours, at least about 8 hours, at least about 9 hours, at
least about 12 hours, at least about 18 hours, or at least about 24
hours.
[0518] In one embodiment, a reconstituted formulation of the
invention is clear and colorless as determined by visual inspection
upon storage at about 5.degree. C. for at least about 1 hour, at
least about 2 hours, at least about 3 hours, at least about 4
hours, at least about 5 hours, at least about 6 hours, at least
about 7 hours, at least about 8 hours, at least about 9 hours, at
least about 12 hours, at least about 15 hours, at least about 18
hours, or at least about 24 hours.
5.3.1.6. Particle Profile
[0519] In certain embodiments, reconstituted formulations of the
invention comprise (or consists of as the aggregate fraction) a
particle profile of less than about 3.4 E +5 particles/ml of
diameter 2-4 .mu.m, less than about 4.0 E +4 particles/ml of
diameter 4-10 .mu.m, less than about 4.2 E +3 particles/ml of
diameter 10-20 .mu.m, less than about 5.0 E +2 particles/ml of
diameter 20-30 .mu.m, less than about 7.5 E +1 particles/ml of
diameter 30-40 .mu.m, and less than about 9.4 particles/ml of
diameter 40-60 .mu.m as determined by a particle multisizer. In
certain embodiments, reconstituted formulations of the invention
contain no detectable particles greater than 40 .mu.m, or greater
than 30 .mu.m.
[0520] In certain embodiments, after storage for about 1 hour,
about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6
hours, about 7 hours, about 8 hours, about 9 hours, about 12 hours,
about 15 hours, about 18 hours, or about 24 hours liquid
formulations of the invention comprise (or consists of as the
aggregate fraction) a particle profile of less than about 3.4 E +5
particles/ml of diameter 2-4 .mu.m, less than about 4.0 E +4
particles/ml of diameter 4-10 .mu.m, less than about 4.2 E +3
particles/ml of diameter 10-20 .mu.m, less than about 5.0 E +2
particles/ml of diameter 20-30 .mu.m, less than about 7.5 E +1
particles/ml of diameter 30-40 .mu.m, and less than about 9.4
particles/ml of diameter 40-60 .mu.m as determined by a particle
multisizer. In certain embodiments, liquid formulations of the
invention contain no detectable particles greater than 40 .mu.m, or
greater than 30 .mu.m.
[0521] Numerous methods useful for determining the degree of
aggregation, and/or types and/or sizes of aggregates present in a
protein formulation (e.g., antibody formulation of the invention)
are known in the art, including but not limited to, size exclusion
chromatography (SEC), high performance size exclusion
chromatography (HPSEC), static light scattering (SLS), Fourier
Transform Infrared Spectroscopy (FTIR), circular dichroism (CD),
urea-induced protein unfolding techniques, intrinsic tryptophan
fluorescence, differential scanning calorimetry, and
1-anilino-8-naphthalenesulfonic acid (ANS) protein binding
techniques. For example, size exclusion chromatography (SEC) may be
performed to separate molecules on the basis of their size, by
passing the molecules over a column packed with the appropriate
resin, the larger molecules (e.g. aggregates) will elute before
smaller molecules (e.g. monomers). The molecules are generally
detected by UV absorbance at 280 nm and may be collected for
further characterization. High pressure liquid chromatographic
columns are often utilized for SEC analysis (HP-SEC). Specific SEC
methods are detailed in the section entitled "Examples" infra.
Alternatively, analytical ultracentrifugation (AUC) may be
utilized. AUC is an orthogonal technique which determines the
sedimentation coefficients (reported in Svedberg, S) of
macromolecules in a liquid sample. Like SEC, AUC is capable of
separating and detecting antibody fragments/aggregates from
monomers and is further able to provide information on molecular
mass. Protein aggregation in the formulations may also be
characterized by particle counter analysis using a coulter counter
or by turbidity measurements using a turbidimeter. Turbidity is a
measure of the amount by which the particles in a solution scatter
light and, thus, may be used as a general indicator of protein
aggregation. In addition, non-reducing polyacrylamide gel
electrophoresis (PAGE) or capillary gel electrophoresis (CGE) may
be used to characterize the aggregation and/or fragmentation state
of antibodies or a fragment thereof in a formulation of the
invention.
5.4. Antibodies Useful in the Formulations of the Invention
[0522] The present invention relates to formulations of BiTE.RTM.
molecules, which formulations exhibit stability, low to
undetectable levels of antibody fragmentation, low to undetectable
levels of aggregation, very little to no loss of the biological
activities of the BiTE.RTM. molecules and very little to no loss of
antibody through absorption to contact surfaces during manufacture,
preparation, storage and use. In certain embodiments, a formulation
of the invention is a lyophilized formulation, which lyophilized
formulation maximizes BiTE.RTM. molecule BiTE.RTM. molecule
recovery from the lyophilized product upon reconstitution.
[0523] The invention further provides methods for preparing
formulations of BiTE.RTM. molecules, which formulations exhibit
stability, low to undetectable levels of antibody fragmentation,
low to undetectable levels of aggregation, very little to no loss
of the biological activities of the BiTE.RTM. molecules and very
little to no loss of antibody through absorption to contact
surfaces during manufacture, preparation, storage and use.
[0524] The present invention provides formulations comprising
BiTE.RTM. molecules.
[0525] Bispecific T cell engagers, or BiTE.RTM.s.TM., are a form of
bispecific antibodies that are based on tandemly arranged
single-chain antibodies (U.S. Pat. No. 7,112,324; reviewed in Wolf
et al., Drug Discov Today. 10(18):1237-44 (2005)). They form a
single polypeptide chain of approximately 55 kDa and are secreted
by Chinese hamster ovary (CHO) cells as a mixture of monomers and
dimers. With one arm, BiTE.RTM.s.TM. bind to the epsilon (c)
subunit of human CD3, a protein component of the signal-transducing
complex of the T cell receptor on T cells. With the other arm,
BiTE.RTM.s.TM. recognize an antigen on target cells (for example,
but not limited to CD19, CD20, epithelial cell adhesion molecule
(Ep-CAM), carcinoembryonic antigen (CEA), EphA2, EphA4, ICOS, IL-5
receptor, INF receptor). T cell activation is only seen when
BiTE.RTM.s.TM. are presented to T cells on the surface of target
cells.
[0526] BiTE.RTM.s.TM. transiently tether T cells and target cells.
T cell activation by BiTE.RTM.s.TM. involves upregulation of CD69,
CD25 and various cell adhesion molecules, de novo expression and
release of cytokines (e.g., IFN-.gamma., TNF-.alpha., IL-6, IL-2,
IL-4 and IL-10), upregulation of granzyme and perforin expression,
and cell proliferation. Redirected target cell lysis by
BiTE.RTM.s.TM. is independent of T cell receptor specificity,
presence of MHC class I and (32 microglobulin, and of any
co-stimulatory stimuli. This independence from regular T cell
signals and recognition molecules may be explained by the induction
through BiTE.RTM.s.TM. of regular cytolytic synapses and maximum
membrane proximity. Displacement of negative regulatory proteins
such as CD45 from BiTE.RTM..TM.-induced synapses may alleviate the
need for co-stimulation.
[0527] BiTE.RTM.s.TM. show redirected lysis in vitro with
previously unstimulated peripheral polyclonal CD8- and CD4-positive
T cells. No activity is seen with naive CD8- or CD4-positive T
cells. CD4 T cells can upregulate granzyme B and perforin
expression when stimulated with BiTE.RTM.s.TM. and thereby
contribute to CD8-mediated target cell lysis. In vitro, redirected
lysis is seen at low picomolar concentrations, suggesting that very
low numbers of BiTE.RTM. molecules need to be bound to target cells
for triggering T cells. In SCID mouse models, sub-.mu.g doses of
BiTE.RTM.s.TM. have been shown to completely prevent tumor
outgrowth (Dreier et al., 2003, J Immunol. 170:4397-4402) and to
eradicate solid tumors up to 200 mm.sup.3 (Schlereth et al., 2005,
Cancer Res. 2005 65(7):2882-89).
[0528] The invention provides formulations comprising BiTE.RTM.
molecules. BiTE.RTM. molecules comprise a first and second binding
domain wherein said first binding domain is specific for CD3 and
said second binding domain is specific for a cell surface antigen
of a target cell. In particular embodiments, the cell surface
antigen of a target cell recognized by a BiTE.RTM. of a formulation
of the invention is selected from the group consisting of a
cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4;
receptors for hormones or growth factors such as, for example,
EGFR, TGF, VEGFR; interferons such as alpha interferon
(.alpha.-IFN), beta interferon (.beta.-IFN) and gamma interferon
(.gamma.-IFN); interferon receptor components such as interferon
receptor 1; CD proteins such as CD2, CD3, CD4, CD8, CD11a, CD14,
CD18, CD19, CD20, CD22, CD23, CD25, CD33, CD34, CD40, CD40L, CD52,
CD63, CD64, CD80 and CD147; T-cell receptors; surface membrane
proteins; homing receptors; addressins; cell adhesion molecules
such as LFA-1, Mac 1, p150.95, VLA-4, ICAM-1, ICAM-3 and VCAM,
a4/p7 integrin, and (Xv/p3 integrin including either a or subunits
thereof, integrin alpha subunits such as CD49a, CD49b, CD49c,
CD49d, CD49e, CD49f, alpha7, alpha8, alpha9, alphaD, CD11a, CD11b,
CD51, CD11c, CD41, alphaIIb, alphaIELb; integrin beta subunits such
as, CD29, CD18, CD61, CD104, beta5, beta6, beta7 and beta8;
Integrin subunit combinations including but not limited to,
.alpha.V.beta.3, .alpha.V.beta.5 and .alpha.4.beta.7; flk2/flt3
receptor; obesity (OB) receptor; mpl receptor; CTLA-4; an Eph
receptor such as EphA2, EphA4, EphB2, etc.; a Human Leukocyte
Antigen (HLA) such as HLA-DR; a glycoprotein receptor such as
GpIb.alpha., GPIIb/IIIa and CD200; cancer antigens including, but
not limited to, ALK receptor (pleiotrophin receptor), pleiotrophin,
KS 1/4 pan-carcinoma antigen; ovarian carcinoma antigen (CAl25);
prostatic acid phosphate; prostate specific antigen (PSA);
melanoma-associated antigen p97; melanoma antigen gp75; high
molecular weight melanoma antigen (HMW-MAA); prostate specific
membrane antigen; carcinoembryonic antigen (CEA); polymorphic
epithelial mucin antigen; human milk fat globule antigen;
colorectal tumor-associated antigens such as: CEA, TAG-72, CO17-1A,
GICA 19-9, CTA-1 and LEA; Burkitt's lymphoma antigen-38.13; CD19;
human B-lymphoma antigen-CD20; CD33; melanoma specific antigens
such as ganglioside GD2, ganglioside G.sub.D3, ganglioside G.sub.M2
and ganglioside GM3; tumor-specific transplantation type
cell-surface antigen (TSTA); virally-induced tumor antigens
including T-antigen, DNA tumor viruses and Envelope antigens of RNA
tumor viruses; oncofetal antigen-alpha-fetoprotein such as CEA of
colon, 5T4 oncofetal trophoblast glycoprotein and bladder tumor
oncofetal antigen; differentiation antigen such as human lung
carcinoma antigens L6 and L20; antigens of fibrosarcoma; human
leukemia T cell antigen-Gp37; neoglycoprotein; sphingolipids;
breast cancer antigens such as EGFR (Epidermal growth factor
receptor); NY-BR-16; NY-BR-16 and HER2 antigen (p185.sup.HER2);
polymorphic epithelial mucin (PEM); malignant human lymphocyte
antigen-APO-1; differentiation antigen such as I antigen found in
fetal erythrocytes; primary endoderm I antigen found in adult
erythrocytes; preimplantation embryos; I(Ma) found in gastric
adenocarcinomas; M18, M39 found in breast epithelium; SSEA-1 found
in myeloid cells; VEP8; VEP9; Myl; VIM-D5; D156-22 found in
colorectal cancer; TRA-1-85 (blood group H); SCP-1 found in testis
and ovarian cancer; C14 found in colonic adenocarcinoma; F3 found
in lung adenocarcinoma; AH6 found in gastric cancer; Y hapten;
Le.sup.y found in embryonal carcinoma cells; TL5 (blood group A);
EGF receptor found in A431 cells; E.sub.1 series (blood group B)
found in pancreatic cancer; FC10.2 found in embryonal carcinoma
cells; gastric adenocarcinoma antigen; CO-514 (blood group
Le.sup.a) found in Adenocarcinoma; NS-10 found in adenocarcinomas;
CO-43 (blood group Le.sup.b); G49 found in EGF receptor of A431
cells; MH2 (blood group ALe.sup.b/Le.sup.y) found in colonic
adenocarcinoma; 19.9 found in colon cancer; gastric cancer mucins;
T.sub.5A.sub.7 found in myeloid cells; R.sub.24 found in melanoma;
4.2, G.sub.D3, D1.1, OFA-1, G.sub.M2, OFA-2, GD2, and M1:22:25:8
found in embryonal carcinoma cells and SSEA-3 and SSEA-4 found in 4
to 8-cell stage embryos; Cutaneous Tcell Lymphoma antigen; MART-1
antigen; Sialy Tn (STn) antigen; Colon cancer antigen NY-CO-45;
Lung cancer antigen NY-LU-12 variant A; Adenocarcinoma antigen
ART1; Paraneoplastic associated brain-testis-cancer antigen
(onconeuronal antigen MA2; paraneoplastic neuronal antigen);
Neuro-oncological ventral antigen 2 (NOVA2); Hepatocellular
carcinoma antigen gene 520; TUMOR-ASSOCIATED ANTIGEN CO-029;
Tumor-associated antigens MAGE-C1 (cancer/testis antigen CT7),
MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b
and MAGE-X2; Cancer-Testis Antigen (NY-EOS-1); YKL-40 and fragments
of any of the above-listed polypeptides.
[0529] In specific embodiments, a BiTE.RTM. molecule BiTE.RTM.
molecule of a formulation of the invention is selected from the
group consisting of: CD19xCD3 BiTE.RTM. (U.S. Pat. No. 7,112,324,
US Patent Application US20070123479A1), CD20xCD3 BiTE.RTM.,
EphA2xCD3 BiTE.RTM. (U.S. patent application Ser. No. 11/645,290,
filed on Dec. 21, 2006), EphA4xCD3 BiTE.RTM., ICOSxCD3 BiTE.RTM.,
IFNR(interferon alpha receptor)xCD3 BiTE.RTM., Ep-CAM (epithelial
cell adhesion molecule)xCD3 BiTE.RTM., CEA (carcinoembryonic
antigen)xCD3 BiTE.RTM. (U.S. patent application Ser. No.
12/158,611, PCT Publication No. WO 2007/071426) and IL-5RxCD3
BiTE.RTM..
5.4.1. BiTE.RTM. Molecules Having Increased Half-Lives
[0530] The present invention provides for formulations of BiTE.RTM.
molecules (e.g., MT103) which have an extended half-life in vivo.
In particular, the present invention provides formulations of
BiTE.RTM. molecules which have a half-life in a mammal (for
example, but not limited to, a human), of greater than 1 hour,
greater than 2 hours, greater than 4 hours, greater than 6 hours,
greater than 8 hours, greater than 10 hours, greater than 12 hours,
greater than 18 hours, greater than 1 day, greater than 2 days,
greater than 3 days, greater than 7 days, greater than 10 days,
greater than 15 days, greater than 25 days, greater than 30 days,
greater than 35 days, greater than 40 days, greater than 45 days,
greater than 2 months, greater than 3 months, greater than 4
months, or greater than 5 months.
[0531] To prolong the serum circulation of BiTE.RTM. molecules in
vivo, for example, inert polymer molecules such as high molecular
weight polyethyleneglycol (PEG) can be attached to the BiTE.RTM.
molecules with or without a multifunctional linker either through
site-specific conjugation of the PEG to the N- or C-terminus of the
antibodies or via epsilon-amino groups present on lysine residues.
Linear or branched polymer derivatization that results in minimal
loss of biological activity will be used. The degree of conjugation
can be closely monitored by SDS-PAGE and mass spectrometry to
ensure proper conjugation of PEG molecules to the BiTE.RTM.
molecules. Unreacted PEG can be separated from BiTE.RTM.
molecule-PEG conjugates by size-exclusion or by ion-exchange
chromatography. PEG-derivatized BiTE.RTM. molecules can be tested
for binding activity as well as for in vivo efficacy using methods
known to those of skill in the art, for example, by immunoassays
described herein.
[0532] Further, BiTE.RTM. molecules can be conjugated to albumin in
order to make the BiTE.RTM. molecules more stable in vivo or have a
longer half life in vivo. The techniques are well known in the art,
see e.g., International Publication Nos. WO 93/15199, WO 93/15200,
and WO 01/77137; and European Patent No. EP 413, 622, all of which
are incorporated herein by reference.
5.5. Methods of Preparing the BiTE.RTM. Molecule Formulations
[0533] The invention further provides a method for preparing a
stable reconstituted formulation comprising reconstituting a
lyophilized mixture of BiTE.RTM. molecules and a lyoprotectant in a
diluent such that the BiTE.RTM. molecule BiTE.RTM. molecule
concentration in the reconstituted formulation is between about 30
micrograms/ml and about 70 micrograms/ml.
[0534] In yet a further embodiment, the invention provides a method
for preparing a formulation comprising the steps of: (a)
lyophilizing a mixture of BiTE.RTM. molecules and a lyoprotectant;
and (b) reconstituting the lyophilized mixture of step (a) in a
diluent such that the reconstituted formulation is stable and has a
protein concentration of between about 30 micrograms/ml and about
70 micrograms/ml.
[0535] The BiTE.RTM. molecule to be formulated is prepared using
techniques which are well established in the art including
synthetic techniques such as recombinant techniques and peptide
synthesis or a combination of these techniques. In certain
embodiments of the invention, the BiTE.RTM. molecule BiTE.RTM.
molecule of choice is MT103.
[0536] After preparation of the BiTE.RTM. molecule BiTE.RTM.
molecule of interest, a "bulk liquid formulation" is produced. The
amount of BiTE.RTM. molecule present in the bulk liquid formulation
is determined taking into account the desired dose volumes, mode(s)
of administration etc. In certain embodiments, a concentration of
between about 20 micrograms/ml and about 300 micrograms/ml
BiTE.RTM. molecule may be used as a starting protein concentration.
The BiTE.RTM. molecule is generally present in solution. For
example, the BiTE.RTM. molecule may be present in a pH-buffered
solution at a pH from about 4-8, and preferably from about 5-7.
Exemplary buffers include histidine, phosphate, Tris, citrate,
succinate and other organic acids. The buffer concentration can be
from about 1 mM to about 20 mM, or from about 3 mM to about 15 mM,
depending, for example, on the buffer and the desired isotonicity
of the formulation (e.g. of the reconstituted formulation).
[0537] The lyoprotectant is added to the pre-lyophilized
formulation. In certain embodiments, the lyoprotectant is a
non-reducing sugar such as sucrose or trehalose. The amount of
lyoprotectant in the pre-lyophilized formulation is generally such
that, upon reconstitution, the resulting formulation will be
isotonic. However, hypertonic reconstituted formulations may also
be suitable. In addition, the amount of lyoprotectant must not be
too low such that an unacceptable amount of degradation/aggregation
of the protein occurs upon lyophilization. Where the lyoprotectant
is a sugar (such as sucrose or trehalose) and the protein is a
BiTE.RTM. molecule, exemplary lyoprotectant concentrations in the
pre-lyophilized formulation are from about 10 mM to about 400 mM.
The ratio of protein to lyoprotectant is selected for each protein
and lyoprotectant combination.
[0538] In certain embodiments, a surfactant is added to the
pre-lyophilized formulation. Alternatively, or in addition, the
surfactant may be added to the lyophilized formulation and/or the
reconstituted formulation. Suitable surfactants include nonionic
surfactants such as polysorbates (e.g. polysorbates 20 or 80);
poloxamers (e.g. poloxamer 188); Triton; sodium dodecyl sulfate
(SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-,
myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-,
linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or
cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,
linoleamidopropyl-, myristamidopropyl-, palnidopropyl-, or
isostearamidopropyl-betaine (e.g. lauroamidopropyl);
myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.), polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc). The amount of surfactant added is such that
it reduces aggregation of the reconstituted protein and minimizes
the formation of particulates after reconstitution. For example,
the surfactant may be present in the pre-lyophilized formulation in
an amount from about 0.001-0.5%, and preferably from about
0.005-0.05%.
[0539] In certain embodiments of the invention, a mixture of the
lyoprotectant (such as sucrose or trehalose) and a bulking agent
(e.g. mannitol or glycine) is used in the preparation of the
pre-lyophilization formulation. The bulking agent may allow for the
production of a uniform lyophilized cake without excessive pockets
therein etc.
[0540] Other pharmaceutically acceptable carriers, excipients or
stabilizers such as those described in Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980) may be included in the
pre-lyophilized formulation (and/or the lyophilized formulation
and/or the reconstituted formulation) provided that they do not
adversely affect the desired characteristics of the formulation.
Acceptable carriers, excipients or stabilizers are nontoxic to
recipients at the dosages and concentrations employed and include;
additional buffering agents; preservatives; co-solvents;
antioxidants including ascorbic acid and methionine; chelating
agents such as EDTA; metal complexes (e.g. Zn-protein complexes);
biodegradable polymers such as polyesters; and/or salt-forming
counterions such as sodium.
[0541] The formulation herein may also contain more than one
BiTE.RTM. molecule as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect the other protein.
[0542] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes, prior to, or following,
lyophilization and reconstitution. Alternatively, sterility of the
entire mixture may be accomplished by autoclaving the ingredients,
except for the BiTE.RTM. molecule, at about 120.degree. C. for
about 30 minutes, for example.
[0543] After the protein, lyoprotectant and other optional
components are mixed together, the formulation is lyophilized. Many
different freeze-dryers are available for this purpose such as
Hull50.TM. (Hull, USA) or GT20.TM. (Leybold-Heraeus, Germany)
freeze-dryers. Freeze-drying is accomplished by freezing the
formulation and subsequently subliming ice from the frozen content
at a temperature suitable for primary drying. Under this condition,
the product temperature is below the eutectic point or the collapse
temperature of the formulation. Typically, the shelf temperature
for the primary drying will range from about -30 to 25.degree. C.
(provided the product remains frozen during primary drying) at a
suitable pressure, ranging typically from about 50 to 250 mTorr.
The formulation, size and type of the container holding the sample
(e.g., glass vial) and the volume of liquid will mainly dictate the
time required for drying, which can range from a few hours to
several days (e.g. 40-60 hrs). A secondary drying stage may be
carried out at about 0-40.degree. C., depending primarily on the
type and size of container and the type of protein employed.
However, it was found herein that a secondary drying step may not
be necessary. For example, the shelf temperature throughout the
entire water removal phase of lyophilization may be from about
15-30.degree. C. (e.g., about 20.degree. C.). The time and pressure
required for secondary drying will be that which produces a
suitable lyophilized cake, dependent, e.g., on the temperature and
other parameters. The secondary drying time is dictated by the
desired residual moisture level in the product and typically takes
at least about 5 hours (e.g. 10-15 hours). The pressure may be the
same as that employed during the primary drying step. Freeze-drying
conditions can be varied depending on the formulation and vial
size.
[0544] In some instances, it may be desirable to lyophilize the
protein formulation in the container in which reconstitution of the
protein is to be carried out in order to avoid a transfer step. The
container in this instance may, for example, be a 3, 5, 10, 20, 50
or 100 cc vial.
[0545] As a general proposition, lyophilization will result in a
lyophilized formulation in which the moisture content thereof is
less than about 5%, and preferably less than about 3%.
[0546] At the desired stage, typically when it is time to
administer the BiTE.RTM. molecule to the patient, the lyophilized
formulation may be reconstituted with a diluent such that the
BiTE.RTM. molecule concentration in the reconstituted formulation
is between about 20 micrograms/ml and about 300 micrograms/ml. High
BiTE.RTM. molecule concentrations in the reconstituted formulation
are considered to be particularly useful where subcutaneous
delivery of the reconstituted formulation is intended. However, for
other routes of administration, such as intravenous administration,
lower concentrations of the BiTE.RTM. molecule in the reconstituted
formulation may be desired. In certain embodiments, the protein
concentration in the reconstituted formulation is significantly
higher than that in the pre-lyophilized formulation.
[0547] Reconstitution generally takes place at a temperature of
about 25.degree. C. to ensure complete hydration, although other
temperatures may be employed as desired. The time required for
reconstitution will depend, e.g., on the type of diluent, amount of
excipient(s) and protein. In certain embodiments diluents include
sterile water, bacteriostatic water for injection (BWFI), a pH
buffered solution (e.g. phosphate-buffered saline), sterile saline
solution, Ringer's solution or dextrose solution. The diluent
optionally contains a preservative. Examples of preservatives have
been described above, with aromatic alcohols such as benzyl or
phenol alcohol being the preferred preservatives. The amount of
preservative employed is determined by assessing different
preservative concentrations for compatibility with the protein and
preservative efficacy testing. For example, if the preservative is
an aromatic alcohol (such as benzyl alcohol), it can be present in
an amount from about 0.1-2.0% and preferably from about 0.5-1.5%,
but most preferably about 1.0-1.2%.
[0548] Preferably, the reconstituted formulation has less than 6000
particles per vial which are >10 micron size.
5.6. Methods of Preparing BiTE.RTM. Molecules
[0549] Generating BiTE.RTM. molecules that specifically bind to an
antigen have been described elsewhere--see Brishwein K et. al.,
Mol. Immunol. 2006 43:1129; Drier T et. al., Int. J. Cancer 2002
100:690; U.S. Pat. No. 7,112,324, each of which is incorporated by
reference in their entireties.
[0550] Antibodies, including antibody fragments thereof, can be
produced by any method known in the art for the synthesis of
antibodies, in particular, by chemical synthesis or by recombinant
expression techniques (see, US Patent Publication
2007/0014724A1).
[0551] Polyclonal antibodies specific for an antigen can be
produced by various procedures well-known in the art. For example,
a human antigen can be administered to various host animals
including, but not limited to, rabbits, mice, rats, etc. to induce
the production of sera containing polyclonal antibodies specific
for the human antigen. Various adjuvants may be used to increase
the immunological response, depending on the host species, and
include but are not limited to, Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and corynebacterium parvum. Such adjuvants are
also well known in the art.
[0552] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N. Y., 1981), and Harlow et al., Using Antibodies: A
laboratory Manual, Cold Spring Harbor Laboratory Press (1999) (said
references incorporated by reference in their entireties). The term
"monoclonal antibody" as used herein is not limited to antibodies
produced through hybridoma technology. The term "monoclonal
antibody" refers to an antibody that is derived from a single
clone, including any eukaryotic, prokaryotic, or phage clone, and
not the method by which it is produced.
[0553] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
Briefly, mice can be immunized with a non-murine antigen and once
an immune response is detected, e.g., antibodies specific for the
antigen are detected in the mouse serum, the mouse spleen is
harvested and splenocytes isolated. The splenocytes are then fused
by well known techniques to any suitable myeloma cells, for example
cells from cell line SP20 available from the ATCC. Hybridomas are
selected and cloned by limited dilution. Additionally, a RIMMS
(repetitive immunization multiple sites) technique can be used to
immunize an animal (Kilpatrack et al., 1997, Hybridoma 16:381-9,
incorporated herein by reference in its entirety). The hybridoma
clones are then assayed by methods known in the art for cells that
secrete antibodies capable of binding a polypeptide of the
invention. Ascites fluid, which generally contains high levels of
antibodies, can be generated by immunizing mice with positive
hybridoma clones.
[0554] The present invention provides methods of generating
monoclonal antibodies as well as antibodies produced by the method
comprising culturing a hybridoma cell secreting an antibody of the
invention wherein the hybridoma is generated by fusing splenocytes
isolated from a mouse immunized with a non-murine antigen with
myeloma cells and then screening the hybridomas resulting from the
fusion for hybridoma clones that secrete an antibody able to bind
to the antigen.
[0555] Antibody fragments which recognize specific particular
epitopes may be generated by any technique known to those of skill
in the art. For example, Fab and F(ab')2 fragments of the invention
may be produced by proteolytic cleavage of immunoglobulin
molecules, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain
the variable region, the light chain constant region and the CH1
domain of the heavy chain. Further, the antibodies of the present
invention can also be generated using various phage display methods
known in the art.
[0556] In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In particular, DNA
sequences encoding VH and VL domains are amplified from animal cDNA
libraries (e.g., human or murine cDNA libraries of affected
tissues). The DNA encoding the VH and VL domains are recombined
together with an scFv linker by PCR and cloned into a phagemid
vector. The vector is electroporated in E. coli and the E. coli is
infected with helper phage. Phage used in these methods are
typically filamentous phage including fd and M13 and the VH and VL
domains are usually recombinantly fused to either the phage gene
III or gene VIII. Phage expressing an antigen binding domain that
binds to a particular antigen can be selected or identified with
antigen, e.g., using labeled antigen or antigen bound or captured
to a solid surface or bead. Examples of phage display methods that
can be used to make the antibodies of the present invention include
those disclosed in Brinkman et al., 1995, J. Immunol. Methods
182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186;
Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et
al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in
Immunology 57:191-280; International application No. PCT/GB91/O1
134; International Publication Nos. WO 90/02809, WO 91/10737, WO
92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and
WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484,
5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908,
5,516,637, 5,780,225, 5,658,727, 5,733,743, 5,969,108, 633,187,
5,824,520, and 5,702,892; each of which is incorporated herein by
reference in its entirety.
[0557] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described below. Techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be
employed using methods known in the art such as those disclosed in
PCT publication No. WO 92/22324; Mullinax et al., 1992,
BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and
Better et al., 1988, Science 240:1041-1043 (said references
incorporated by reference in their entireties).
[0558] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be appropriate to use
humanized antibodies. Completely human antibodies and humanized
antibodies are particularly desirable for therapeutic treatment of
human subjects. Human antibodies can be made by a variety of
methods known in the art including phage display methods described
above using antibody libraries derived from human immunoglobulin
sequences. See also U.S. Pat. Nos. 4,444,887 and 4,716,111; and
International Publication Nos. WO 98/46645, WO 98/50433, WO
98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;
each of which is incorporated herein by reference in its
entirety.
[0559] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then be bred to
produce homozygous offspring which express human antibodies. The
transgenic mice are immunized in the normal fashion with a selected
antigen, e.g., all or a portion of a polypeptide of the invention.
Monoclonal antibodies directed against the antigen can be obtained
from the immunized, transgenic mice using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar
(1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
International Publication Nos. WO 98/24893, WO 96/34096, and WO
96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425,
5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, which
are incorporated by reference herein in their entirety. In
addition, companies such as Abgenix, Inc. (Freemont, Calif.) and
Genpharm (San Jose, Calif.) can be engaged to provide human
antibodies directed against a selected antigen using technology
similar to that described above.
[0560] A humanized antibody is an antibody or its variant or
fragment thereof which is capable of binding to a predetermined
antigen and which comprises a framework region having substantially
the amino acid sequence of a human immunoglobulin and a CDR having
substantially the amino acid sequence of a non-human immuoglobulin.
A humanized antibody comprises substantially all of at least one,
and typically two, variable domains (Fab, Fab', F(ab')2, Fabc, Fv)
in which all or substantially all of the CDR regions correspond to
those of a non-human immunoglobulin (i.e., donor antibody) and all
or substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. In one embodiment, a humanized
antibody also comprises at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin.
Ordinarily, the antibody will contain both the light chain as well
as at least the variable domain of a heavy chain. The antibody also
may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy
chain. The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including IgG.sub.1, IgG.sub.2, IgG.sub.3 and lgG.sub.4.
Usually the constant domain is a complement fixing constant domain
where it is desired that the humanized antibody exhibit cytotoxic
activity, and the class is typically IgG.sub.1. Where such
cytotoxic activity is not desirable, the constant domain may be of
the IgG.sub.2 class. The humanized antibody may comprise sequences
from more than one class or isotype, and selecting particular
constant domains to optimize desired effector functions is within
the ordinary skill in the art. The framework and CDR regions of a
humanized antibody need not correspond precisely to the parental
sequences, e.g., the donor CDR or the consensus framework may be
mutagenized by substitution, insertion or deletion of at least one
residue so that the CDR or framework residue at that site does not
correspond to either the consensus or the import antibody. Such
mutations, however, will not be extensive. Usually, at least 75% of
the humanized antibody residues will correspond to those of the
parental framework and CDR sequences, more often 90%, and greater
than 95%. Humanized antibody can be produced using variety of
techniques known in the art, including but not limited to,
CDR-grafting (European Patent No. EP 239,400; International
publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,
5,530,101, and 5,585,089), veneering or resurfacing (European
Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular
Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein
Engineering 7(6):805-814; and Roguska et al., 1994, PNAS
91:969-973), chain shuffling (U.S. Pat. No. 5,565,332), and
techniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat.
No. 5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119-25
(2002), Caldas et al., Protein Eng. 13(5):353-60 (2000), Morea et
al., Methods 20(3):267-79 (2000), Baca et al., J. Biol. Chem.
272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10):895-904
(1996), Couto et al., Cancer Res. 55 (23 Supp): 5973s-5977s (1995),
Couto et al., Cancer Res. 55(8):1717-22 (1995), Sandhu J S, Gene
150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol.
235(3):959-73 (1994). Often, framework residues in the framework
regions will be substituted with the corresponding residue from the
CDR donor antibody to alter, preferably improve, antigen binding.
These framework substitutions are identified by methods well known
in the art, for example, but not limited to, by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and
Riechmann et al., 1988, Nature 332:323, which are incorporated
herein by reference in their entireties).
[0561] Single domain antibodies, for example, antibodies lacking
the light chains, can be produced by methods well-known in the art.
See Riechmann et al., 1999, J. Immuno. 231:25-38; Nuttall et al.,
2000, Curr. Pharm. Biotechnol. 1(3):253-263; Muylderman, 2001, J.
Biotechnol. 74(4):277302; U.S. Pat. No. 6,005,079; and
International Publication Nos. WO 94/04678, WO 94/25591, and WO
01/44301, each of which is incorporated herein by reference in its
entirety.
5.7. Recombinant Expression of Bispecific Antibodies
[0562] Bispecific antibodies (e.g., BiTE.RTM..TM.) may be produced
by any methods known to one of skill in the art including, but not
limited to, chemical synthesis or recombinant expression techniques
described in U.S. Pat. No. 7,112,324 which is incorporated by
reference herein in its entirety.
[0563] Polynucleotides encoding BiTE.RTM. molecules can be used
alone or as part of a vector to express the BiTE.RTM. molecule in
cells. The polynucleotides or vectors containing the DNA
sequence(s) encoding BiTE.RTM. molecules is introduced into host
cells which in turn produce the polypeptide of interest (e.g.,
MT103). Accordingly, recombinant expression of BiTE.RTM. molecules
requires construction of an expression vector containing a
polynucleotide that encodes the antibody. Polynucleotides encoding
a particular BiTE.RTM. molecule may be obtained and sequenced by
any method known in the art. For example, a polynucleotide encoding
BiTE.RTM. molecules used in the methods of the invention may be
assembled from chemically synthesized oligonucleotides (e.g., as
described in Kutmeier et al., 1994, BioTechniques 17:242), which,
briefly, involves the synthesis of overlapping oligonucleotides
containing fragments of the sequence encoding the polypeptide,
annealing and ligating of those oligonucleotides, and then
amplification of the ligated oligonucleotides by PCR.
[0564] Once a polynucleotide encoding BiTE.RTM. molecules has been
obtained, the vector for the production of the antibody molecule
may be produced by recombinant DNA technology using techniques well
known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding
nucleotide sequence are described herein. Methods which are well
known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination.
[0565] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce BiTE.RTM. molecules (e.g.,
MT103). A variety of host-expression vector systems may be utilized
to express BiTE.RTM. molecules (see, e.g., U.S. Pat. No.
5,807,715). Such host-expression systems represent vehicles by
which the coding sequences of interest may be produced and
subsequently purified, but also represent cells which may, when
transformed or transfected with the appropriate nucleotide coding
sequences, express an antibody molecule of the invention in situ.
These include but are not limited to microorganisms such as
bacteria (e.g., E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing antibody coding sequences; yeast (e.g.,
Saccharomyces Pichia) transformed with recombinant yeast expression
vectors containing antibody coding sequences; insect cell systems
infected with recombinant virus expression vectors (e.g.,
baculovirus) containing antibody coding sequences; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing antibody coding sequences; or mammalian cell
systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., metallothionein promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter). Bacterial cells such as Escherichia
coli may be used for the expression of a recombinant antibody
molecule. Eukaryotic cells may also be used for the expression of a
recombinant antibody molecule. For example, mammalian cells such as
Chinese hamster ovary cells (CHO), in conjunction with a vector
such as the major intermediate early gene promoter element from
human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., 1986, Gene 45:101; and Cockett et al.,
1990, BioTechnology 8:2). In a specific example, the expression of
nucleotide sequences encoding BiTE.RTM. molecules is regulated by a
constitutive promoter, inducible promoter or tissue specific
promoter. Alternatively, the polynucleotides of the invention may
be expressed in a transgenic plant expression system, such as, for
instance, the LEX System.TM. disclosed in U.S. Pat. No. 6,040,498,
international application published on Feb. 18, 1999 (WO 99/07210),
and international application published on Feb. 7, 2002 (WO
02/10414). Another transgenic plant expression system that may be
utilized is the Plantibodies.TM. technology described in U.S. Pat.
Nos. 5,202,422; 5,639,947; 5,959,177; and 6,417,429.
[0566] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
molecule being expressed. For example, when a large quantity of
such a protein is to be produced, for the generation of
pharmaceutical compositions of an BiTE.RTM. molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited to, the E. coli expression vector pUR278
(Ruther et al., 1983, EMBO 12:1791), in which the BiTE.RTM.
molecule coding sequence may be ligated individually into the
vector in frame with the lac Z coding region so that a fusion
protein is produced; pIN vectors (Inouye & Inouye, 1985,
Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.
Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be
used to express foreign polypeptides as fusion proteins with
glutathione 5-transferase (GST). In general, such fusion proteins
are soluble and can easily be purified from lysed cells by
adsorption and binding to matrix glutathione-agarose beads followed
by elution in the presence of free glutathione. The pGEX vectors
are designed to include thrombin or factor Xa protease cleavage
sites so that the cloned target gene product can be released from
the GST moiety. Another microbial system that may be used to
express the polynucleotides of the invention is the Pfenex
Expression Technology is based on novel strains of Pseudomonas
fluorescens, as described in Squires et al., BioProcess Int'l p. 54
December 2004; and Squires et al., Specialty Chemicals July/August
2004.
[0567] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The
BiTE.RTM. molecule coding sequence may be cloned individually into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter).
[0568] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the BiTE.RTM. molecule coding sequence of
interest may be ligated to an adenovirus transcription/translation
control complex, e.g., the late promoter and tripartite leader
sequence. This chimeric gene may then be inserted in the adenovirus
genome by in vitro or in vivo recombination. Insertion in a
non-essential region of the viral genome (e.g., region E1 or E3)
will result in a recombinant virus that is viable and capable of
expressing the BiTE.RTM. molecule in infected hosts (e.g., see
Logan & Shenk, 1984, PNAS 8 1:355-359). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see, e.g., Bittner et al., 1987, Methods in
Enzymol. 153:516-544).
[0569] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERO, BHK, HeLa,
COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, NS1, and
T47D, NS0 (a murine myeloma cell line that does not endogenously
produce any immunoglobulin chains), CRL7O3O and HsS78BsT-cells.
[0570] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which express the BiTE.RTM. molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compositions that interact directly or indirectly
with the BiTE.RTM. molecule.
[0571] A number of selection systems may be used, including but not
limited to, the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), glutamine synthase, hypoxanthine guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-,
gs-, hgprt- or aprt- cells, respectively. Also, antimetabolite
resistance can be used as the basis of selection for the following
genes: dhfr, which confers resistance to methotrexate (Wigler et
al., 1980, PNAS 77:357; O'Hare et al., 1981, PNAS 78:1527); gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg,
1981, PNAS 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87; Tolstoshev,
1993, Ann. Rev. Pharmacol. Toxicol. 32:573; Mulligan, 1993, Science
260:926; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191;
May, 1993, TIB TECH 11:155-); and hygro, which confers resistance
to hygromycin (Santerre et al., 1984, Gene 30:147). Methods
commonly known in the art of recombinant DNA technology may be
routinely applied to select the desired recombinant clone, and such
methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol.
Biol. 150:1, which are incorporated by reference herein in their
entireties.
[0572] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the BiTE.RTM.
molecule will also increase (Crouse et al., 1983, Mol. Cell. Biol.
3:257).
[0573] Once BiTE.RTM. molecules (e.g., BiTE.RTM.) has been produced
by recombinant expression, it may be purified by any method known
in the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange chromatography,
affinity chromatography, and size exclusion chromatography),
centrifugation, differential solubility, or by any other standard
technique for the purification of proteins. Further, BiTE.RTM.
molecules may be fused to heterologous polypeptide sequences known
in the art to facilitate protein purification.
5.8. BiTE.RTM. Molecule Purification and Isolation
[0574] Recombinantly produced BiTE.RTM. molecule s (e.g.,
BiTE.RTM..TM.) may be purified by any methods known to one of skill
in the art including, but not limited to, chromatography techniques
described in U.S. Pat. No. 7,112,324 which is incorporated by
reference herein in its entirety.
[0575] When using recombinant techniques, the BiTE.RTM. molecule
can be produced intracellularly, in the periplasmic space, or
directly secreted into the medium. If the BiTE.RTM. molecule is
produced intracellularly, as a first step, the particulate debris,
either host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology,
10:163-167 (1992) describe a procedure for isolating BiTE.RTM.
molecule which are secreted into the periplasmic space of E. coli.
Briefly, cell paste is thawed in the presence of sodium acetate (pH
3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
min. Cell debris can be removed by centrifugation. Where the
BiTE.RTM. molecule mutant is secreted into the medium, supernatants
from such expression systems are generally first concentrated using
a commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0576] The BiTE.RTM. molecule composition prepared from the cells
can be purified using, for example, hydroxylapatite chromatography,
hydrophobic interaction chromatography, ion exchange
chromatography, gel electrophoresis, dialysis, and/or affinity
chromatography either alone or in combination with other
purification steps. The suitability of protein A as an affinity
ligand depends on the species and isotype of any immunoglobulin Fc
domain that is present in the antibody mutant. Protein A can be
used to purify antibodies that are based on human .gamma.1,
.gamma.2, or .gamma.4 heavy chains (Lindmark et al., J. Immunol.
Methods, 62:1-13 (1983)). Protein G is recommended for all mouse
isotypes and for human .gamma.3 (Guss et al., EMBO J., 5:15671575
(1986)). The matrix to which the affinity ligand is attached is
most often agarose, but other matrices are available. Mechanically
stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
antibody comprises a CH3 domain, the Bakerbond ABX resin (J. T.
Baker, Phillipsburg, N.J.) is useful for purification. Other
techniques for protein purification such as fractionation on an
ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin, SEPHAROSE
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0577] Following any preliminary purification step(s), the mixture
comprising the BiTE.RTM. molecules of interest and contaminants may
be subjected to low pH hydrophobic interaction chromatography using
an elution buffer at a pH between about 2.5-4.5, and performed at
low salt concentrations (e.g., from about 0-0.25 M salt).
5.9. Administration and Dosing
[0578] Administration of formulations of the invention to a human
patient can be by any route, including but not limited to
intravenous, intradermal, transdermal, subcutaneous, intramuscular,
inhalation (e.g., via an aerosol), buccal (e.g., sub-lingual),
topical (i.e., both skin and mucosal surfaces, including airway
surfaces), intrathecal, intraarticular, intraplural, intracerebral,
intra-arterial, intraperitoneal, oral, intralymphatic, intranasal,
rectal or vaginal administration, by perfusion through a regional
catheter, or by direct intralesional injection. In one embodiment,
formulations of the invention are administered by intravenous push
or intravenous infusion given over a defined period (e.g., 0.5 to 4
hours). In another embodiment, formulations of the invention are
administered by continuous intravenous infusion given over a
defined period (e.g., 4 to 40 days). Compositions of the invention
can be delivered by peristaltic means or in the form of a depot,
although the most suitable route in any given case will depend, as
is well known in the art, on such factors as the species, age,
gender and overall condition of the subject, the nature and
severity of the condition being treated and/or on the nature of the
particular formulation (i.e., dosage) that is being administered.
In particular embodiments, the route of administration is via bolus
or continuous infusion over a period of time, once or twice a week.
In other particular embodiments, the route of administration is by
subcutaneous injection, optionally once or twice weekly. In one
embodiment, compositions, and/or methods of the invention are
administered on an outpatient basis.
[0579] In certain embodiments, a formulation of the present
invention is a lyophilized formulation. A lyophilized formulation
is reconstituted into a liquid formulation by dissolving the
lyophilized cake in a diluent. In certain embodiments, diluent that
may be used for the reconstitution of a lyophilized formulation of
the invention include, but are not limited to, sterile water,
bacteriostatic water for injection (BWFI), a pH buffered solution
(e.g. phosphate-buffered saline), sterile saline solution, Ringer's
solution or dextrose solution. In an alternative embodiment,
diluents can include aqueous solutions of salts and/or buffers.
[0580] In certain embodiments, a reconstituted formulation of the
invention may be further diluted prior to administration to a
patient. A reconstituted formulation may be diluted by at least
about 2 fold, at least about 3 fold, at least about 5 fold, at
least about 10 fold, at least about 20 fold, at least about 30
fold, at least about 40 fold, at least about 50 fold, at least
about 60 fold, at least about 70 fold, at least about 80 fold, at
least about 90 fold, at least about 100 fold, at least about 150
fold, at least about 200 fold, at least about 250 fold, at least
about 300 fold, at least about 500 fold, or at least about 1000
fold.
[0581] In certain embodiments, a reconstituted formulation of the
invention may be diluted prior to administration to a patient. A
reconstituted formulation of the invention may be diluted in, for
example, but not limited to, a pH buffered solution (e.g.
phosphate-buffered saline), sterile saline solution, Ringer's
solution or dextrose solution. The solution used for dilution of a
reconstituted formulation of the invention may further comprise
additional excipients, such as, but not limited to, proteins (for
example, but not limited to, serum albumin), amino acids (for
example, but not limited to, aspartic acid, glutamic acid, lysine,
arginine, glycine), surfactants (for example, but not limited to,
SDS, Tween 20, Tween 80, polysorbate, polysorbate 80 and nonionic
surfactants), saccharides (for example, but not limited to,
glucose, sucrose, maltose and trehalose), polyols (for example, but
not limited to, mannitol and sorbitol), fatty acids and
phospholipids (for example, but not limited to, alkyl sulfonates
and caprylate). In further embodiments, a reconstituted formulation
of the invention may be diluted with a buffered saline solution
comprising lysine HCl and polysorbate 80. In a specific embodiment,
a reconstituted formulation of the invention may be diluted into a
saline solution comprising about 25 mM lysine HCl and 0.02%
polysorbate 80.
[0582] In certain embodiments, a formulation of the invention is
administered subcutaneously. The bioavailability of subcutaneously
delivered BiTE.RTM. molecules is generally lower than that of
intravenously delivered BiTE.RTM. molecules. The present invention
provides BiTE.RTM. formulations that result in high bioavailability
of BiTE.RTM. molecules upon subcutaneous delivery of the
formulation. In certain embodiments, the bioavailability of
BiTE.RTM. molecules following subcutaneous delivery of a
formulation of the invention is between about 10% and about 90%,
between about 10% and about 75%, between about 10% and about 60%,
between about 20% and about 60%, or between about 10% and about
50%, of the bioavailability of the BiTE.RTM. molecules following
intravenous delivery of the same dose of the formulation. In
certain embodiments, the bioavailability of BiTE.RTM. molecules
following subcutaneous delivery of a formulation of the invention
is at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, or at least about 90% of the
bioavailability of the BiTE.RTM. molecules following intravenous
delivery of the same dose of the formulation. In certain
embodiments, the bioavailability of BiTE.RTM. molecules following
subcutaneous delivery of a formulation of the invention is about
10%, about 20%, about 30%, about 40%, about 50%, about 60%, about
70%, about 80%, or about 90% of the bioavailability of the
BiTE.RTM. molecules following intravenous delivery of the same dose
of the formulation. In a specific embodiment, the bioavailability
of BiTE.RTM. molecules following subcutaneous delivery of a
formulation of the invention is about 10% of the bioavailability of
the BiTE.RTM. molecules following intravenous delivery of the same
dose of the formulation. In a specific embodiment, the
bioavailability of BiTE.RTM. molecules following subcutaneous
delivery of a formulation of the invention is about 10% of the
bioavailability of the BiTE.RTM. molecules following intravenous
delivery of the same dose of the formulation. In a specific
embodiment, the bioavailability of BiTE.RTM. molecules following
subcutaneous delivery of a formulation of the invention is about
20% of the bioavailability of the BiTE.RTM. molecules following
intravenous delivery of the same dose of the formulation. In a
specific embodiment, the bioavailability of BiTE.RTM. molecules
following subcutaneous delivery of a formulation of the invention
is about 30% of the bioavailability of the BiTE.RTM. molecules
following intravenous delivery of the same dose of the formulation.
In a specific embodiment, the bioavailability of BiTE.RTM.
molecules following subcutaneous delivery of a formulation of the
invention is about 40% of the bioavailability of the BiTE.RTM.
molecules following intravenous delivery of the same dose of the
formulation. In a specific embodiment, the bioavailability of
BiTE.RTM. molecules following subcutaneous delivery of a
formulation of the invention is about 50% of the bioavailability of
the BiTE.RTM. molecules following intravenous delivery of the same
dose of the formulation. In a specific embodiment, the
bioavailability of BiTE.RTM. molecules following subcutaneous
delivery of a formulation of the invention is about 60% of the
bioavailability of the BiTE.RTM. molecules following intravenous
delivery of the same dose of the formulation.
[0583] In certain embodiments, the dose of a formulation comprising
BiTE.RTM. molecules (e.g., MT103) is measured in units of mg/kg of
patient body weight. In other embodiments, the dose of a
formulation comprising BiTE.RTM. molecules (e.g., MT103) is
measured in units of mg/kg of patient lean body weight (i.e., body
weight minus body fat content). In yet other embodiments, the dose
of a formulation comprising BiTE.RTM. molecules (e.g., MT103) is in
units of mg/m.sup.2 of patient body surface area. In yet other
embodiments, the dose of a composition comprising a formulation
comprising BiTE.RTM. molecules (e.g., MT103) is measured in units
of mg per dose administered to a patient. Any measurement of dose
can be used in conjunction with compositions and methods of the
invention and dosage units can be converted by means standard in
the art.
[0584] Those skilled in the art will appreciate that dosages can be
selected based on a number of factors including the age, sex,
species and condition of the subject (e.g., stage of B cell
malignancy), the desired degree of cellular depletion, the disease
to be treated and/or the particular BiTE.RTM. molecule being used
and can be determined by one of skill in the art. For example,
effective amounts of compositions of the invention may be
extrapolated from dose-response curves derived in vitro test
systems or from animal model (e.g., the cotton rat or monkey) test
systems. Models and methods for evaluation of the effects of
BiTE.RTM. molecules are known in the art (Wooldridge et al., Blood,
89(8): 2994-2998 (1997)), incorporated by reference herein in its
entirety). In specific embodiments, for particular B cell
malignancies, therapeutic regimens standard in the art for antibody
therapy can be used with formulations and methods of the
invention.
[0585] Examples of dosing regimens that can be used in methods of
the invention include, but are not limited to, daily, three times
weekly (intermittent), weekly, or every 14 days. In certain
embodiments, dosing regimens include, but are not limited to,
monthly dosing or dosing every 6-8 weeks.
[0586] Those skilled in the art will appreciate that dosages may be
higher and/or frequency of administration greater for initial
treatment as compared with maintenance regimens.
[0587] In certain embodiments, dosages of BiTE.RTM. molecules
(e.g., MT103) (optionally in a pharmaceutically acceptable carrier
as part of a pharmaceutical composition) are at least about 0.0005,
0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1,
0.25, 0.375, 0.5, 1, 2.5, 5, 10, 20, 37.5, or 50 mg/m.sup.2 and/or
less than about 500, 475, 450, 425, 400, 375, 350, 325, 300, 275,
250, 225, 200, 175, 150, 125, 100, 75, 60, 50, 37.5, 20, 15, 10, 5,
2.5, 1, 0.5, 0.375, 0.2, 0.1, 0.09, 0.08, 0.075, 0.05, 0.04, 0.03,
0.02, or 0.01 mg/m.sup.2. In certain embodiments, the dosage is
between about 0.0005 to about 200 mg/m.sup.2, between about 0.001
and 150 mg/m.sup.2, between about 0.01 and 100 mg/m.sup.2, between
about 0.05 and 50 mg/m.sup.2, between about 0.05 and 25 mg/m.sup.2,
between about 0.05 and 10 mg/m.sup.2, and between about 0.05 and 5
mg/m.sup.2. In related embodiments, the dosage of BiTE.RTM.
molecules (e.g., MT103) used is at least about 0.0001, 0.0005,
0.001, 0.0025, 0.005, 0.0075, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,
0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5,
0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1,
1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 1.55, 1.6, 1.65, 1.7,
1.75, 1.8, 1.85, 1.9, 1.95, 2, 3, 5 mg/kg of body weight of a
patient. In certain embodiments, the dose of BiTE.RTM. molecules
used is at least about 0.1 to 1, 0.5 to 1.5, 1 to 2, or 1.5 to 2.5
micrograms/kg of body weight of a patient. In certain embodiments,
the dose of a bispecific used is at least about 0.1 to 2, 0.3 to 5,
or 0.5 to 10 micrograms/kg of body weight of a patient. In other
embodiments, the dose of BiTE.RTM. molecules used is at least about
0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 5, or 10 micrograms/kg of body
weight of a patient. In certain embodiments, a single dosage unit
of the BiTE.RTM. molecule (optionally in a pharmaceutically
acceptable carrier as part of a pharmaceutical formulation) can be
at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,
60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,
94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,
122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,
148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,
174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,
200, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,
228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, or 250
micrograms/m.sup.2. In other embodiments, dose is up to 1 g per
single dosage unit.
[0588] In specific embodiments of the invention, CD19xCD3
bispecific antibodies (e.g., MT103) bind to B cells and may result
in efficient (i.e., at low dosage) depletion of B cells. Higher
degrees of binding may be achieved where the density of human CD19
on the surface of a patient's B cells is high. In specific
embodiments, dosages of MT103 (e.g., MT103) (optionally in a
pharmaceutically acceptable carrier as part of a pharmaceutical
composition) are at least about 0.0005, 0.001, 0.01, 0.02, 0.03,
0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.25, 0.375, 0.5, 1, 2.5,
5, 10, 20, 37.5, or 50 mg/m.sup.2 and/or less than about 500, 475,
450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150,
125, 100, 75, 60, 50, 37.5, 20, 15, 10, 5, 2.5, 1, 0.5, 0.375, 0.2,
0.1, 0.09, 0.08, 0.075, 0.05, 0.04, 0.03, 0.02, or 0.01 mg/m.sup.2.
In specific embodiments, the dosage is between about 0.0005 to
about 200 mg/m.sup.2, between about 0.001 and 150 mg/m.sup.2,
between about 0.01 and 100 mg/m.sup.2, between about 0.05 and 50
mg/m.sup.2, between about 0.05 and 25 mg/m.sup.2, between about
0.05 and 10 mg/m.sup.2, and between about 0.05 and 5 mg/m.sup.2. In
related specific embodiments, the dosage of MT103 used is at least
about 0.0001, 0.0005, 0.001, 0.0025, 0.005, 0.0075, 0.01, 0.02,
0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25,
0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85,
0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45,
1.5, 1.55, 1.6, 1.65, 1.7, 1.75, 1.8, 1.85, 1.9, 1.95, 2, 3, 5
mg/kg of body weight of a patient. In certain specific embodiments,
the dose of MT103 used is at least about 0.1 to 1, 0.5 to 1.5, 1 to
2, or 1.5 to 2.5 micrograms/kg of body weight of a patient. In
certain specific embodiments, the dose of MT103 used is at least
about 0.1 to 2, 0.3 to 5, or 0.5 to 10 micrograms/kg of body weight
of a patient. In other specific embodiments, the dose of MT103 used
is at least about 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 5, or 10
micrograms/kg of body weight of a patient. In certain specific
embodiments, a single dosage unit of MT1103 (optionally in a
pharmaceutically acceptable carrier as part of a pharmaceutical
formulation) can be at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,
84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,
114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,
140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,
166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,
192, 194, 196, 198, 200, 204, 206, 208, 210, 212, 214, 216, 218,
220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,
246, 248, or 250 micrograms/m.sup.2. In other specific embodiments,
dose is up to 1 g per single dosage unit.
[0589] In some embodiments of methods of this invention,
formulations of this invention can be administered at BiTE.RTM.
molecules dose lower than about 375 mg/m.sup.2; at a dose lower
than about 37.5 mg/m.sup.2; at a dose lower than about 0.375
mg/m.sup.2; at a dose lower than about 0.0375 mg/m.sup.2; and/or at
a dose between about 0.015 mg/m.sup.2 and about 125 mg/m.sup.2. In
certain embodiments of methods of the invention, dosage regimens
comprise low doses, administered at repeated intervals. For
example, in one embodiment, formulations of the invention can be
administered at a dose lower than about 0.375 mg/m.sup.2 at
intervals of approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, or
200 days. In further embodiments of methods of the invention,
dosage regimens comprise low doses, administered via continuous
intravenous infusion. For example, in one embodiment, compositions
of the invention can be administered at a dose lower than about
0.375 mg/m.sup.2 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,
35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200
days.
[0590] In specific embodiments of methods of this invention, MT103
formulations of this invention can be administered at BiTE.RTM.
molecules dose lower than about 375 mg/m.sup.2; at a dose lower
than about 37.5 mg/m.sup.2; at a dose lower than about 0.375
mg/m.sup.2; at a dose lower than about 0.0375 mg/m.sup.2; and/or at
a dose between about 0.015 mg/m.sup.2 and about 125 mg/m.sup.2. In
certain specific embodiments of methods of the invention, dosage
regimens comprise low doses, administered at repeated intervals.
For example, in one embodiment, MT103 formulations of the invention
can be administered at a dose lower than about 0.375 mg/m.sup.2 at
intervals of approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, or
200 days. In further specific embodiments of methods of the
invention, dosage regimens comprise low doses, administered via
continuous intravenous infusion. For example, in one embodiment,
compositions of the invention can be administered at a dose lower
than about 0.375 mg/m.sup.2 for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, or
200 days.
[0591] In specific embodiments, the specified dosage can result in
B cell depletion in the human treated using MT103 formulations and
methods of the invention for a period of at least about 1, 2, 3, 5,
7, 10, 14, 20, 30, 45, 60, 75, 90, 120, 150 or 180 days or longer.
In certain embodiments, pre-B cells (not expressing surface
immunoglobulin) are depleted. In certain embodiments, mature B
cells (expressing surface immunoglobluin) are depleted. In other
embodiments, all non-malignant types of B cells can exhibit
depletion. Any of these types of B cells can be used to measure B
cell depletion. B cell depletion can be measured in bodily fluids
such as blood serum, or in tissues such as bone marrow. In certain
embodiments of methods of the invention, B cells are depleted by at
least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in comparison to B
cell levels in the patient being treated before use of compositions
and methods of the invention. In other embodiments of methods of
the invention, B cells are depleted by at least 30%, 40%, 50%, 60%,
70%, 80%, 90%, or 100% in comparison to typical standard B cell
levels for humans. In related embodiments, the typical standard B
cell levels for humans are determined using patients comparable to
the patient being treated with respect to age, sex, weight, and
other factors.
[0592] In various embodiments, the therapies (e.g., prophylactic or
therapeutic agents) are administered as a continuous infusion, less
than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at
about 1 hour apart, at about 1 to about 2 hours apart, at about 2
hours to about 3 hours apart, at about 3 hours to about 4 hours
apart, at about 4 hours to about 5 hours apart, at about 5 hours to
about 6 hours apart, at about 6 hours to about 7 hours apart, at
about 7 hours to about 8 hours apart, at about 8 hours to about 9
hours apart, at about 9 hours to about 10 hours apart, at about 10
hours to about 11 hours apart, at about 11 hours to about 12 hours
apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours
apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48
hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72
hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours
apart, or 96 hours to 120 hours part. In specific embodiments, two
or more therapies are administered within the same patient
visit.
[0593] In certain embodiments, BiTE.RTM. molecule of a formulation
of the invention and one or more other therapies (e.g.,
prophylactic or therapeutic agents) are cyclically administered.
Cycling therapy involves the administration of a first therapy
(e.g., a first prophylactic or therapeutic agent) for a period of
time, followed by the administration of a second therapy (e.g., a
second prophylactic or therapeutic agent) for a period of time,
optionally, followed by the administration of a third therapy
(e.g., prophylactic or therapeutic agent) for a period of time and
so forth, and repeating this sequential administration, i.e., the
cycle in order to reduce the development of resistance to one of
the therapies, to avoid or reduce the side effects of one of the
therapies, and/or to improve the efficacy of the therapies.
[0594] In certain embodiments, the administration of BiTE.RTM.
molecules of a formulation of the invention may be repeated and the
administrations may be separated by at least 1 day, 2 days, 3 days,
5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3
months, or at least 6 months. In other embodiments, the
administration of a therapy (e.g., a prophylactic or therapeutic
agent) other than BiTE.RTM. molecules of a formulation of the
invention may be repeated and the administration may be separated
by at least at least 1 day, 2 days, 3 days, 5 days, 10 days, 15
days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6
months.
[0595] In certain embodiments of the invention, the dose can be
escalated or reduced to maintain a constant dose in the blood or in
a tissue, such as, but not limited to, bone marrow. In related
embodiments, the dose is escalated or reduced by about 2%, 5%, 8%,
10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% in order
to maintain a desired level of BiTE.RTM. molecules of formulations
and methods of the invention.
[0596] In certain embodiments, the dosage can be adjusted and/or
the infusion rate can be reduced based on patient's immunogenic
response to BiTE.RTM. molecules formulated according to the methods
of the invention.
[0597] Bioavailability of a formulation measures the fraction of an
administered dose of unchanged drug that reaches the systemic
circulation. Bioavailability of intravenously delivered drug is
deemed 100%. The bioavailability of drug substance administered via
other routes (e.g., subcutaneous injection) is below 100% due to
incomplete absorption and first-pass metabolism. Absolute
bioavailability compares the bioavailability (estimated as area
under the curve, or AUC) of the active drug in systemic circulation
following non-intravenous administration (e.g., subcutaneous
administration), with the bioavailability of the same drug
following intravenous administration. In order to determine
absolute bioavailability of a drug, a pharmacokinetic study must be
done to obtain a plasma drug concentration as a function of time
plot for the drug after both intravenous (IV) and non-intravenous
(e.g., subcutaneous) administration. The absolute bioavailability
is the dose-corrected area under curve (AUC) non-intravenous
divided by AUC intravenous.
[0598] The bioavailability of a bispecific antibody formulated
according to a composition or method described herein may be
determined using any methods known to one of skill in the art. For
example, the serum levels of a bispecific antibody may be monitored
by an ELISA assay. Bioavailability may be determined in a human
subject or in any one of the suitable non-human experimental
systems including, but not limited to non-human primates (e.g.,
cyano) and rodents (e.g., mouse).
[0599] In one embodiment, a formulation of the invention is
suitable for subcutaneous delivery. In one embodiment, the
bioavailability of a bispecific antibody formulated according to a
composition or method described herein is between about 10% and
about 100%, between about 10% and about 90%, between about 10% and
about 80%, between about 10% and about 70%, between about 10% and
about 60%, between about 10% and about 50%, between about 10% and
about 40%, between about 10% and about 30%, between about 10% and
about 20%, between about 20% and about 80%, between about 20% and
about 60%, between about 20% and about 40%, between about 30% and
about 100%, or between about 30% and about 60%.
[0600] In another embodiment, the bioavailability of a bispecific
antibody formulated according to a composition or method described
herein is between 10% and 100%, between 10% and 90%, between 10%
and 80%, between 10% and 70%, between 10% and 60%, between 10% and
50%, between 10% and 40%, between 10% and 30%, between 10% and 20%,
between 20% and 80%, between 20% and 60%, between 20% and 40%,
between 30% and 100%, or between 30% and 60%. In a further
embodiment, the bioavailability of a bispecific antibody formulated
according to a composition or method described herein is at least
about 10%, at least about 20%, at least about 30%, at least about
40%, at least about 60%, at least about 70%, at least about 80%, or
at least about 90%. In a further embodiment, the bioavailability of
a bispecific antibody formulated according to a composition or
method described herein is at least 10%, at least 20%, at least
30%, at least 40%, at least 60%, at least 70%, at least 80%, or at
least 90%. In a further embodiment, the bioavailability of a
bispecific antibody formulated according to a composition or method
described herein is about 10%, about 20%, about 30%, about 40%,
about 60%, about 70%, about 80%, or about 90%. In a further
embodiment, the bioavailability of a bispecific antibody formulated
according to a composition or method described herein is 10%, 20%,
30%, 40%, 60%, 70%, 80%, or 90%.
5.9.1. Toxicity Testing
[0601] The tolerance, toxicity and/or efficacy of the formulations
and/or treatment regimens of the present invention can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population), the ED50 (the dose
therapeutically effective in 50% of the population), and IC50 (the
dose effective to achieve a 50% inhibition). In a specific
embodiment, the dose is a dose effective to achieve at least a 60%,
70%, 80%, 90%, 95%, or 99% depletion of circulating B cells or
circulating immunoglobulin, or both. The dose ratio between toxic
and therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD50/ED50. Therapies that exhibit large
therapeutic indices may be preferred.
[0602] Data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosages of the
formulations and/or treatment regimens for use in humans. The
dosage of such agents may lie within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage may vary within this range depending upon the dosage
form employed and the route of administration utilized. For any
therapy used in methods of the invention, a therapeutically
effective dose can be estimated by appropriate animal models.
Depending on the species of the animal model, the dose can be
scaled for human use according to art-accepted formulas, for
example, as provided by Freireich et al., Quantitative comparison
of toxicity of anticancer agents in mouse, rat, monkey, dog, and
human, Cancer Chemotherapy Reports, NCI 1966 40:219-244. Data
obtained from cell culture assays can be useful for predicting
potential toxicity. Animal studies can be used to formulate a
specific dose to achieve a circulating plasma concentration range
that includes the IC50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Plasma drug levels may
be measured, for example, by high performance liquid
chromatography, ELISA, or by cell based assays.
5.10. Patient Diagnosis, Staging and Therapeutic Regimens in
Oncology
[0603] According to certain aspects of the invention, the treatment
regimen and dose used with CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) formulations and methods of the invention is chosen based on
a number of factors including, but not limited to, the stage of the
B cell disease or disorder being treated. Appropriate treatment
regimens can be determined by one of skill in the art for
particular stages of a B cell disease or disorder in a patient or
patient population. Dose response curves can be generated using
standard protocols in the art in order to determine the effective
amount of anti-CD19 BiTE.RTM. formulations of the invention for
treating patients having different stages of a B cell disease or
disorder. In general, patients having more advanced stages of a B
cell disease or disorder will require higher doses and/or more
frequent doses which may be administered over longer periods of
time in comparison to patients having an early stage B cell disease
or disorder.
[0604] CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulations and
methods of the invention may be practiced to treat B cell diseases,
including B cell malignancies. The term "B cell malignancy"
includes any malignancy that is derived from a cell of the B cell
lineage. Exemplary B cell malignancies include, but are not limited
to: B cell subtype non-Hodgkin's lymphoma (NHL) including low
grade/follicular NHL, small lymphocytic (SL) NHL, intermediate
grade/follicular NHL, intermediate grade diffuse NHL, high grade
immunoblastic NHL, high grade lymphoblastic NHL, high grade small
non-cleaved cell NHL; mantle-cell lymphoma, and bulky disease NHL;
Burkitt's lymphoma; multiple myeloma; pre-B acute lymphoblastic
leukemia and other malignancies that derive from early B cell
precursors; common acute lymphocytic leukemia (ALL); chronic
lymphocytic leukemia (CLL) including immunoglobulin-mutated CLL and
immunoglobulin-unmutated CLL; hairy cell leukemia; Null-acute
lymphoblastic leukemia; Waldenstrom's Macroglobulinemia; diffuse
large B cell lymphoma (DLBCL) including germinal center B cell-like
(GCB) DLBCL, activated B cell-like (ABC) DLBCL, and type 3 DLBCL;
pro-lymphocytic leukemia; light chain disease; plasmacytoma;
osteosclerotic myeloma; plasma cell leukemia; monoclonal gammopathy
of undetermined significance (MGUS); smoldering multiple myeloma
(SMM); indolent multiple myeloma (IMM); Hodgkin's lymphoma
including classical and nodular lymphocyte pre-dominant type;
lymphoplasmacytic lymphoma (LPL); and marginal-zone lymphoma
including gastric mucosal-associated lymphoid tissue (MALT)
lymphoma.
[0605] In a further embodiment the invention can be employed to
treat mature B cell malignancies (i.e., express Ig on the cell
surface) including but not limited to follicular lymphoma,
mantle-cell lymphoma, Burkitt's lymphoma, multiple myeloma, diffuse
large B-cell lymphoma (DLBCL) including germinal center B cell-like
(GCB) DLBCL, activated B cell-like (ABC) DLBCL, and type 3 DLBCL,
Hodgkin's lymphoma including classical and nodular lymphocyte
pre-dominant type, lymphoplamacytic lymphoma (LPL), marginal-zone
lymphoma including gastric mucosal-associated lymphoid tissue
(MALT) lymphoma, and chronic lymphocytic leukemia (CLL) including
immunoglobulin-mutated CLL and immunoglobulin-unmutated CLL.
[0606] Further, CD19 is expressed earlier in B cell development
than, for example, CD20, and is therefore particularly suited for
treating pre-B cell and immature B cell malignancies (i.e., do not
express Ig on the cell surface), for example, in the bone marrow.
Illustrative pre-B cell and immature B cell malignancies include,
but are not limited to, acute lymphoblastic leukemia.
[0607] In other particular embodiments, the invention can be
practiced to treat extranodal tumors.
5.10.1. Diagnosis and Staging of B Cell Malignancies
[0608] The progression of cancer, such as a B cell disease or
disorder capable of tumor formation (e.g., non-Hodgkin lymphoma,
diffuse large B cell lymphoma, follicular lymphoma, and Burkitt
lymphoma) is typically characterized by the degree to which the
cancer has spread through the body and is often broken into the
following four stages which are prognostic of outcome. Stage I: The
cancer is localized to a particular tissue and has not spread to
the lymph nodes. Stage II: The cancer has spread to the nearby
lymph nodes, i.e., metastasis. Stage III: The cancer is found in
the lymph nodes in regions of the body away from the tissue of
origin and may comprise a mass or multiple tumors as opposed to
one. Stage IV: The cancer has spread to a distant part of the body.
The stage of a cancer can be determined by clinical observations
and testing methods that are well known to those of skill in the
art. The stages of cancer described above are traditionally used in
conjunction with clinical diagnosis of cancers characterized by
tumor formation, and can be used in conjunction with the
formulations and methods of the present invention to treat B cell
diseases and disorders. Typically early stage disease means that
the disease remains localized to a portion of a patient's body or
has not metastasized.
[0609] With respect to non-tumor forming B cell diseases and
disorders such as, but not limited to, multiple myeloma, the
criteria for determining the stage of disease differs. The
Durie-Salmon Staging System has been widely used. In this staging
system, clinical stage of disease (stage I, II, or III) is based on
several measurements, including levels of M protein, the number of
lytic bone lesions, hemoglobin values, and serum calcium levels.
Stages are further divided according to renal (kidney) function
(classified as A or B). According to the Durie-Salmon Staging
System Stage I (low cell mass) is characterized by all of the
following: Hemoglobin value >10 g/dL; Serum calcium value normal
or .ltoreq.12 mg/dL; Bone x-ray, normal bone structure (scale 0) or
solitary bone plasmacytoma only; and Low M-component production
rate: IgG value <5 g/dL, IgA value <3 g/d, Bence Jones
protein <4 g/24 h. Stage I patients typically have no related
organ or tissue impairment or symptoms. Stage II (intermediate cell
mass) is characterized by fitting neither stage I nor stage III.
Stage III (high cell mass) is characterized by one or more of the
following: Hemoglobin value <8.5 g/dL; Serum calcium value
>12 mg/dL; Advanced lytic bone lesions (scale 3); High
M-component production rate: IgG value >7 g/dL, IgA value >5
g/dL, Bence Jones protein >12 g/24 h Subclassification (either A
or B), where A is Relatively normal renal function (serum
creatinine value <2.0 mg/dL) and B is Abnormal renal function
(serum creatinine value .gtoreq.2.0 mg/dL).
[0610] Another staging system for myeloma is the International
Staging System (ISS) for myeloma. This system can more effectively
discriminate between staging groups and is based on easily measured
serum levels of beta 2-microglobulin (.beta.2-M) and albumin.
According to the ISS for myeloma, Stage I is characterized by
.beta.2-M<3.5 and Albumin .gtoreq.3.5, Stage II is characterized
by .beta.2-M<3.5 and albumin <3.5 or .beta.2-M 3.5-5.5, and
Stage III is characterized by .beta.2-M>5.5 (Multiple Myeloma
Research Foundation, New Canaan, Conn.).
[0611] The stage of a B cell malignancy in a patient is a clinical
determination. As indicated above, with respect to solid tumors,
the spread, location, and number of tumors are the primary factors
in the clinical determination of stage. Determination of stage in
patients with non-tumor forming B cell malignancies can be more
complex requiring serum level measurements as described above.
[0612] The descriptions of stages of B cell diseases and disorders
above are not limiting. Other characteristics known in the art for
the diagnosis of B cell diseases and disorders can be used as
criteria for patients to determine stages of B cell diseases or
disorders.
5.10.2. Clinical Criteria for Diagnosing B Cell Malignancies
[0613] Diagnostic criteria for different B cell malignancies are
known in the art. Historically, diagnosis is typically based on a
combination of microscopic appearance and immunophenotype. More
recently, molecular techniques such as gene-expression profiling
have been applied to develop molecular definitions of B cell
malignancies (see, e.g., Shaffer et al., Nature 2:920-932 (2002)).
Exemplary methods for clinical diagnosis of particular B cell
malignancies are provided below. Other suitable methods will be
apparent to those skilled in the art.
5.11. Patient Diagnosis and Therapeutic Regimens in
Transplantation
[0614] According to certain aspects of the invention, the treatment
regimen and dose used with formulations and methods of the
invention is chosen based on a number of factors including, for
example, clinical manifestation that place a patient at risk for
developing a humoral rejection, or clinical evidence that such a
rejection is developing. The terms "humoral" and
"antibody-mediated" are used interchangeably herein.
[0615] The criteria for assessing the risk that a patient will
develop a humoral rejection are established according to the
knowledge and skill in the art. In one embodiment, a positive
complement dependent cytotoxicity or antiglobulin enhanced
complement dependent cytotoxicity crossmatch indicates that a
patient is at high risk for humoral rejection. In one embodiment, a
positive crossmatch or a prior positive complement dependent
cytotoxicity or anti-globulin enhanced complement dependent
cytotoxicity crossmatch indicates that a patient is at an
intermediate risk for humoral rejection. In one embodiment, a
negative crossmatch indicates that a patient is at a low risk for
humoral rejection.
[0616] In another embodiment, a transplant recipient in need of
prophylaxis against graft rejection may be identified as a patient
or patient population having detectable circulating anti-HLA allo
antibodies prior to transplantation. In another example, the
patient or patient population is identified as having panel
reactive antibodies prior to transplantation. The presence of
detectable circulating anti-HLA allo antibodies in a transplant
recipient post-transplantation can also be used to identify the
patient or patient population in need of treatment for humoral
rejection according to the invention. The patient or patient
population in need of treatment for humoral rejection can also be
identified according to other clinical criteria that indicate that
a transplant recipient is at risk for developing a humoral
rejection or has already developed a humoral rejection. For
example, a transplant recipient in need of treatment of humoral
rejection may be identified as a patient or population in an early
stage of humoral rejection, such as a latent humoral response
characterized by circulating anti-donor allo antibodies. An early
stage of humoral rejection may also be a silent reaction
characterized by circulating anti-donor allo antibodies and C4d
deposition, or a subclinical rejection characterized by circulating
anti-donor allo antibodies, C4d deposition, and tissue pathology.
In later stages, the recipient is identified as a patient or
patient population presenting with clinical indications of humoral
rejection characterized according to the knowledge and skill in the
art, for example, by circulating anti-donor allo antibodies, C4d
deposition, tissue pathology, and graft dysfunction.
[0617] The present invention provides therapeutic formulations,
methods and regimens effective to reduce the incidence, severity,
or duration of GVHD, a rejection episode, or post-transplant
lymphoproliferative disorder. In certain embodiments, CD19xCD3
BiTE.RTM. molecule (e.g., MT103) formulations and methods of the
invention are effective to attenuate the host response to ischemic
reperfusion injury of a solid tissue or organ graft. In one
embodiment, CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulations
and methods of the invention are effective to prolong survival of a
graft in a transplant recipient.
[0618] The present invention encompasses grafts that are
autologous, allogeneic, or xenogeneic to the recipient. The types
of grafts encompassed by the invention include tissue and organ
grafts, including but not limited to, bone marrow grafts,
peripheral stem cell grafts, skin grafts, arterial and venous
grafts, pancreatic islet cell grafts, and transplants of the
kidney, liver, pancreas, thyroid, and heart. The terms "graft" and
"transplant" are used interchangeably herein. In one embodiment,
the autologous graft is a bone marrow graft, an arterial graft, a
venous graft or a skin graft. In one embodiment, the allograft is a
bone marrow graft, a corneal graft, a kidney transplant, a
pancreatic islet cell transplant, or a combined transplant of a
kidney and pancreas. In one embodiment, the graft is a xenograft,
wherein the possible animal donors include, but are not limited to
pigs. The formulations and methods of the present invention may
also be used to suppress a deleterious immune response to a
non-biological graft or implant, including but not limited to an
artificial joint, a stent, or a pacemaker device. CD19xCD3
BiTE.RTM. molecule (e.g., MT103) formulations and methods of the
invention may be used to treat or prevent GVHD, humoral rejection,
or post-transplant lymphoproliferative disorder without regard to
the particular indications initially giving rise to the need for
the transplant or the particular type of tissue transplanted.
[0619] Therapeutic formulations and regimens of the present
invention are described for treating human subjects diagnosed with
autoimmune diseases or disorders, including but not limited to,
rheumatoid arthritis, SLE, ITP, pemphigus-related disorders,
diabetes, and scleroderma.
[0620] Appropriate treatment regimens can be determined by one of
skill in the art for the particular patient or patient population.
In particular embodiments, the treatment regimen is a
pre-transplant conditioning regimen, a post-transplant maintenance
regimen, or post-transplant treatment regimen for an acute or a
chronic rejection. In certain embodiments, the particular regimen
is varied for a patient who is assessed as being at a high or
intermediate risk of developing a humoral response, compared with
the regimen for a patient who is assessed as being at a low risk of
developing a humoral response.
[0621] In certain embodiments, the particular regimen is varied
according to the stage of humoral rejection, with more aggressive
therapy being indicated for patients at later stages of rejection.
The stages of humoral rejection may be classified according to the
knowledge and skill in the art. For example, the stages of humoral
rejection may be classified as one of stages I to IV according to
the following criteria: Stage I Latent Response, characterized by
circulating anti-donor allo antibodies, especially anti-HLA
antibodies; Stage II Silent Reaction, characterized by circulating
anti-donor allo antibodies, especially anti-HLA antibodies, and C4d
deposition, but without histologic changes or graft dysfunction;
Stage III Subclinical Rejection: characterized by circulating
anti-donor allo antibodies, especially anti-HLA antibodies, C4d
deposition, and tissue pathology, but without graft dysfunction;
Stage IV Humoral Rejection: characterized by circulating anti-donor
allo antibodies, especially anti-HLA antibodies, C4d deposition,
tissue pathology, and graft dysfunction.
[0622] Dose response curves can be generated using standard
protocols in the art in order to determine the effective amount of
CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulations of the
invention for use in a particular regimen, for example, in
conditioning regimens prior to transplantation, and in
post-transplantation regimens for prophylaxis and treatment of
GVHD, humoral rejection, or post-transplantation
lymphoproliferative disorders. In general, patients at high risk
for developing a humoral rejection and those already exhibiting one
or more clinical indicators of rejection will require higher doses
and/or more frequent doses which may be administered over longer
periods of time in comparison to patients who are not at high risk
or who do not exhibit any indications of active rejection.
[0623] CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulations and
methods of the invention may be practiced to treat or prevent GVHD,
humoral rejection, or post-transplantation lymphoproliferative
disorders, either alone or in combination with other therapeutic
agents or treatment regimens. Other therapeutic regimens for the
treatment or prevention of GVHD, humoral rejection, or
post-transplantation lymphoproliferative disorders may comprise,
for example, one or more of anti-lymphocyte therapy, steroid
therapy, antibody depletion therapy, immunosuppression therapy, and
plasmapheresis.
[0624] Anti-lymphocyte therapy may comprise the administration to
the transplant recipient of anti-thymocyte globulins, also referred
to as thymoglobulin. Anti-lymphocyte therapy may also comprise the
administration of one or more monoclonal antibodies directed
against T cell surface antigens. Examples of such antibodies
include, without limitation, CAMPATH.TM.-1H (alemtuzumab),
CAMPATH.TM.-1G, CAMPATH.TM.-1M, SIMULECT.TM. (basiliximab), and
ZENAPAX.TM. (daclizumab). In a specific embodiment, the
anti-lymphocyte therapy comprises one or more additional antibodies
directed against B cells, including, without limitation,
RITUXAN.TM. (rituximab).
[0625] Steroid therapy may comprise administration to the
transplant recipient of one or more steroids selected from the
group consisting of cortisol, prednisone, methyl prednisolone,
dexamethazone, and indomethacin. One or more of the steroids may be
corticosteroids, including without limitation, cortisol,
prednisone, and methylprednisolone.
[0626] Antibody depletion therapy may include, for example,
administration to the transplant recipient of intravenous
immunoglobulin. Antibody depletion therapy may also comprise
immunoadsorption therapy applied to the graft ex vivo, prior to
transplantation. Immunoadsorption may be accomplished using any
suitable technique, for example, protein A affinity, or antibody
based affinity techniques using antibodies directed against T cell
or B cell surface markers such as anti-CD3 antibodies, anti-CD20
antibodies, and anti-CD19 antibodies.
[0627] Immunosuppression therapy may comprise the administration of
one or more immunosuppressive agents such as inhibitors of cytokine
transcription (e.g., cyclosporin A, tacrolimus), nucleotide
synthesis (e.g., azathiopurine, mycophenolate mofetil), growth
factor signal transduction (e.g., sirolimus, rapamycin), and the T
cell interleukin 2 receptor (e.g., daclizumab, basiliximab). In a
particular embodiment, an immunosuppressant agent used in
combination with formulations and methods of the invention includes
one or more of the following: adriamycin, azathiopurine, busulfan,
cyclophosphamide, cyclosporin A ("CyA"), cytoxin, fludarabine,
5-fluorouracil, methotrexate, mycophenolate mofetil (MOFETIL),
nonsteroidal anti-inflammatories (NSAIDs), rapamycin, and
tacrolimus (FK506). Immunosuppressive agents may also comprise
inhibitors of complement, for example, soluble complement
receptor-1, anti-C5 antibody, or a small molecule inhibitor of C1s,
for example as described in Buerke et al. (J. Immunol., 167:5375-80
(2001).
[0628] In one embodiment, formulations and methods of the invention
are used in combination with one or more therapeutic regimens for
suppressing humoral rejection, including, without limitation,
tacrolimus and mycophenolate mofetil therapy, immunoadsorption,
intravenous immunoglobulin therapy, and plasmapheresis.
5.11.1. Diagnosis and Clinical Criteria
[0629] The present invention provides CD19xCD3 BiTE.RTM. molecule
(e.g., MT103) formulations and methods for treating and preventing
GVHD, humoral rejection, and post-transplant lymphoproliferative
disorder in human transplant recipients. Formulations and methods
of the invention can be used regardless of the particular
indications which gave rise to the need for a transplant.
Similarly, the use of formulations and methods of the invention for
the treatment and prevention of GVHD, humoral rejection, and
post-transplant lymphoproliferative disorders is not limited by the
particular type of tissue which is intended for transplantation or
which has been transplanted.
[0630] In one embodiment, the invention provides CD19xCD3 BiTE.RTM.
molecule (e.g., MT103) formulations and methods for the prevention
of humoral rejection in a human transplant recipient wherein the
transplant recipient is identified as a patient or patient
population at increased risk for developing a humoral rejection.
Such patients may also be referred to as "sensitized." The criteria
for the identification of sensitized patients is known to the
skilled practitioner. Such criteria may include, for example,
patients having detectable levels of circulating antibodies against
HLA antigens, e.g., anti-HLA allo antibodies. Such criteria may
also include patients who have undergone previous transplantations,
a pregnancy, or multiple blood transfusions. Patients who are at an
increased risk for humoral rejection also include those having
imperfect donor-recipient HLA matching, and those transplantations
which are ABO-incompatible. Sensitized individuals are candidates
for pretreatment or conditioning prior to transplantation.
Sensitized individuals are also candidates for post-transplantation
maintenance regimens for the prevention of humoral rejection.
[0631] In one embodiment, CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
formulations and methods of the invention comprise or are used in
combination with a therapeutic regimen for the treatment of an
acute or chronic rejection. In particular embodiments, the
rejection is characterized as a Stage I, a Stage II, a Stage III,
or a Stage IV humoral rejection.
[0632] In one embodiment, CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
formulations and methods of the invention comprise or are used in
combination with a therapeutic regimen for the treatment of an
early stage humoral rejection. In particular embodiments, the early
stage humoral rejection is a Stage I, II, or III rejection.
Clinical indications of an early stage humoral rejection are
determined according to the knowledge and skill in the art and may
include, for example, the development in the patient of circulating
donor-specific anti-HLA antibodies, the presence of complement
markers of antibody activity such as C4d and C3d deposits in graft
biopsies, and the presence of anti-HLA antibodies in graft
biopsies. Other indicators of an early stage humoral rejection are
known to the skilled practioner and may include, for example, the
development of antiendothelial antibodies, especially antivimentin
antibodies, and the development of nonclassical MHC class I-related
chain A (MICA) allo antibodies.
[0633] In one embodiment, CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
formulations and methods of the invention comprise or are used in
combination with a therapeutic regimen for the treatment of humoral
rejection characterized in part by graft dysfunction. In particular
embodiments, the patient or patient population in need of treatment
for humoral rejection is identified according to criteria known in
the art for graft dysfunction. Examples of such criteria for
particular types of grafts are provided in the sections that
follow. In other embodiments, the patient or patient population in
need of treatment for humoral rejection is identified according to
other criteria that are particular to the type of tissue graft,
such as histological criteria. Examples of such criteria are also
provided in the sections that follow.
5.11.2. Bone Marrow Transplants
[0634] CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulations and
methods of the invention are useful for treating or preventing
GVHD, humoral rejection, and post-transplant lymphoproliferative
disorder in a bone marrow transplant recipient. In one embodiment,
formulations and methods of the invention comprise or are used in
combination with a pre-transplant conditioning regimen.
[0635] In one embodiment, the indication is a B cell associated
autoimmune condition and formulations and methods of the invention
may be used to eliminate the deleterious B cells from the patient
without the need for chemotherapy or radiation therapy conditioning
regimens. In one embodiment, formulations of the invention are
administered in combination with a chemotherapy or radiation
therapy regimen, which regimen comprises a lower dose of one or
more chemotherapeutic agents, or a lower dose of radiation, than
the dose that is administered in the absence of formulations of the
invention.
[0636] A patient or patient population in need of, or likely to
benefit from, a bone marrow transplant is identified according to
the knowledge and skill in the art. Examples of patients that may
be candidates for bone marrow transplantation include patients who
have undergone chemotherapy or radiation therapy for the treatment
of a cancer or an autoimmune disease or disorder, and patients who
are unable to clear a viral infection residing in cells of the
immune system.
5.11.3. Post-Transplant Lymphoproliferative Disorder
[0637] The immunosuppression necessary for successful
transplantation can give rise to a post-transplant
lymphoproliferative disorder of B cell origin. Generally, a
post-transplant lymphoproliferative disorder is associated with
Epstein-Barr virus infected cells. Post-transplant
lymphoproliferative disorder (PTLD) can range in severity from a
benign self-limiting mononucleosis-like syndrome to an aggressive
non-Hodgkins lymphoma. Formulations and methods of the present
invention may be used to treat PTLD arising from any transplant.
The transplant may be a solid organ transplant, for example a heart
transplant, a liver transplant, a kidney transplant, or a combined
kidney-pancreas transplant. In one embodiment, formulations and
methods of the invention are used to treat PTLD as part of a
therapeutic regimen that includes a temporary cessation or
reduction of other immunosuppressive therapy.
[0638] In one embodiment, CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
formulations are administered as part of a therapeutic regimen
including one or more of the following: high dose intravenous gamma
globulin, a cytokine, an anti-viral agent, and an anti-CD20
monoclonal antibody. The therapeutic regimen may include a
temporary cessation or reduction of immunosuppression therapy. In
one embodiment, intravenous gamma globulin is administered at a
daily dose of 0.4 g/kg for 1 to 5 days, preferably for 3 days, and
the cytokine is interferon alpha administered for at least 7 days.
In one embodiment, one or more cytokines is used in the regimen. In
one embodiment, one or more anti-viral agents is used in the
regimen. The anti-viral agent may be selected from any suitable
anti-viral agent known to those of skill in the art. In one
embodiment, the anti-viral agent is aciclovir or ganciclovir. The
anti-viral agent may be administered for at least one or two weeks.
The anti-viral agent may also be administered for longer periods,
for example, 1 month, 2 months, 3 months, 4 months, or 5
months.
5.12. Patient Diagnosis and Therapeutic Regimens in Autoimmune
Disease
[0639] According to certain aspects of the invention, the treatment
regimen and dose used with CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) formulations and methods of the invention is chosen based on
a number of factors including, but not limited to, the stage of the
autoimmune disease or disorder being treated. Appropriate treatment
regimens can be determined by one of skill in the art for
particular stages of an autoimmune disease or disorder in a patient
or patient population. Dose response curves can be generated using
standard protocols in the art in order to determine the effective
amount of CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulations of
the invention for treating patients having different stages of a
autoimmune disease or disorder. In general, patients having more
activity of a autoimmune disease or disorder will require higher
doses and/or more frequent doses which may be administered over
longer periods of time in comparison to patients having less
activity of an autoimmune disease or disorder.
[0640] CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulations and
methods may be practiced to treat an autoimmune disease or
disorder. The term "autoimmune disease or disorder" refers to a
condition in a subject characterized by cellular, tissue and/or
organ injury caused by an immunologic reaction of the subject to
its own cells, tissues and/or organs. The term "inflammatory
disease" is used interchangeably with the term "inflammatory
disorder" to refer to a condition in a subject characterized by
inflammation, including, but not limited to chronic inflammation.
Autoimmune disorders may or may not be associated with
inflammation. Moreover, inflammation may or may not be caused by an
autoimmune disorder. Thus, certain disorders may be characterized
as both autoimmune and inflammatory disorders. Exemplary autoimmune
diseases or disorders include, but are not limited to: alopecia
areata, ankylosing spondylitis, antiphospholipid syndrome,
autoimmune Addison's disease, autoimmune diseases of the adrenal
gland, autoimmune hemolytic anemia, autoimmune hepatitis,
autoimmune oophoritis and orchitis, autoimmune thrombocytopenia,
Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac
sprue-dermatitis, chronic fatigue immune dysfunction syndrome
(CFIDS), chronic inflammatory demyelinating polyneuropathy,
Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold
agglutinin disease, Crohn's disease, discoid lupus, essential mixed
cryoglobulinemia, diabetes, eosinophilic fascites,
fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,
Guillain-Barre, Hashimoto's thyroiditis, Henoch-Schonlein purpura,
idiopathic pulmonary fibrosis, idiopathic/autoimmune
thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis,
lichen planus, lupus erthematosus, Meniere's disease, mixed
connective tissue disease, multiple sclerosis, type 1 or
immune-mediated diabetes mellitus, myasthenia gravis,
pemphigus-related disorders (e.g., pemphigus vulgaris), pernicious
anemia, polyarteritis nodosa, polychrondritis, polyglandular
syndromes, polymyalgia rheumatica, polymyositis and
dermatomyositis, primary agammaglobulinemia, primary biliary
cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon,
Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma,
Sjogren's syndrome, stiff-man syndrome, systemic lupus
erythematosis (SLE), Sweet's syndrome, Still's disease, lupus
erythematosus, takayasu arteritis, temporal arteristis/giant cell
arteritis, ulcerative colitis, uveitis, vasculitides such as
dermatitis herpetiformis vasculitis, vitiligo, and Wegener's
granulomatosis. Examples of inflammatory disorders include, but are
not limited to, asthma, encephilitis, inflammatory bowel disease,
chronic obstructive pulmonary disease (COPD), allergic disorders,
septic shock, pulmonary fibrosis, undifferentitated
spondyloarthropathy, undifferentiated arthropathy, arthritis,
inflammatory osteolysis, graft versus host disease, urticaria,
Vogt-Koyanagi-Hareda syndrome and chronic inflammation resulting
from chronic viral or bacteria infections.
[0641] CD19 is expressed on immature B cells, therefore an
anti-CD19 BiTE.RTM. molecule (e.g., CD19xCD3 BiTE.RTM. molecule
(e.g., MT103)) may be particularly suited for depleting pre-B cells
and immature B cells, e.g., in the bone marrow.
5.12.1. Diagnosis of Autoimmune Diseases or Disorders
[0642] The diagnosis of an autoimmune disease or disorder is
complicated in that each type of autoimmune disease or disorder
manifests differently among patients. This heterogeneity of
symptoms means that multiple factors are typically used to arrive
at a clinical diagnosis. Generally, clinicians use factors, such
as, but not limited to, the presence of auto antibodies, elevated
cytokine levels, specific organ dysfunction, skin rashes, joint
swelling, pain, bone remodeling, and/or loss of movement as
primarily indicators of an autoimmune disease or disorder. For
certain autoimmune diseases or disorders, such as RA and SLE,
standards for diagnosis are known in the art. For certain
autoimmune diseases or disorders, stages of disease have been
characterized and are well known in the art. These art recognized
methods for diagnosing autoimmune diseases and disorders as well as
stages of disease and scales of activity and/or severity of disease
that are well known in the art can be used to identify patients and
patient populations in need of treatment for an autoimmune disease
or disorder using formulations and methods of the invention.
5.12.2. Clinical Criteria for Diagnosing Autoimmune Diseases or
Disorders
[0643] Diagnostic criteria for different autoimmune diseases or
disorders are known in the art. Historically, diagnosis is
typically based on a combination of physical symptoms. More
recently, molecular techniques such as gene-expression profiling
have been applied to develop molecular definitions of autoimmune
diseases or disorders. Exemplary methods for clinical diagnosis of
particular autoimmune diseases or disorders are provided below.
Other suitable methods will be apparent to those skilled in the
art.
[0644] In certain embodiments, patients with low levels of
autoimmune disease activity or patients with an early stage of an
autoimmune disease (for diseases where stages are recognized) can
be identified for treatment using CD19xCD3 BiTE.RTM. molecule
(e.g., MT103) formulations and methods. The early diagnosis of
autoimmune disease is difficult due to the general symptoms and
overlap of symptoms among diseases. In such embodiments, a patient
treated at an early stage or with low levels of an autoimmune
disease activity has symptoms comprising at least one symptom of an
autoimmune disease or disorder. In related embodiments, a patient
treated at an early stage or with low levels of an autoimmune
disease has symptoms comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, or 15 symptoms of an autoimmune disease or
disorder. The symptoms may be of any autoimmune diseases and
disorders or a combination thereof. Examples of autoimmune disease
and disorder symptoms are described below.
5.13. Determining CD19 Density in a Sample or Subject
[0645] While not required, assays for CD19 density can be employed
to further characterize the patient's diagnosis. Methods of
determining the density of anti-CD19 antibody binding to cells are
known to those skilled in the art (See, e.g., Sato et al., J.
Immunology 165:6635-6643 (2000); which discloses a method of
assessing cell surface density of specific CD antigens). Other
standard methods include Scatchard analysis. For example, an
anti-CD19 antibody or fragment can be isolated, radiolabeled, and
the specific activity of the radiolabeled antibody determined. The
antibody is then contacted with a target cell expressing CD19. The
radioactivity associated with the cell can be measured and, based
on the specific activity, the amount of antibody or antibody
fragment bound to the cell determined.
[0646] Fluorescence activated flow cytometry can also be employed.
Generally, the antibody or antibody fragment is bound to a target
cell expressing CD19. A second reagent that binds to the antibody
is then added, for example, a flourochrome labeled
anti-immunoglobulin antibody. Flourochrome staining can then be
measured and used to determine the density of antibody or antibody
fragment binding to the cell.
[0647] As another suitable method, the antibody or antibody
fragment can be directly labeled with a detectable label, such as a
fluorophore, and bound to a target cell. The ratio of label to
protein is determined and compared with standard beads with known
amounts of label bound thereto. Comparison of the amount of label
bound to the cell with the known standards can be used to calculate
the amount of antibody bound to the cell.
[0648] In yet another aspect, the present invention provides a
method for detecting in vitro or in vivo the presence and/or
density of CD19 in a sample or individual. This can also be useful
for monitoring disease and effect of treatment and for determining
and adjusting the dose of the CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) formulation to be administered. The in vivo method can be
performed using imaging techniques such as PET (positron emission
tomography) or SPECT (single photon emission computed tomography).
One could also label an anti-CD19 antibody with Indium using a
covalently attached chelator. The resulting antibody can be imaged
using standard gamma cameras the same way as ZEVALIN.TM. (Indium
labeled anti-CD20 mAb) (Biogen Idec, Cambridge Mass.) is used to
image CD20 antigen.
[0649] In one embodiment, the in vivo method can be performed by
contacting a sample to be tested, optionally along with a control
sample, with a human anti-CD19 antibody under conditions that allow
for formation of a complex between the antibody and the human CD19
antigen. Complex formation is then detected (e.g., using
fluorescent activated flow cytometry or Western blotting). When
using a control sample along with the test sample, a complex is
detected in both samples and any statistically significant
difference in the formation of complexes between the samples is
indicative of the presence of human CD19 in the test sample.
[0650] In other embodiments, mean florescence intensity can be used
as a measure of CD19 density. In such embodiments, B cells are
removed from a patient and stained with a CD19 antibodies that have
been labeled with a florescent label and the fluorescence intensity
is measured using flow cytometry. Fluorescence intensities can be
measured and expressed as an average of intensity per B cell. Using
such methods, mean florescence intensities that are representative
of CD19 density can be compared for a patient before and after
treatment using methods and formulations of the invention, or
between patients and normal levels of hCD 19 on B cells.
[0651] In patients where the density of CD19 expression on B cells
has been determined, the density of CD19 may influence the
determination and/or adjustment of the dosage and/or treatment
regimen used with CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
formulations and methods of the invention. For example, where
density of CD19 is high, it may be possible to use a lower dosage
of an CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulation of the
invention. In certain embodiments, where the patient treated using
formulations and methods of the invention has a low CD19 density, a
higher dosage of an CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
formulation of the invention may be used. In other embodiments,
where the patient treated using formulations and methods of the
invention has a low CD19 density, a low dosage of an CD19xCD3
BiTE.RTM. molecule (e.g., MT103) formulation of the invention may
be used. In certain embodiments, where the patient treated using
formulations and methods of the invention has a high CD19 density,
a higher dosage of an CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
formulation of the invention may be used. In certain embodiments,
CD19 density can be compared to CD20 density in a patient, CD19
density can be compared to an average CD19 density for humans or
for a particular patient population, or CD19 density can be
compared to CD19 levels in the patient prior to therapy or prior to
onset of a B cell disease or disorder. In certain embodiments, the
patient treated using formulations and methods of the invention has
a B cell malignancy where CD19 is present on the surface of B
cells.
5.14. Immunotherapeutic Protocols
[0652] CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulations used
in therapeutic regimen/protocols, referred to herein as "anti-CD19
immunotherapy", can be used as a single agent therapy or in
combination with other therapeutic agents or regimens. CD19xCD3
BiTE.RTM. molecule (e.g., MT103) formulations can be administered
prior to, concurrently with, or following the administration of one
or more therapeutic agents. Therapeutic agents that can be used in
combination therapeutic regimens with formulations of the invention
include any substance that inhibits or prevents the function of
cells and/or causes destruction of cells. Examples include, but are
not limited to, radioactive isotopes, chemotherapeutic agents, and
toxins such as enzymatically active toxins of bacterial, fungal,
plant or animal origin, or fragments thereof.
[0653] The therapeutic regimens described herein, or any desired
treatment regimen can be tested for efficacy using a transgenic
animal model which expresses human CD19 antigen in place of native
CD19 antigen. Thus, an CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
formulation treatment regimen can be tested in an animal model to
determine efficacy before administration to a human.
[0654] CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulations and
methods may be practiced to treat B cell diseases, including B cell
malignancies. The term "B cell malignancy" includes any malignancy
that is derived from a cell of the B cell lineage. Exemplary B cell
malignancies include, but are not limited to: B cell subtype
non-Hodgkin's lymphoma (NHL) including low grade/follicular, NHL,
small lymphocytic (SL) NHL, intermediate grade/follicular NHL,
intermediate grade diffuse NHL, high grade immunoblastic NHL, high
grade lymphoblastic NHL, high grade small non-cleaved cell NHL;
mantle-cell lymphoma, and bulky disease NHL; Burkitt's lymphoma;
multiple myeloma; pre-B acute lymphoblastic leukemia and other
malignancies that derive from early B cell precursors; common acute
lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL)
including immunoglobulin-mutated CLL and immunoglobulin-unmutated
CLL; hairy cell leukemia; Null-acute lymphoblastic leukemia;
Waldenstrom's Macroglobulinemia; diffuse large B cell lymphoma
(DLBCL) including germinal center B cell-like (GCB) DLBCL,
activated B cell-like (ABC) DLBCL, and type 3 DLBCL;
pro-lymphocytic leukemia; light chain disease; plasmacytoma;
osteosclerotic myeloma; plasma cell leukemia; monoclonal gammopathy
of undetermined significance (MGUS); smoldering multiple myeloma
(SMM); indolent multiple myeloma (IMM); Hodgkin's lymphoma
including classical and nodular lymphocyte pre-dominant type;
lymphoplasmacytic lymphoma (LPL); and marginal-zone lymphoma
including gastric mucosal-associated lymphoid tissue (MALT)
lymphoma.
[0655] In a further embodiment the invention can be employed to
treat mature B cell malignancies (i.e., express Ig on the cell
surface) including but not limited to follicular lymphoma,
mantle-cell lymphoma, Burkitt's lymphoma, multiple myeloma, diffuse
large B-cell lymphoma (DLBCL) including germinal center B cell-like
(GCB) DLBCL, activated B cell-like (ABC) DLBCL, and type 3 DLBCL,
Hodgkin's lymphoma including classical and nodular lymphocyte
pre-dominant type, lymphoplasmacytic lymphoma (LPL), marginal-zone
lymphoma including gastric mucosal-associated lymphoid tissue
(MALT) lymphoma, and chronic lymphocytic leukemia (CLL) including
immunoglobulin-mutated CLL and immunoglobulin-unmutated CLL.
[0656] Further, CD19 is expressed earlier in B cell development
than, for example, CD20, and is therefore particularly suited for
treating pre-B cell and immature B cell malignancies (i.e., do not
express Ig on the cell surface), for example, in the bone marrow.
Illustrative pre-B cell and immature B cell malignancies include
but are not limited to acute lymphoblastic leukemia
[0657] In other particular embodiments, the invention can be
practiced to treat extranodal tumors.
5.15. Anti-CD19 Immunotherapy
[0658] In accordance with the present invention "anti-CD19
immunotherapy" encompasses the administration of any of the
CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulations of the
invention in accordance with any therapeutic regimen described
herein.
[0659] Anti-CD19 immunotherapy encompasses the administration of an
CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulation as a single
agent therapeutic for the treatment of a B cell malignancy.
Anti-CD19 immunotherapy encompasses methods of treating an early
stage disease resulting from a B cell malignancy. Anti-CD19
immunotherapy encompasses methods of treating a B cell malignancy
wherein an CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulation
mediates depletion of B cells. Anti-CD19 immunotherapy encompasses
methods of treating a B cell malignancy wherein an CD19xCD3
BiTE.RTM. molecule (e.g., MT103) formulation is administered before
the patient has received any treatment for the malignancy, whether
that therapy is chemotherapy, radio chemical based therapy or
surgical therapy.
[0660] In one embodiment, a human subject having a B cell
malignancy can be treated by administering a CD19xCD3 BiTE.RTM.
molecule (e.g., MT103) formulation that may be able to mediate B
cell depletion.
[0661] In one embodiment, the dose of CD19xCD3 BiTE.RTM. molecule
(e.g., MT103) formulation used should be sufficient to deplete
circulating B cells. Progress of the therapy can be monitored in
the patient by analyzing blood samples. Other signs of clinical
improvement can be used to monitor therapy.
[0662] Methods for measuring depletion of B cells that can be used
in connection with formulations and methods of the invention are
well known in the art and include, but are not limited to the
following embodiments. In one embodiment, circulating B cells
depletion can be measured with flow cytometry using a reagent other
than an anti-CD19 BiTE.RTM. molecule that binds to B cells to
define the amount of B cells. In other embodiments, B cell levels
in the blood can be monitored using standard serum analysis. In
such embodiments, B cell depletion is indirectly measured by
defining the amount of an antibody known to be produced by B cells.
The level of that antibody is then monitored to determine the
depletion and/or functional depletion of B cells. In another
embodiment, B cell depletion can be measured by immunochemical
staining to identify B cells. In such embodiments, B cells or
tissues or serum comprising B cells extracted from a patient can be
placed on microscope slides, labeled and examined for presence or
absence. In related embodiments, a comparison is made between B
cells extracted prior to therapy and after therapy to determine
differences in the presence of B cells.
[0663] Tumor burden can be measured and used in connection with
formulations and methods of the invention. Methods for measuring
tumor burden are well known in the art and include, but are not
limited to the following embodiments. In certain embodiments, PET
scans can be used to measure metabolic activity and identify areas
of higher activity which are indicative of tumors. CT scans and MRI
can also be used to examine soft tissue for the presence and size
of tumors. In other embodiments, bone scan can be used to measure
tumor volume and location. In yet other embodiments, tumor burden
can be measured by examining the blood flow into and out of a tumor
using doppler technology (e.g., ultrasound). In such embodiments,
changes in blood flow over time or deviations from normal blood
flow in the appropriate tissue of a patient can be used to
calculate an estimate to tumor burden. Such methods for measuring
tumor burden can be used prior to and following methods of
treatment of the invention.
[0664] In embodiments of the invention where an CD19xCD3 BiTE.RTM.
molecule (e.g., MT103) formulation is administered as a single
agent therapy, the invention contemplates use of different
treatment regimens.
[0665] According to certain aspects of the invention, the dose of
CD19xCD3 BiTE.RTM. molecule (e.g., MT103) used is at least about
0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,
5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,
12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5,
19, 19.5, 20, 20.5 .mu.g/kg of body weight of a patient. In certain
embodiments, the dose of CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
used is at least about 0.01 to 0.1, 0.05 to 0.2, 0.1 to 0.5, or 0.2
to 1 .mu.g/kg of body weight of a patient. In certain embodiments,
the dose of CD19xCD3 BiTE.RTM. molecule (e.g., MT103) used is at
least about 0.5 to 2, 1 to 5, or 3 to 10 .mu.g/kg of body weight of
a patient.
[0666] In certain embodiments, the dose comprises at least about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450,
500 .mu.g/m.sup.2 of CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
administered daily for 10 to 50 consecutive days. In certain
embodiments, the dose is at least about 10, 12, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, 200
.mu.g/m.sup.2 of CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
administered daily for 10 to 50 consecutive days.
[0667] In one embodiment, the above doses are single dose
injections. In other embodiments, the doses are administered over a
period of time. In specific embodiments, the doses are administered
by continuous IV. In other embodiments, the doses are administered
multiple times over a period of time. The period of time may be
measured in hours, days, weeks, or months. Multiple doses of
CD19xCD3 BiTE.RTM. molecule (e.g., MT103) can be administered at
intervals suitable to achieve a therapeutic benefit while balancing
toxic side effects.
[0668] In certain embodiments, CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) formulations of the invention are administered to a human
patient as long as the patient is responsive to therapy. In other
embodiments, formulations of the invention are administered to a
human patient as long as the patient's disease does not progress.
In related embodiments, formulations of the invention are
administered to a human patient until a patient's disease does not
progress or has not progressed for a period of time, then the
patient is not administered formulations of the invention unless
the disease reoccurs or begins to progress again. For example, a
patient can be treated with any of the above doses for about 10 to
50 days, during which time the patient is monitored for disease
progression. If disease progression stops or reverses, then the
patient will not be administered formulations of the invention
until that patient relapses, i.e., the disease being treated
reoccurs or progresses. Upon this reoccurrence or progression, the
patient can be treated again with the same dosing regimen initially
used or using other doses described above.
[0669] In certain embodiments, CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) formulations of the invention can be administered as a
loading dose followed by multiple lower doses (maintenance doses)
over a period of time. In such embodiments, the doses may be timed
and the amount adjusted to maintain effective B cell depletion. The
maintenance doses can be continued indefinitely, until toxicity is
present, until platelet count decreases, until there is no disease
progression, until the patient exhibits immunogenicity, or until
disease progresses to a terminal state. In yet other embodiments,
formulations of the invention are administered to a human patient
until the disease progresses to a terminal stage.
5.16. Combination with Chemotherapeutic Agents
[0670] Anti-CD19 immunotherapy using CD19xCD3 BiTE.RTM. molecule
(e.g., MT103) can be used in conjunction with other therapies
including but not limited to, chemotherapy, radioimmunotherapy
(RIT), chemotherapy and external beam radiation (combined modality
therapy, CMT), or combined modality radioimmunotherapy (CMRIT)
alone or in combination, etc. In certain embodiments, a CD19xCD3
BiTE.RTM. molecule (e.g., MT103) therapy of the present invention
can be administered in conjunction with CHOP
(Cyclophosphamide-Hydroxydoxorubicin-Oncovin
(vincristine)-Prednisolone), the most common chemotherapy regimen
for treating non-Hodgkin's lymphoma. As used herein, the term
"administered in conjunction with" means that an anti-CD19
immunotherapy can be administered before, during, or subsequent to
the other therapy employed.
[0671] In certain embodiments, a CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) immunotherapy is in conjunction with a cytotoxic
radionuclide or radiotherapeutic isotope. For example, an
alpha-emitting isotope such as .sup.225Ac, .sup.224Ac, .sup.211At,
.sup.212Bi, .sup.213Bi, .sup.212Pb, .sup.224Ra, or .sup.223Ra. The
cytotoxic radionuclide may also be a beta-emitting isotope such as
.sup.186Re, .sup.188Re, .sup.90Y, .sup.131I, .sup.67Cu, .sup.177Lu,
.sup.153Sm, .sup.166Ho, or .sup.64Cu. Further, the cytotoxic
radionuclide may emit Auger and low energy electrons and include
the isotopes .sup.125I, .sup.123I or .sup.77Br. In other
embodiments the isotope may be .sup.198Au, .sup.32P, and the like.
In certain embodiments, the amount of the radionuclide administered
to the subject is between about 0.001 mCi/kg and about 10
mCi/kg.
[0672] In some embodiments, the amount of the radionuclide
administered to the subject is between about 0.1 mCi/kg and about
1.0 mCi/kg. In other embodiments, the amount of the radionuclide
administered to the subject is between about 0.005 mCi/kg and 0.1
mCi/kg.
[0673] In certain embodiments, a CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) immunotherapy is in conjunction with a chemical toxin or
chemotherapeutic agent. The chemical toxin or chemotherapeutic
agent may be selected from the group consisting of an enediyne such
as calicheamicin and esperamicin; duocarmycin, methotrexate,
doxorubicin, melphalan, chlorambucil, ARA-C, vindesine, mitomycin
C, cis-platinum, etoposide, bleomycin and 5-fluorouracil.
[0674] Suitable chemical toxins or chemotherapeutic agents that can
be used in combination therapies with a CD19xCD3 BiTE.RTM. molecule
(e.g., MT103) immunotherapy include members of the enediyne family
of molecules, such as calicheamicin and esperamicin. Chemical
toxins can also be taken from the group consisting of duocarmycin
(see, e.g., U.S. Pat. No. 5,703,080 and U.S. Pat. No. 4,923,990),
methotrexate, doxorubicin, melphalan, chlorambucil, ARA-C,
vindesine, mitomycin C, cis-platinum, etoposide, bleomycin and
5-fluorouracil. Examples of chemotherapeutic agents also include
Adriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside
("Ara-C"), Cyclophosphamide, Thiotepa, Taxotere (docetaxel),
Busulfan, Cytoxin, Taxol, Methotrexate, Cisplatin, Melphalan,
Vinblastine, Bleomycin, Etoposide, Ifosfamide, Mitomycin C,
Mitoxantrone, Vincreistine, Vinorelbine, Carboplatin, Teniposide,
Daunomycin, Carminomycin, Aminopterin, Dactinomycin, Mitomycins,
Esperamicins (see, U.S. Pat. No. 4,675,187), Melphalan and other
related nitrogen mustards.
[0675] In other embodiments, for example, "CVB" (1.5 g/m.sup.2
cyclophosphamide, 200-400 mg/m.sup.2 etoposide, and 150-200
mg/m.sup.2 carmustine) can be used in combination therapies of the
invention. CVB is a regimen used to treat non-Hodgkin's lymphoma.
Patti et al., Eur. J. Haematol. 51:18 (1993). Other suitable
combination chemotherapeutic regimens are well-known to those of
skill in the art. See, for example, Freedman et al., "Non-Hodgkin's
Lymphomas," in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et
al. (eds.), pp. 2028-2068 (Lea & Febiger 1993). As an
illustration, first generation chemotherapeutic regimens for
treatment of intermediate-grade non-Hodgkin's lymphoma include
C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone)
and CHOP (cyclophosphamide, doxorubicin, vincristine, and
prednisone). A useful second generation chemotherapeutic regimen is
m-BACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide,
vincristine, dexamethasone and leucovorin), while a suitable third
generation regimen is MACOP-B (methotrexate, doxorubicin,
cyclophosphamide, vincristine, prednisone, bleomycin and
leucovorin). Additional useful drugs include phenyl butyrate and
brostatin-1. In a multimodal therapy, both chemotherapeutic drugs
and cytokines are co-administered with a CD19xCD3 BiTE.RTM.
molecule (e.g., MT103) formulation according to the present
invention. The cytokines, chemotherapeutic drugs and CD19xCD3
BiTE.RTM. molecule (e.g., MT103) formulation can be administered in
any order, or together.
[0676] Other toxins that may be used in formulations and methods of
the invention include poisonous lectins, plant toxins such as
ricin, abrin, modeccin, botulina and diphtheria toxins.
[0677] Suitable toxins and chemotherapeutic agents are described in
REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.
1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF
THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985). Other
suitable toxins and/or chemotherapeutic agents are known to those
of skill in the art.
[0678] In certain embodiments, administration of formulations and
methods of the invention may enable the postponement of toxic
therapy and may help avoid unnecessary side effects and the risks
of complications associated with chemotherapy and delay development
of resistance to chemotherapy. In certain embodiments, toxic
therapies and/or resistance to toxic therapies is delayed in
patients administered formulations and methods of the invention
delay for up to about 6 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
years.
5.17. Combination with Therapeutic Antibodies
[0679] A CD19xCD3 BiTE.RTM. molecule (e.g., MT103) immunotherapy
described herein may be administered in combination with other
antibodies, including, but not limited to, anti-CD20 mAb, anti-CD52
mAb, anti-CD22 antibody, and anti-CD20 antibodies, such as
RITUXAN.TM. (C2B8; RITUXIMAB.TM.; IDEC Pharmaceuticals). Other
examples of therapeutic antibodies that can be used in combination
with methods of the invention or used in formulations of the
invention include, but are not limited to, HERCEPTIN.TM.
(Trastuzumab; Genentech), MYLOTARG.TM. (Gemtuzumab ozogamicin;
Wyeth Pharmaceuticals), CAMPATH.TM. (Alemtuzumab; Berlex),
ZEVALIN.TM. (Ipritumomab tiuxetan; Biogen Idec), BEXXAR.TM.
(Tositumomab; GlaxoSmithKline Corixa), ERBITUX.TM. (Cetuximab;
Imclone), and AVASTIN.TM. (Bevacizumab; Genentech).
[0680] In certain embodiments, a CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) and an anti-CD20 and/or an anti-CD22 mAb and/or an anti-CD52
mAb can be administered, optionally in the same pharmaceutical
formulation, in any suitable ratio.
5.18. Combination with Immunoregulatory Agents
[0681] A CD19xCD3 BiTE.RTM. molecule (e.g., MT103) immunotherapy
may also be in conjunction with an immunoregulatory agent. The term
"immunoregulatory agent" as used herein for combination therapy
refers to substances that act to suppress, mask, or enhance the
immune system of the host. This would include substances that
suppress cytokine production, downregulate or suppress self-antigen
expression, or mask the MHC antigens. Examples of such agents
include 2-amino-6-aryl-5-substituted pyrimidines (see, U.S. Pat.
No. 4,665,077), azathioprine (or cyclophosphamide, if there is an
adverse reaction to azathioprine); bromocryptine; glutaraldehyde
(which masks the MHC antigens, as described in U.S. Pat. No.
4,120,649); anti-idiotypic antibodies for MHC antigens and MHC
fragments; cyclosporin A; steroids such as glucocorticosteroids,
e.g., prednisone, methylprednisolone, and dexamethasone; cytokine
or cytokine receptor antagonists including anti-interferon-.gamma.,
-.beta., or -.alpha. antibodies; anti-tumor necrosis factor-.alpha.
antibodies; anti-tumor necrosis factor-0 antibodies;
anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;
anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T
antibodies, for example anti-CD3 or anti-CD4/CD4a antibodies;
soluble peptide containing a LFA-3 binding domain (WO 90/08187
published Jul. 26, 1990); streptokinase; TGF-.beta.;
streptodornase; RNA or DNA from the host; FK506; RS-61443;
deoxyspergualin; rapamycin; T-cell receptor (U.S. Pat. No.
5,114,721); T-cell receptor fragments (Offner et al., Science
251:430-432 (1991); WO 90/11294; and WO 91/01133); and T-cell
receptor antibodies (EP 340,109) such as T10B9. Examples of
cytokines include, but are not limited to 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.; mullerian-inhibiting
substance; mouse gonadotropin-associated peptide; inhibin; activin;
vascular endothelial growth factor; integrin; thrombopoiotin (TPO);
nerve growth factors such as NGF-.alpha.; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-.alpha. and
TGF-.alpha.; insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors; interferons; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CgP (GM-CSP); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis
factor such as TNF-.alpha. or TNF-.beta.; 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. In certain embodiments, the methods further
include administering to the subject one or more immunomodulatory
agents, for example a cytokine. Suitable cytokines may be selected
from the group consisting of interleukin-1 (IL-1), IL-2, IL-3,
IL-12, IL-15, IL-18, G-CSF, GM-CSF, thrombopoietin, and .gamma.
interferon.
[0682] These immunoregulatory agents are administered at the same
time or at separate times from a CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103). The choice of immunoregulatory agent will depend on many
factors, including the type of disorder being treated, as well as
the patient's history, but the agent frequently may be selected
from cyclosporin A, a glucocorticosteroid (for example prednisone
or methylprednisolone), azathioprine, bromocryptine, heterologous
anti-lymphocyte globulin, or a mixture thereof.
5.19. Combination with Other Therapeutic Agents
[0683] Agents that act on the tumor neovasculature can also be used
in conjunction with a CD19xCD3 BiTE.RTM. molecule (e.g., MT103) and
include tubulin-binding agents such as combrestatin A4 (Griggs et
al., Lancet Oncol. 2:82, (2001)) and angiostatin and endostatin
(reviewed in Rosen, Oncologist 5:20 (2000), incorporated by
reference herein). Immunomodulators suitable for use in combination
with CD19xCD3 bispecific antibodies (e.g., MT103) include, but are
not limited to, of a-interferon, .gamma.-interferon, and tumor
necrosis factor alpha (TNF.alpha.). In certain embodiments, the
therapeutic agents used in combination therapies using formulations
and methods of the invention are peptides.
[0684] In certain embodiments, a CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) immunotherapy is in conjunction with one or more
calicheamicin molecules. The calicheamicin family of antibiotics
are capable of producing double-stranded DNA breaks at
sub-picomolar concentrations. Structural analogues of calicheamicin
which may be used include, but are not limited to, .gamma.1.sup.I,
.gamma.2.sup.I, .gamma.3.sup.I, N-acetyl-.gamma.1.sup.I, PSAG and
011 Hinman et al., Cancer Research 53:3336-3342 (1993) and Lode et
al., Cancer Research 58: 2925-2928 (1998)).
[0685] In certain embodiments, a treatment regimen includes
compounds that mitigate the cytotoxic effects of a CD19xCD3
BiTE.RTM. molecule (e.g., MT103) formulation. Such compounds
include analgesics (e.g., acetaminophen), bisphosphonates,
antihistamines (e.g., chlorpheniramine maleate), and steroids
(e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol,
cortisone, prednisone, dehydrotestosterone, glucocorticoids,
mineralocorticoids, estrogen, testosterone, progestins).
[0686] In certain embodiments, the therapeutic agent used in
combination with a CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
immunotherapy is a small molecule (i.e., inorganic or organic
compounds having a molecular weight of less than about 2500
daltons). For example, libraries of small molecules may be
commercially obtained from Specs and BioSpecs B.V. (Rijswijk, The
Netherlands), Chembridge Corporation (San Diego, Calif.), Comgenex
USA Inc. (Princeton, N.J.), and Maybridge Chemicals Ltd. (Cornwall
PL34 OHW, United Kingdom).
[0687] In certain embodiments a CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) immunotherapy can be administered in combination with an
anti-bacterial agent. Non-limiting examples of anti-bacterial
agents include proteins, polypeptides, peptides, fusion proteins,
antibodies, nucleic acid molecules, organic molecules, inorganic
molecules, and small molecules that inhibit and/or reduce a
bacterial infection, inhibit and/or reduce the replication of
bacteria, or inhibit and/or reduce the spread of bacteria to other
cells or subjects. Specific examples of anti-bacterial agents
include, but are not limited to, antibiotics such as penicillin,
cephalosporin, imipenem, axtreonam, vancomycin, cycloserine,
bacitracin, chloramphenicol, erythromycin, clindamycin,
tetracycline, streptomycin, tobramycin, gentamicin, amikacin,
kanamycin, neomycin, spectinomycin, trimethoprim, norfloxacin,
rifampin, polymyxin, amphotericin B, nystatin, ketocanazole,
isoniazid, metronidazole, and pentamidine.
[0688] In certain embodiments a CD19xCD3 BiTE.RTM. molecule (e.g.,
MT103) immunotherapy can be administered in combination with an
anti-fungal agent. Specific examples of anti-fungal agents include,
but are not limited to, azole drugs (e.g., miconazole, ketoconazole
(NIZORAL.RTM.), caspofungin acetate (CANCIDAS.RTM.), imidazole,
triazoles (e.g., fluconazole (DIFLUCAN.RTM.)), and itraconazole
(SPORANOX.RTM.)), polyene (e.g., nystatin, amphotericin B
(FUNGIZONE.RTM.), amphotericin B lipid complex ("ABLC")
(ABELCET.RTM.), amphotericin B colloidal dispersion ("ABCD")
(AMPHOTEC.RTM.), liposomal amphotericin B (AMBISONE.RTM.)),
potassium iodide (KI), pyrimidine (e.g., flucytosine
(ANCOBON.RTM.), and voriconazole (VFEND.RTM.)). Administration of
anti bacterial and anti-fungal agents can mitigate the effects or
escalation of infectious disease that may occur in methods of the
invention where a patient's B cells are significantly depleted.
[0689] In certain embodiments of the invention, a CD19xCD3
BiTE.RTM. molecule (e.g., MT103) immunotherapy can be administered
in combination with one or more of the agents described above to
mitigate the toxic side effects that may accompany administration
of formulations of the invention. In other embodiments, a CD19xCD3
BiTE.RTM. molecule (e.g., MT103) immunotherapy can be administered
in combination with one or more agents that are well known in the
art for use in mitigating the side effects of antibody
administration, chemotherapy, toxins, or drugs.
[0690] In certain embodiments of the invention where a CD19xCD3
BiTE.RTM. molecule (e.g., MT103) immunotherapy is administered to
treat multiple myeloma, formulations of the invention may be
administered in combination with or in treatment regimens with
high-dose chemotherapy (melphalan, melphalan/prednisone (MP),
vincristine/doxorubicin/dexamethasone (VAD), liposomal
doxorubicin/vincristine, dexamethasone (DVd), cyclophosphamide,
etoposide/dexamethasone/cytarabine, cisplatin (EDAP)), stem cell
transplants (e.g., autologous stem cell transplantation or
allogeneic stem cell transplantation, and/or mini-allogeneic
(non-myeloablative) stem cell transplantation), radiation therapy,
steroids (e.g., corticosteroids, dexamethasone,
thalidomide/dexamethasone, prednisone, melphalan/prednisone),
supportive therapy (e.g., bisphosphonates, growth factors,
antibiotics, intravenous immunoglobulin, low-dose radiotherapy,
and/or orthopedic interventions), THALOMID.TM. (thalidomide,
Celgene), and/or VELCADE.TM. (bortezomib, Millennium).
[0691] In embodiments of the invention where a CD19xCD3 BiTE.RTM.
molecule (e.g., MT103) immunotherapy is administered in combination
with another antibody or antibodies and/or agent, the additional
antibody or antibodies and/or agents can be administered in any
sequence relative to the administration of the CD19xCD3 BiTE.RTM.
molecule (e.g., MT103). For example, the additional antibody or
antibodies can be administered before, concurrently with, and/or
subsequent to administration of a CD19xCD3 BiTE.RTM. molecule
(e.g., MT103) to the human subject. The additional antibody or
antibodies can be present in the same pharmaceutical formulation as
a CD19xCD3 BiTE.RTM. molecule (e.g., MT103), and/or present in a
different pharmaceutical formulation. The dose and mode of
administration of a CD19xCD3 BiTE.RTM. molecule (e.g., MT103) and
the dose of the additional antibody or antibodies can be the same
or different, in accordance with any of the teachings of dosage
amounts and modes of administration as provided in this application
and as are well known in the art.
5.20. Kits
[0692] The present invention provides kits that can be used in the
above-described methods. The invention provides a pharmaceutical
pack or kit comprising a lyophilized formulation of the invention
in one or more containers. In one embodiment, a kit comprises a
formulation of the invention, in one or more containers. In another
embodiment, a kit comprises a formulation of the invention, in one
or more containers, and further comprises a suitable diluent (e.g.
BWFI), in one or more containers. In a further embodiment, a kit
comprises a formulation of the invention, in one or more
containers, and one or more other prophylactic or therapeutic
agents. The kit may further comprise instructions for method of
administration of a reconstituted formulation of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0693] The invention provides a pharmaceutical pack or kit
comprising one or more containers filled with a lyophilized
formulation of a CD19xCD3 BiTE.RTM. molecule (e.g., MT103) for the
prevention, treatment, management or amelioration of B cell
diseases and disorders, such as, but not limited to, B cell
malignancies, autoimmune diseases, graft-versus-host disease
(GVHD), humoral rejection, and post-transplantation
lymphoproliferative disorder, or one or more symptoms thereof, in a
human subject. In one embodiment, a kit comprises a CD19xCD3
BiTE.RTM. molecule (e.g., MT103) lyophilized formulation of the
invention, in one or more containers. In another embodiment, a kit
comprises a CD19xCD3 BiTE.RTM. molecule (e.g., MT103) formulation
of the invention, in one or more containers, and further comprises
a suitable diluent (e.g. BWFI), in one or more containers. In a
further embodiment, a kit comprises a CD19xCD3 BiTE.RTM. molecule
(e.g., MT103) formulation of the invention, in one or more
containers, and one or more other prophylactic or therapeutic
agents useful for the prevention, treatment, management or
amelioration of B cell diseases and disorders, such as, but not
limited to, B cell malignancies, autoimmune diseases,
graft-versus-host disease (GVHD), humoral rejection, and
post-transplantation lymphoproliferative disorder, or one or more
symptoms thereof, in a human subject. In a specific embodiment, the
lyophilized formulations of the invention are formulated in single
dose vials as a sterile lyophilized cake consisting of MT103,
citrate, trehalose dehydrate, lysine HCl, and polysorbate 80. The
formulations of the invention may be supplied in 3 cc USP Type I
borosilicate amber vials (e.g., West Pharmaceutical Services--Part
No. 6800-0675). The kit may further comprise instructions for
method of administration of a reconstituted formulation of a
CD19xCD3 BiTE.RTM. molecule (e.g., MT103) described herein.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
5.21. Articles of Manufacture
[0694] The present invention also encompasses a finished packaged
and labeled pharmaceutical product. This article of manufacture
includes the appropriate unit dosage form in an appropriate vessel
or container such as a glass vial or other container that is
hermetically sealed. The unit dosage form is provided as a sterile
lyophilized powder comprising BiTE.RTM. molecules wherein said
lyophilized powder is suitable for reconstitution and subsequent
parenteral administration. In one embodiment, a unit dosage form is
provided as a sterile lyophilized powder comprising a CD19xCD3
BiTE.RTM. molecule wherein said lyophilized powder is suitable for
reconstitution and subsequent parenteral administration. In a
specific embodiment, a unit dosage form is provided as a sterile
lyophilized powder comprising MT103 wherein said lyophilized powder
is suitable for reconstitution and subsequent parenteral
administration. In one embodiment, the reconstituted unit dosage
form is suitable for intravenous, intramuscular, intranasal, oral,
topical or subcutaneous delivery. Thus, the invention encompasses
sterile solutions suitable for each delivery route.
[0695] As with any pharmaceutical product, the packaging material
and container are designed to protect the stability of the product
during storage and shipment. Further, the products of the invention
include instructions for use or other informational material that
advise the physician, technician or patient on how to appropriately
prevent or treat the disease or disorder in question. In other
words, the article of manufacture includes instruction means
indicating or suggesting a dosing regimen including, but not
limited to, actual doses, monitoring procedures, and other
monitoring information.
[0696] Specifically, the invention provides an article of
manufacture comprising packaging material, such as a box, bottle,
tube, vial, container, sprayer, insufflator, intravenous (i.v.)
bag, envelope and the like; and at least one unit dosage form of a
pharmaceutical agent contained within said packaging material,
wherein said pharmaceutical agent comprises a lyophilized
formulation containing BiTE.RTM. molecules (e.g., MT103). The
packaging material includes instruction means which indicate that
said BiTE.RTM. molecule can be used to prevent, treat, manage or
ameliorate one or more symptoms associated with a B cell disease
and disorder, such as, but not limited to, B cell malignancy,
autoimmune disease, graft-versus-host disease (GVHD), humoral
rejection, and post-transplantation lymphoproliferative disorder in
a human subject by administering specific doses and using specific
dosing regimens as described herein.
[0697] The invention also provides an article of manufacture
comprising packaging material, such as a box, bottle, tube, vial,
container, sprayer, insufflator, intravenous (i.v.) bag, envelope
and the like; and at least one unit dosage form of each
pharmaceutical agent contained within said packaging material,
wherein one pharmaceutical agent comprises a lyophilized
formulation containing a CD19xCD3 BiTE.RTM. molecule (e.g., MT103)
and the other pharmaceutical agent comprises a prophylactic or
therapeutic agent other than an antibody, and wherein said
packaging material includes instruction means which indicate that
said agents can be used to prevent, treat and/or manage one or more
symptoms associated with a B cell disease and disorder, such as,
but not limited to, B cell malignancy, autoimmune disease,
graft-versus-host disease (GVHD), humoral rejection, and
post-transplantation lymphoproliferative disorder in a human
subject by administering specific doses and using specific dosing
regimens as described herein.
[0698] The present invention provides that the adverse effects that
may be reduced or avoided by the methods of the invention are
indicated in informational material enclosed in an article of
manufacture for use in preventing, treating and/or managing one or
more symptoms associated with a B cell disease and disorder, such
as, but not limited to, B cell malignancy, autoimmune disease,
graft-versus-host disease (GVHD), humoral rejection, and
post-transplantation lymphoproliferative disorder in a human
subject. Adverse effects that may be reduced or avoided by the
methods of the invention include, but are not limited to, vital
sign abnormalities (fever, tachycardia, bardycardia, hypertension,
hypotension), hematological events (anemia, lymphopenia,
leukopenia, thrombocytopenia), headache, chills, dizziness, nausea,
asthenia, back pain, chest pain (chest pressure), diarrhea,
myalgia, pain, pruritus, psoriasis, rhinitis, sweating, injection
site reaction, and vasodilatation.
5.22. Specific Embodiments
[0699] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0700] 1. A sterile liquid pharmaceutical formulation comprising a
bispecific antibody and lysine, wherein the bispecific antibody
comprises a first and second binding domains, wherein the first
binding domain specifically binds the CD3 T cell surface
antigen.
[0701] 2. The formulation of claim 1, wherein the formulation is a
reconstituted liquid formulation.
[0702] 3. The formulation of any one of claim 1 or 2, wherein the
first binding domain comprises the amino acid sequence of residues
283-525 of SEQ ID NO:5.
[0703] 4. The formulation of any one of claims 1-3, wherein the
second binding domain specifically binds an antigen selected from
the group consisting of: CD19, CD20, CD22, EphA2, EphA4, INFR,
ICOS, Ep-CAM, CEA, and IL-5 receptor.
[0704] 5. The formulation of claim 4, wherein the second binding
domain specifically binds CD19.
[0705] 6. The formulation of claim 5, wherein the second binding
domain comprises the amino acid sequence of residues 28-277 of SEQ
ID NO:5.
[0706] 7. The formulation of claim 5, wherein the bispecific
antibody comprises the amino acid sequence of residues 28-525 of
SEQ ID NO:5.
[0707] 8. The formulation of any one of claims 1-7, wherein the
formulation is an aqueous formulation.
[0708] 9. The formulation of any one of claims 1-8, wherein the
formulation is isotonic, hypertonic, or hypotonic.
[0709] 10. The formulation of any one of claim 1 or 2-9, wherein
the formulation is suitable for lyophilization.
[0710] 11. The formulation of any one of claims 1-10, wherein the
formulation comprises between about 1 microgram/ml and about 300
micrograms/ml of the bispecific antibody.
[0711] 12. The formulation of any one of claims 1-11, wherein the
formulation further comprises a buffering agent selected from the
group consisting of: histidine, citrate, phosphate, glycine, and
acetate.
[0712] 13. The formulation of any one of claims 1-12, wherein the
formulation further comprises a carbohydrate excipient selected
from the group consisting of: sucrose, trehalose, lactose,
mannitol, and raffinose.
[0713] 14. The formulation of any one of claims 1-13, wherein the
formulation further comprises a surfactant selected from the group
consisting of: Polysorbate 20, Polysorbate 40, Polysorbate 60, or
Polysorbate 80.
[0714] 15. The formulation of any one of claims 1-11, wherein the
formulation further comprises citrate, trehalose, and Polysorbate
80.
[0715] 16. The formulation of claim 15, wherein the formulation
comprises between about 5 mM and about 125 mM citrate, between
about 25 mM and about 400 mM lysine, between about 3% and about 50%
trehalose, and between about 0.01% and about 1% Polysorbate 80, and
has a pH of between about 4.0 and about 8.0.
[0716] 17. The formulation of any one of claims 1-16, wherein the
formulation comprises about 55 micrograms/ml bispecific antibody,
about 25 mM citrate, about 200 mM lysine, about 15% trehalose and
about 0.1% Polysorbate 80, and has a pH of about 7.0.
[0717] 18. The formulation of any one of claims 1-16, wherein the
formulation comprises about 150 micrograms/ml bispecific antibody,
about 30 mM citrate, about 75 mM lysine, about 6.5% trehalose and
about 0.02% Polysorbate 80 and has a pH of about 7.0.
[0718] 19. The formulation of any one of claims 1-16, wherein the
formulation comprises about 150 micrograms/ml bispecific antibody,
about 30 mM citrate, about 75 mM lysine, about 6% trehalose and
about 0.02% Polysorbate 80 and has a pH of about 7.0.
[0719] 20. The formulation of any one of claims 1-16, wherein the
formulation comprises about 160 micrograms/ml bispecific antibody,
about 25 mM citrate, about 200 mM lysine, about 15% trehalose and
about 0.1% Polysorbate 80, and has a pH of about 7.0.
[0720] 21. The formulation of any one of claims 1-16, wherein the
bispecific antibody retains at least 90% of binding ability to CD19
and CD3 antigens during storage at about 5.degree. C. for at least
about 1 month as compared to a reference antibody representing the
antibody prior to said storage.
[0721] 22. The formulation of any one of claims 1-16, wherein less
than 5% of the bispecific antibody forms an aggregate as determined
by HPSEC upon storage at about 5.degree. C. for about 1 month.
[0722] 23. The formulation of any one of claims 1-16, wherein less
than 5% of the bispecific antibody forms a dimer as determined by
HPSEC upon storage at about 5.degree. C. for about 1 month.
[0723] 24. The formulation of any one of claims 1-16, wherein less
than 5% of the bispecific antibody forms an aggregate as determined
by HPSEC upon shaking a vial filled to half of its volume with a
sample of said formulation for 4 hours at a speed of 400 shakes per
minute.
[0724] 25. The formulation of any one of claims 1-16, wherein less
than 5% of the antibody forms an aggregate as determined by HPSEC
upon subjecting the formulation to three freeze/thaw cycles.
[0725] 26. The formulation of any one of claims 1-16, wherein less
than 5% of the antibody forms a dimer as determined by HPSEC upon
shaking a vial filled to half of its volume with a sample of said
formulation for 4 hours at a speed of 400 shakes per minute.
[0726] 27. The formulation of any one of claims 1-16, wherein less
than 5% of the antibody forms a dimer as determined by HPSEC upon
subjecting the formulation to three freeze/thaw cycles.
[0727] 28. The formulation of any one of claims 1-16, wherein at
least about 95% of the antibody is recovered by reconstituting a
lyophilized cake generated from the formulation.
[0728] 29. The formulation of any one of claims 1-16, wherein the
formulation is clear and colorless as determined by visual
inspection upon storage at about 5.degree. C. for at least about 1
month.
[0729] 30. A sterile lyophilized pharmaceutical formulation
comprising a bispecific antibody and lysine, wherein the bispecific
antibody comprises a first and second binding domains, wherein the
first binding domain specifically binds the CD3 T cell surface
antigen.
[0730] 31. The formulation of claim 30, wherein the first binding
domain comprises the amino acid sequence of residues 283-525 of SEQ
ID NO:5.
[0731] 32. The formulation of any one of claim 30 or 31, wherein
the second binding domain specifically binds an antigen selected
from the group consisting of: CD19, CD20, CD22, EphA2, EphA4, INFR,
ICOS, Ep-CAM, CEA, and IL-5 receptor.
[0732] 33. The formulation of claim 32, wherein the second binding
domain specifically binds CD19.
[0733] 34. The formulation of claim 33, wherein the second binding
domain comprises the amino acid sequence of residues 28-277 of SEQ
ID NO:5.
[0734] 35. The formulation of claim 33, wherein the bispecific
antibody comprises the amino acid sequence of residues 28-525 of
SEQ ID NO:5.
[0735] 36. The formulation of any one of claims 30-35, wherein the
formulation further comprises a buffering agent selected from the
group consisting of: histidine, citrate, phosphate, glycine, and
acetate.
[0736] 37. The formulation of any one of claims 30-36, wherein the
formulation further comprises a carbohydrate excipient selected
from the group consisting of: sucrose, trehalose, lactose,
mannitol, and raffinose.
[0737] 38. The formulation of any one of claims 30-37, wherein the
formulation further comprises a surfactant selected from the group
consisting of: Polysorbate 20, Polysorbate 40, Polysorbate 60, or
Polysorbate 80.
[0738] 39. The formulation of any one of claims 30-35, wherein the
formulation further comprises citrate, trehalose, and Polysorbate
80.
[0739] 40. The formulation of any one of claims 30-39, wherein at
least about 90% of the bispecific antibody is recovered by
reconstituting the lyophilized formulation upon storage at about
5.degree. C. for at least about 1 year.
[0740] 41. The formulation of any one of claims 30-39, wherein less
than 5% of the bispecific antibody forms an aggregate as determined
by HPSEC upon reconstitution following storage of the lyophilized
formulation at about 5.degree. C. for at least about 1 year.
[0741] 42. The formulation of any one of claims 30-39, wherein less
than 5% of the bispecific antibody forms a dimer as determined by
HPSEC upon reconstitution following storage of the lyophilized
formulation at about 5.degree. C. for at least about 1 year.
[0742] 43. The formulation of any one of claims 30-39, wherein
following storage of the lyophilized formulation at about 5.degree.
C. for at least about 1 month, upon reconstitution the bispecific
antibody retains at least 90% of binding ability to CD19 and CD3
antigens as compared to a reference antibody representing the
antibody prior to said storage.
[0743] 44. A sterile liquid reconstituted pharmaceutical
formulation comprising a bispecific antibody and lysine, wherein
the bispecific antibody comprises a first and second binding
domains, wherein the first binding domain specifically binds the
CD3 T cell surface antigen.
[0744] 45. The formulation of claim 44, wherein the first binding
domain comprises the amino acid sequence of residues 283-525 of SEQ
ID NO:5.
[0745] 46. The formulation of any one of claim 44 or 45, wherein
the second binding domain specifically binds an antigen selected
from the group consisting of: CD19, CD20, CD22, EphA2, EphA4, INFR,
ICOS, Ep-CAM, CEA, and IL-5 receptor.
[0746] 47. The formulation of claim 46, wherein the second binding
domain specifically binds CD19.
[0747] 48. The formulation of claim 47, wherein the second binding
domain comprises the amino acid sequence of residues 28-277 of SEQ
ID NO:5.
[0748] 49. The formulation of claim 47, wherein the bispecific
antibody comprises the amino acid sequence of residues 28-525 of
SEQ ID NO:5.
[0749] 50. The formulation of any one of claims 44-49, wherein the
formulation is an aqueous formulation.
[0750] 51. The formulation of any one of claims 44-50, wherein the
formulation is isotonic, hypertonic, or hypotonic.
[0751] 52. The formulation of any one of claims 44-51, wherein the
formulation comprises between about 1 microgram/ml and about 300
micrograms/ml of the bispecific antibody.
[0752] 53. The formulation of any one of claims 44-52, wherein the
formulation further comprises a buffering agent selected from the
group consisting of: histidine, citrate, phosphate, glycine, and
acetate.
[0753] 54. The formulation of any one of claims 44-53, wherein the
formulation further comprises a carbohydrate excipient selected
from the group consisting of: sucrose, trehalose, lactose,
mannitol, and raffinose.
[0754] 55. The formulation of any one of claims 44-54, wherein the
formulation further comprises a surfactant selected from the group
consisting of: Polysorbate 20, Polysorbate 40, Polysorbate 60, or
Polysorbate 80.
[0755] 56. The formulation of any one of claims 44-52, wherein the
formulation further comprises citrate, trehalose, and Polysorbate
80.
[0756] 57. The formulation of any one of claims 44-56, wherein the
formulation comprises between about 5 mM and about 125 mM citrate,
between about 25 mM and about 400 mM lysine, between about 3% and
about 50% trehalose, and between about 0.01% and about 1%
Polysorbate 80, and has a pH between about 4.0 and 8.0.
[0757] 58. The formulation of any one of claims 44-56, wherein the
formulation comprises about 55 micrograms/ml bispecific antibody,
about 25 mM citrate, about 200 mM lysine, about 15% trehalose and
about 0.1% Polysorbate 80, and has a pH of about 7.0.
[0758] 59. The formulation of any one of claims 44-56, wherein the
formulation comprises about 150 micrograms/ml bispecific antibody,
about 30 mM citrate, about 75 mM lysine, about 6.5% trehalose and
about 0.02% Polysorbate 80 and has a pH of about 7.0.
[0759] 60. The formulation of any one of claims 44-56, wherein the
formulation comprises about 150 micrograms/ml bispecific antibody,
about 30 mM citrate, about 75 mM lysine, about 6% trehalose and
about 0.02% Polysorbate 80 and has a pH of about 7.0.
[0760] 61. The formulation of any one of claims 44-56, wherein the
formulation comprises about 160 micrograms/ml bispecific antibody,
about 25 mM citrate, about 200 mM lysine, about 15% trehalose and
about 0.1% Polysorbate 80, and has a pH of about 7.0.
[0761] 62. The formulation of any one of claims 44-61, wherein the
bispecific antibody retains at least 90% of binding ability to the
CD19 and CD3 antigens during storage at about 5.degree. C. for at
least about 12 hours as compared to a reference antibody
representing the antibody prior to said storage.
[0762] 63. The formulation of any one of claims 44-61, wherein less
than 5% of the bispecific antibody forms an aggregate as determined
by HPSEC upon storage at about 5.degree. C. for about 12 hours.
[0763] 64. The formulation of any one of claims 44-61, wherein less
than 5% of the bispecific antibody forms a dimer as determined by
HPSEC upon storage at about 5.degree. C. for about 12 hours.
[0764] 65. The formulation of any one of claims 44-61, wherein the
formulation is clear and colorless as determined by visual
inspection upon storage at about 5.degree. C. for at least about 12
hours.
[0765] 66. The formulation of any one of claim 1-29 or 44-65,
wherein the formulation is an injectable formulation.
[0766] 67. The formulation of claim 66, wherein the formulation is
for intravenous, subcutaneous, or intramuscular administration.
[0767] 68. The formulation of claim 67, wherein the formulation is
suitable for subcutaneous administration.
[0768] 69. The formulation of claim 68, wherein the bioavailability
of the bispecific antibody following subcutaneous delivery of the
formulation is between about 10% and about 50% as compared to the
bioavailability of the bispecific antibody following intravenous
delivery of the same dose of the formulation.
[0769] 70. The pharmaceutical unit dosage form of a formulation of
any one of claims 1-69, wherein the antibody formulation is
administered intravenously, subcutaneously, or intramuscularly.
[0770] 71. A sealed container containing the formulation of any one
of claims 1-69.
[0771] 72. A kit comprising the formulation of any one of claims
1-69.
[0772] 73. A method of preventing, managing, treating or
ameliorating B cell disorders or diseases such as, B cell
malignancies, autoimmune diseases, graft-versus-host disease
(GVHD), humoral rejection, and post-transplantation
lymphoproliferative disorder in a human subject, said methods
comprising administering to a subject in need thereof a
prophylactically or therapeutically effective amount of the
formulation of any one of claims 1-69.
[0773] 74. The method of claim 73, wherein the disease or disorder
is a B cell malignancy.
[0774] 75. A method of making the formulation of any one of claims
1-69, comprising: combining the bispecific antibody with
lysine.
[0775] 76. A method of stabilizing a bispecific antibody,
comprising: combining said antibody with lysine, wherein the
bispecific antibody comprises a first and second binding domains,
wherein the first binding domain specifically binds the CD3 T cell
surface antigen.
6. EXAMPLES
6.1. Development of MT103 Lyophilized Formulation I
[0776] All chemicals used were USP grade or higher. Trehalose
dehydrate was obtained from Ferro Pfanstiehl; polysorbate 80,
arginine HCl, lysine HCl, citrate, sodium sulphate and potassium
phosphate may be obtained from JT Baker or Sigma; NaOH and HCl was
obtained from VWR.
6.1.1. Analytical Methods
[0777] Visual Inspection: Visual inspection of lyophilized cakes
was performed by visually examining the cakes for their general
shape and for any visible signs of collapse, melt back, flakiness,
cracks, shrinkage, or other apparent defects. Reconstituted vials
were inspected for presence of particulates.
[0778] Size Exclusion Chromatography (SEC): SEC was used to
determine the level of aggregate present in a sample and to
determine the concentration of MT103 protein in the sample. SEC
analysis was performed by injecting 2 to 100 .mu.L of the sample on
an Agilent HPLC system with a TosoHaas G3000 SW.sub.xL column
(7.8.times.300 mm) using fluorescence detection (Excitation at 280
nm, Emission at 345 nm) at a flow rate of 1 mL per minute. The
mobile phase consisted of 0.1 M KH.sub.2PO.sub.4, 0.2 M
Na.sub.2SO.sub.4 at pH 6.6 in HPLC grade water. MT103 was
quantitated using a three point bracketed standard curve generated
by injecting a working reference standard on each run. The SEC
assay served as a surrogate tool for A280 measurements because the
concentration of the working reference standard was previously
correlated with UV absorbance at 280 nm (A280) using the extinction
coefficient determined from amino acid analysis
(.epsilon.=2.16).
[0779] Moisture Analysis: Residual moisture content of lyophilized
cakes was determined by Coulometric Karl Fisher titration. Briefly,
lyophilized cakes were weighed and dissolved in 1 ml anhydrous
methanol. A fixed weight of the dissolved sample was transferred
into a coulometric Karl Fisher titrator with a diaphragm-free cell.
The residual moisture content of the lyophilized sample is
expressed as % w/w.
[0780] Determination of protein recovery: Vials containing
lyophilized MT103 were accurately weighed before and after
reconstitution. The MT103 protein solution was then transferred to
another vial and the original vial was thoroughly rinsed, dried and
weighed. Concentration of reconstituted MT103 protein was
determined by SEC analysis. The protein recovery was calculated
using the experimentally determined net weight of the reconstituted
cakes in each vial as described above, and the concentration
determined by SEC.
6.1.2. Formulation
[0781] Buffer Preparation: Buffers were prepared with USP or
multi-compendial grade chemicals. All pH adjustments were done with
either 1 N HCl or 1 N NaOH as appropriate. All buffers were
filtered through 0.22 .mu.m filters and stored at 2 to 8.degree. C.
and were used within two weeks of preparation.
[0782] Dialysis: Buffer exchanges were performed using Pierce
dialysis cassettes with capacities ranging from 0.5 to 12 mL as
appropriate for the volume to be dialyzed. All exchanges were
performed prior to addition of polysorbate 80. At a minimum, two
serial exchanges were performed, each with at least a 100-fold
excess of the target formulation buffer over a minimum period of 4
hours. Complete exchange was confirmed by matching the osmolality
and pH of the dialyzed protein solution recovered from the dialysis
cassette with that of the target formulation buffer. Protein
recovered from the dialysis cassettes was immediately formulated
with polysorbate 80 where appropriate, and filtered through 0.22
.mu.m syringe filters and stored at 2 to 8.degree. C. until
used.
[0783] Evaluation of stability to freeze-thaw stress: Samples (0.5
to 1.0 mL) were transferred into 1 mL polypropylene cryovials and
subjected to three freeze-thaw cycles. Vials of the matching
formulation that served as a control were maintained at 2 to
8.degree. C. and did not undergo any freeze-thaw. Each freeze-thaw
cycle consisted of freezing in a -70.degree. C. environmental
chamber for a minimum of 8 hours and thawing at 2 to 8.degree. C.
until completely thawed. Following the final thaw, the samples were
gently mixed and analyzed by SEC.
[0784] Evaluation of stability to shaking stress: Shaking studies
were performed at room temperature in a thermally insulated
container on a laboratory benchtop shaker (VWR Digital Mini
Vortexer) at the maximum setting of 10 (approx. 400 shakes per
minute) over a period of 2 to 4 hours. The vials were placed
horizontally with the length of the vials along the direction of
shaking with the objective of maximizing turbulence, leading to an
increase in the surface area of the air-liquid interface. Vials of
the matching formulation that served as a control were maintained
at 2 to 8.degree. C. and did not undergo any shaking. Samples from
the shaken vials and the unshaken controls were then analyzed by
SEC.
[0785] Fill/Finish procedure: All glass vials were triple rinsed
with deionized water and sterilized prior to use. The rubber
stoppers were purchased pre-washed and were sterilized prior to
use. All fills were done manually using Eppendorf repeat pipettor
(model) and were carried out inside a laminar flow hood. All
solutions were filtered inside the laminar flow hood through 0.22
.mu.m sterilizing filter before the fills. Vials were each filled
with 0.6 mL of the respective formulations (setting #3 when using a
10 mL Eppendorf disposable cartridge insert). Vials were then
stoppled and transferred onto lyophilization trays for transfer to
the shelves on the lyophilizer. Following lyophilization, the vials
were capped with 13 mm aluminum overseals either manually or with a
semi-automated capper.
[0786] Lyophilization: During the course of these studies two
lyophilization cycle variations were evaluated. The cycles are
defined in Table 1 below. Evaluations were performed using an
external condenser lyophilizer (Quantum 20.times. retrofitted by
Hull).
TABLE-US-00001 TABLE 1 Lyophilization cycle variations tested. Non
annealing Annealing cycle cycle Description (L1) (L2) Total Lyo
Cycle Length 96 hr 103 hr Load Temp 20.degree. C. 20.degree. C.
Thermal Initial Temp 8.degree. C. 8.degree. C. Treatment Freeze
Temp -40.degree. C. Phase Ramp rate 0.3.degree. C./min Hold Time at
freeze 150 min temp Annealing Temp -25.degree. C. Annealing ramp
rate 0.5.degree. C./min Hold Time at Annealing 300 min temp Final
Freeze Temp -40.degree. C. -40.degree. C. Ramp rate 0.3.degree.
C./min 0.5.degree. C./min Hold Time at Final 150 min 60 min Freeze
temp Condenser temp set -50.degree. C. -50.degree. C. Vacuum set 60
mT 60 mT Primary Temp Set -35.degree. C. -35.degree. C. Drying Hold
Time 68 hr 68 hr Secondary Temp Set 20.degree. C. 20.degree. C.
Drying Ramp rate 0.3.degree. C./min 0.3.degree. C./min Hold Time 19
hr 19 hr Stoppering Backfill Nitrogen Nitrogen
6.1.3. Overview of Formulation Studies
[0787] 17 different formulations were evaluated in sequential
campaigns, with each campaign consisting of three to five
formulations lyophilized in the same run. A non-annealing
lyophilization cycle (L1, Table 1) was used as the default cycle
for all formulations except where the annealing L2 cycle is
specifically indicated. All formulations discussed in this report
were formulated at a target concentration of 50 to 55 .mu.g of
MT103 per mL. All formulations were prepared with 25 mM citrate as
the buffering agent, and none of the formulations contained lysine
acetate or PEG 3350. Formulations in select campaigns were
evaluated for stability to freeze-thaw, shaking stresses and
accelerated stability in the liquid form. Lyophilized formulations
were evaluated for visual appearance, aggregate and concentration
(SEC-HPLC), accelerated stability, and protein recovery. Residual
moisture content was determined for selected formulations; moisture
content was within the .gtoreq.2% specification. The various
formulations evaluated are listed in FIGS. 1, 9, and 11, and Table
2.
TABLE-US-00002 TABLE 2 Summary of formulations analysed.
Formulation Composition ID # Each formulation consists of 25 mM
citrate PLUS the following: 0 15% trehalose dihydrate ph 7 1 15%
trehalose dihydrate 0.1% polysorbate 80 pH 7 2 15% trehalose
dihydrate 0.1% polysorbate 80, 100 mM arginine HCl pH 7 3, 14 15%
trehalose dihydrate 0.1% polysorbate 80, 500 mM arginine HCl pH 7 4
15% trehalose dihydrate, 0.1% polysorbate 80, 100 mM NaCl pH 7 5
15% trehalose dihydrate, 0.1% polysorbate 80, 500 mM NaCl pH 7 6,
15, 16 15% trehalose dihydrate, 0.1% polysorbate 80, 500 mM lysine
HCl pH 7 7 15% trehalose dihydrate, 0.1% polysorbate 80, 625 mM
lysine base pH 7 8 5% mannitol, 0.1% polysorbate 80 pH 7 9 5%
trehalose dihydrate, 0.1% polysorbate 80, 500 mM arginine HCl pH 7
10 5% Mannitol pH 7 11 15% trehalose dihydrate, 0.1% polysorbate
80, 100 mM arginine HCl pH 6 12 15% trehalose dihydrate, 0.1%
polysorbate 80, 100 mM arginine HCl pH 7 13 15% trehalose
dihydrate, 0.1% polysorbate 80, 500 mM arginine HCl pH 6 17, 30, 33
15% trehalose dihydrate, 0.1% polysorbate 80, 300 mM lysine HCl pH
7 18, 27, 31 15% trehalose dihydrate, 0.1% polysorbate 80, 100 mM
lysine HCl pH 7 19 15% trehalose dihydrate, 0.175% polysorbate 80,
100 mM lysine HCl pH 7 20, 28 15% trehalose dihydrate, 0.25%
polysorbate 80, 100 mM lysine HCl pH 7 21 10% trehalose dihydrate,
0.1% polysorbate 80, 500 mM lysine HCl pH 7 22 10% trehalose
dihydrate, 0.1% polysorbate 80, 300 mM lysine HCl pH 7 23 10%
trehalose dihydrate, 0.1% polysorbate 80, 100 mM lysine HCl pH 7 24
10% trehalose dihydrate, 0.175% polysorbate 80, 100 mM lysine HCl
pH 7 25 10% trehalose dihydrate, 0.25% polysorbate 80, 100 mM
lysine HCl pH 7 26 15% trehalose dihydrate, 0.25% polysorbate 80 pH
7 29, 32 15% trehalose dihydrate, 0.1% polysorbate 80, 200 mM
lysine HCl pH 7
[0788] The first campaign focused on assessing the need for the
presence of trehalose dihydrate and polysorbate 80. A formulation
containing arginine hydrochloride (500 mM) was evaluated as a
replacement for lysine acetate. A formulation containing only 5%
mannitol was utilized as a negative control.
[0789] The second campaign focused on an evaluation of arginine
hydrochloride (100 mM or 500 mM), lysine hydrochloride (500 mM) and
the effect of pH (pH 6 or pH 7).
[0790] The third campaign focused on determining the optimal level
of lysine hydrochloride (100, 300 and 500 mM) and polysorbate 80
(0.1, 0.175, 0.25% w/v) in presence of 25 mM citrate at pH 7 and
15% trehalose dihydrate. These studies were repeated in placebo
formulations in presence of 25 mM citrate at pH 7 and 10% trehalose
dihydrate in an attempt to reduce the amount of the sugar.
[0791] A subsequent campaign focused on further narrowing the level
of lysine hydrochloride by evaluating 100, 200 or 300 mM lysine
hydrochloride in presence of 25 mM citrate at pH 7 and 15%
trehalose dihydrate, and 0.1% polysorbate 80. Both non-annealing
(L1) and annealing (L2) cycles were studied for these
formulations.
[0792] An optimization of the lyophilization cycle was then
performed to determine if the durations of primary and secondary
cycles could be shortened without any product impact. This was
accomplished by pulling vials from the lyophilizer at various time
points prior to completion of the primary and secondary cycles
using a sample thief.
[0793] Finally, the lyophilization of the selected formulation was
repeated using the selected cycle to confirm reproducibility.
6.1.4. Excipient Screening
[0794] Formulations evaluated in the excipient screening study are
listed in FIG. 1.
[0795] FIG. 2 shows that the excipients polysorbate 80 and
trehalose markedly improve the recovery of MT103 from lyophilized
formulations. Addition of 15% trehalose (in formulation F0)
improves protein recovery from 40 percent (formulation F10) to 70
percent. A further 20 percent improvement in recovery is obtained
upon inclusion of 0.1% polysorbate 80 (formulation F1) with the 15%
trehalose. Inclusion of 100 or 500 mM arginine HCl did not further
improve recovery, however, it contributed towards the formation of
very elegant lyophilized cakes.
[0796] Upon evaluating the excipients in a liquid stability,
several interesting findings came to light. Arginine HCl induced
the formation of protein aggregates in the liquid state. The rate
of aggregation increased with an increase in arginine HCl
concentration from 100 mM to 500 mM (FIGS. 3 and 4). The rate of
aggregation also increased with an increase in protein
concentration from 50 to 300 .mu.g/mL (FIGS. 3 and 4).
[0797] Quantitation of protein recovery from the formulations in a
freeze-thaw study demonstrated that formulations containing
trehalose and polysorbate 80 (F3) conferred better protection to
the protein during the freezing stresses compared to formulations
lacking polysorbate (F1) or both polysorbate and trehalose (F10).
Presence of arginine HCl did not contribute any additional benefit.
Identical findings were obtained in a shaking study (FIG. 6).
[0798] Evaluation of recovery in liquid bulks in a short-term
liquid stability study at 40.degree. C. over a period of 7 days
further confirmed the above findings.
[0799] In a post-reconstitution evaluation of stability of MT103
over 48 hours was performed.
6.1.5. Formulation Optimization
[0800] Selection of formulation pH: Formulations evaluated in the
pH screening study are listed in FIG. 9. The pH evaluation was
limited to within a narrow range of 6 and 7 because it was
desirable to be as close to neutral as possible. Higher pHs were
avoided due to the enhanced likelihood of deamidation at NG motifs
in the MT103 sequence. The present efforts were aimed at
determining if recovery could be improved marginally by a small
change in the formulation pH. Formulations pairs F11-F12 and
F13-F14 compared the effect of pH in presence of either 100 or 500
mM Arginine HCl, respectively, while the other excipients were held
constant. Lysine HCl was introduced in this set for evaluation as a
potential replacement for arginine HCl due to the earlier
observation that arginine HCl promoted rapid aggregation in liquid
MT103 formulations.
[0801] There did not appear to be any significant effect of the
formulation pH on recoveries (FIG. 10). However, as before, a
significant increase in the aggregate levels was seen with the
formulations containing 500 mM arginine HCl (F13 and F14)
irrespective of the pH. It was determined that this increase
occurred in the liquid state prior to lyophilization, and there was
no further change after lyophilization (data not shown).
Interestingly, formulation F15 with lysine HCl at a comparable
concentration (500 mM) showed no increase in aggregate levels while
providing comparable recoveries to F14. However, F15 exhibited
signs of significant collapse, suggesting that it may not yield
visually acceptable cakes.
[0802] Based on the evidence obtained thus far, it appeared that
arginine HCl promoted aggregation in the liquid state and was
therefore undesirable as an excipient. Lysine HCl was found to be a
suitable replacement that prevented aggregation in liquid
formulations, but needed further lyophilization optimization. pH 7,
which is closer to physiologic, was selected as the pH of choice
for the formulation because there was no difference between pH 6
and 7 on MT103 recovery or aggregate levels.
[0803] Selection of Trehalose concentration: Two levels of
trehalose (10% and 15%) were evaluated using blank (placebo)
formulations to conserve protein. It was assumed that due to the
extremely high trehalose/protein ratios which were far in excess of
levels minimally recommended for effective cryo- and lyo-protection
of protein, the effects would be limited to visual appearance of
the cakes, and there would be no impact on protein recovery or
aggregation.
TABLE-US-00003 TABLE 3 Formulations analysed in trehalose
concentration screen. Formulation Composition ID # Each formulation
consists of 25 mM citrate PLUS the following: B16 15% trehalose
dihydrate, 0.1% polysorbate 80, 500 mM lysine HCl pH 7 B17 15%
trehalose dihydrate, 0.1% polysorbate 80, 300 mM lysine HCl pH 7
B18 15% trehalose dihydrate, 0.1% polysorbate 80, 100 mM lysine HCl
pH 7 B19 15% trehalose dihydrate, 0.175% polysorbate 80, 100 mM
lysine HCl pH 7 B20 15% trehalose dihydrate, 0.25% polysorbate 80,
100 mM lysine HCl pH 7 B21 10% trehalose dihydrate, 0.1%
polysorbate 80, 500 mM lysine HCl pH 7 B22 10% trehalose dihydrate,
0.1% polysorbate 80, 300 mM lysine HCl pH 7 B23 10% trehalose
dihydrate, 0.1% polysorbate 80, 100 mM lysine HCl pH 7 B24 10%
trehalose dihydrate, 0.175% polysorbate 80, 100 mM lysine HCl pH 7
B25 10% trehalose dihydrate, 0.25% polysorbate 80, 100 mM lysine
HCl pH 7
[0804] Upon visually examining four placebo formulation pairs which
were otherwise perfectly excipient matched except for containing
10% or 15% trehalose (B17-B22; B18-B23; B19-B24; B20-B25), it was
observed that the cake height for formulations with the 10%
trehalose was low at less than 4 mm. This effect was attributed to
the lower total solids content, combined with the lack of a
suitable bulking agent, and a low 0.6 mL initial fill volume. The
formulations with 15% trehalose had cake heights of approx 5 mm or
greater, and looked more aesthetic. The formulation containing 10%
trehalose with a lysine HCl concentration of 300 mM (B22) showed
total collapse but the matching 15% trehalose formulation (B17) had
a well formed cake, presumably due to the extra bulk. At a higher
lysine content of 500 mM, both the 10% trehalose (B16) and the 15%
trehalose (B21) formulations completely collapsed. This suggested
that higher trehalose content could provide enough additional bulk
to protect against collapse in presence of lysine HCl
concentrations up to 300 mM. Mechanistically, this phenomenon can
be explained by the fact that pure lysine HCl has a Tg' well below
-50.degree. C. (Lueckel, et al, 1997), and the additional
trehalose, with a Tg' of -28.degree. C., helps in raising the
collapse temperature of the mixture to above that of the effective
primary drying temperature during lyophilization.
[0805] Based on these observations, 15% trehalose concentration was
selected because it allowed for successful lyophilization with the
use of as much as 300 mM lysine HCl.
[0806] Selection of Polysorbate 80 concentration: The initial
excipient screen demonstrated that the presence of the non-ionic
surfactant polysorbate 80 in formulation F1 at a concentration of
0.1% significantly improved recovery by approximately 20% when
compared to formulation F0 that lacked the polysorbate 80. FIGS. 12
to 15 show that 0.25% polysorbate 80 (F27) does not provide any
additional benefit over 0.1% polysorbate 80 (F28). In order to
address the concerns that 0.1% polysorbate could have potential
adverse effects on the formation of lyophilized cakes due to its
ability to act as a plasticizer, matched placebo formulations
differing only in the concentration of polysorbate 80 (0.1, 0.175
and 0.25%) were lyophilized and visually inspected (B18, B19, B20
in Table 4). No visible differences were observed in the
lyophilized cakes. There were no differences in the moisture levels
of the cakes as determined by thermogravimetric analysis. Moisture
levels for the two bracketing formulations B18 and B20 were
determined to be comparable at 1.7%.+-.0.6 for B18 (N=5) and
1.7.+-.0.8 for B20 (N=4). Therefore it was concluded that 0.1%
polysorbate 80 was an acceptable concentration and had no adverse
effects on cake formation. Lower polysorbate 80 concentrations may
be evaluated during future optimization of this formulation.
TABLE-US-00004 TABLE 4 Formulations analysed in polysorbate
concentration optimization screen. Formulation Composition ID #
Each formulation consists of 25 mM citrate PLUS the following: F0
15% trehalose dihydrate ph 7 F1 15% trehalose dihydrate 0.1%
polysorbate 80 pH 7 B18 15% trehalose dihydrate, 0.1% polysorbate
80, 100 mM lysine HCl pH 7 B19 15% trehalose dihydrate, 0.175%
polysorbate 80, 100 mM lysine HCl pH 7 B20 15% trehalose dihydrate,
0.25% polysorbate 80, 100 mM lysine HCl pH 7
[0807] Lysine hydrochloride: The effect of lysine HCl in inhibiting
aggregation in liquid formulations was further evaluated by
analyzing the formulations listed in FIG. 11. FIGS. 12-15
demonstrate that lysine does not contribute to stabilization of
MT103 against the stresses of freeze-thaw or shaking as evidenced
by no changes seen in aggregate levels or recovery either in
absence of lysine HCl or in presence of 100, 200 or 300 mM lysine
HCl. Consequently, it was selected as an excipient solely to
minimize the tendency of MT103 to aggregate in liquid bulks. Based
on the results shown in FIGS. 12-15, lysine HCl concentrations
between 100 and 300 mM provide identical results. Higher
concentrations had shown a tendency to lead to collapsed
lyophilized cakes, and absence of lysine HCl was shown to promote
aggregation.
[0808] Stability of each of the three lysine containing liquid
formulations with 0.1% polysorbate 80 (F27, F29, F30) was evaluated
over 80 days and found to be stable with no decrease in recovery
and no significant increase in aggregate levels at 4.degree. C. in
polypropylene 1 mL tubes (FIGS. 16 and 17).
[0809] Stability of each of the three lysine containing lyophilized
formulations with 0.1% polysorbate 80 (F27, F29, F30) was evaluated
over 2 to 4 weeks and found to be stable with no significant
changes in the aggregate levels or recoveries (FIGS. 18 to 21). The
residual moistures for selected formulations (F27, F30) were 0.7%
and 1.2%, respectively, as determined by the Karl Fisher
method.
[0810] Based on the above evaluations, the formulation F29
comprising 25 mM citrate, 15% trehalose dihydrate, 200 mM lysine
HCl, 0.1% polysorbate 80 was selected. F29 formulation afforded
adequate protection to MT103 from freeze-thaw stresses and shaking
stresses, and also provided acceptable MT103 stability and recovery
from liquid and lyophilized formulations.
6.1.6. Lyophilization Cycle Development
[0811] Evaluation of annealing vs. non-annealing cycle: The effect
of annealing was evaluated on all three formulations (the lead
formulation and the two backups) by lyophilizing the same batch of
formulations (F31, F32, F33) containing 100, 200, and 300 mM lysine
HCl with a non-annealing (L1) and annealing (L2) lyophilization
cycle. The lyophilized formulations were visually examined and
their stability was evaluated at 4, and 40.degree. C. over a period
of approx 8 weeks (Tables 5 to 8). Recoveries of MT103 were
assessed immediately after lyophilization and also at various time
points for both 4 and 40.degree. C. conditions (Tables 9 and 10).
Based on these data, the non-annealed formulation with 200 mM
lysine HCl was selected.
TABLE-US-00005 TABLE 5 Stability of formulations F31, F32, F32
lyophilized in large lyophilizer, using an annealing cycle.
Lyophilized vials were stored at 4.degree. C. ID Composition Signal
Type Pre-lyo Day 0 Day 22 Day 35 Day 59 F31 25 mM citrate, 15%
trehalose, % Dimer 1.31 0.58 0.73 1.18 1.15 100 mM Lysine HCl, 0.1%
% Monomer 98.69 99.42 99.27 98.82 98.85 Tween80, pH 7 Conc (ug/mL)
58.2 52.4 49.8 56.7 52.0 F32 25 mM citrate, 15% trehalose, % Dimer
1.25 0.76 0.84 1.41 1.18 200 mM Lysine HCl, 0.1% % Monomer 98.75
99.24 99.16 98.59 98.82 Tween80, pH 7 Conc (ug/mL) 58.3 53.2 51.3
56.5 52.8 F33 25 mM citrate, 15% trehalose, % Dimer 1.26 0.77 0.94
1.49 1.31 300 mM Lysine HCl, 0.1% % Monomer 98.74 99.23 99.06 98.51
98.69 Tween80, pH 7 Conc (ug/mL) 58.3 53.4 51.1 55.3 52.3
TABLE-US-00006 TABLE 6 Stability of formulations F31, F32, F32
lyophilized in large lyophilizer, using an annealing cycle.
Lyophilized vials were stored at 40.degree. C. ID Composition
Signal Type Pre-lyo Day 0 Day 22 Day 35 Day 59 F31 25 mM citrate,
15% trehalose, % Dimer 1.31 0.58 0.85 1.17 1.3 100 mM Lysine HCl,
0.1% % Monomer 98.69 99.42 99.15 98.83 98.7 Tween80, pH 7 Conc.
(ug/mL) 58.2 52.4 51.2 54.1 52.7 F32 25 mM citrate, 15% trehalose,
% Dimer 1.25 0.76 0.85 1.3 1.19 200 mM Lysine HCl, 0.1% % Monomer
98.75 99.24 99.15 98.7 98.81 Tween80, pH 7 Conc (ug/mL) 58.3 53.2
50.9 54.7 51.9 F33 25 mM citrate, 15% trehalose, % Dimer 1.26 0.77
0.92 1.33 1.27 300 mM Lysine HCl, 0.1% % Monomer 98.74 99.23 99.08
98.67 98.73 Tween80, pH 7 Conc. (ug/mL) 58.3 53.4 51.0 53.9
51.6
TABLE-US-00007 TABLE 7 Stability of formulations F31, F32, F32
lyophilized in large lyophilizer, using a non-annealing cycle.
Lyophilized vials were stored at 4.degree. C. ID Composition Signal
Type Pre-lyo Day 0 Day 15 Day 28 Day 52 F31 25 mM citrate, 15%
trehalose, % Dimer 0.96 0.72 0.94 1.2 1.08 100 mM Lysine HCl, 0.1%
% Monomer 99.04 99.28 99.06 98.8 98.92 Tween80, pH 7 Conc (ug/mL)
55.7 58.2 50.6 52.8 51.8 F32 25 mM citrate, 15% trehalose, % Dimer
0.87 0.85 0.93 1.32 1.42 200 mM Lysine HCl, 0.1% % Monomer 99.13
99.15 99.07 98.68 98.58 Tween80, pH 7 Conc (ug/mL) 56.2 53.7 50.8
54.2 53.0 F33 25 mM citrate, 15% trehalose, % Dimer 0.75 0.85 0.99
1.43 1.32 300 mM Lysine HCl, 0.1% % Monomer 99.25 99.15 99.01 98.57
98.68 Tween80, pH 7 Conc (ug/mL) 55.5 53.0 50.8 54.3 52.4
TABLE-US-00008 TABLE 8 Stability of formulations F31, F32, F32
lyophilized in large lyophilizer, using a non-annealing cycle.
Lyophilized vials were stored at 40.degree. C. ID Composition
Signal Type Pre-lyo Day 0 Day 15 Day 28 Day 52 F31 25 mM citrate,
15% trehalose, % Dimer 0.96 0.72 0.87 1.77 1.07 100 mM Lysine HCl,
0.1% % Monomer 99.04 99.28 99.13 98.23 98.93 Tween80, pH 7 Conc
(ug/mL) 55.7 58.2 50.5 53.6 52.2 F32 25 mM citrate, 15% trehalose,
% Dimer 0.87 0.85 1.01 1.36 1.19 200 mM Lysine HCl, 0.1% % Monomer
99.13 99.15 98.99 98.64 98.81 Tween80, pH 7 Conc (ug/mL) 56.2 53.7
51.0 54.0 52.0 F33 25 mM citrate, 15% trehalose, % Dimer 0.75 0.85
0.99 1.49 1.28 300 mM Lysine HCl, 0.1% % Monomer 99.25 99.15 99.01
98.51 98.72 Tween80, pH 7 Conc (ug/mL) 55.5 53.0 50.8 53.0 51.9
TABLE-US-00009 TABLE 9 Protein recovery from formulations F31, F32,
F32 lyophilized in large lyophilizer, using a non-annealing cycle.
Recovery of MT103 from cakes lyophilized in a Non-Annealing Cycle
(L1) Percent Recovery Time Temperature F31 F32 F33 Initial N/A 95.8
96.4 98.2 7 week 4.degree. C. (N = 1) 94.6 95.9 96.4 40.degree. C.
(N = 3) 95.1 94.1 95.1
TABLE-US-00010 TABLE 9 Protein recovery from formulations F31, F32,
F32 lyophilized in large lyophilizer, using an annealing cycle.
Recovery of MT103 from cakes lyophilized in a Annealing Cycle (L2)
Percent Recovery Time Temperature F31 F32 F33 Initial N/A 96.4 98.5
97.4 8 week 4.degree. C. (N = 1) 94.6 95.8 96.1 40.degree. C. (N =
3) 96.1 93.9 94.9
[0812] Post-reconstitution stability of the MT103 formulation F29
was determined using multiple independent vials (N=3). Protein
recovery from reconstituted vials stored on bench after
reconstitution was assessed over 3 days (FIG. 22).
6.2. Development of MT103 Lyophilized Formulation II
[0813] Further studies were performed to characterize the MT103
formulations described in the previous section. In particular, the
studies described herein elucidated: 1) the acceptable pH range for
MT103 formulations, 2) the threshold concentrations of stabilizing
excipients. The short term stability of liquid and lyophilized
MT103 formulations comprising threshold concentration of
stabilizing excipients was also determined.
6.2.1. Optimal pH Range of MT103 Formulations
[0814] Liquid formulations consisting of 110 .mu.g/ml MT103, 10 mM
citrate, 10 mM TrisHCl, 0.1% polysorbate 80 and 200 mM of a basic
amino acid selected from the group consisting of histidine, lysine
and arginine were prepared following the standard protocols.
Aliquots of each formulation were adjusted to pH 3.0, 4.5, 6.0,
7.0, or 8.5. Aggregate content of each MT103 formulation tested was
determined by SEC-HPLC following storage at 5.degree. C. or
25.degree. C. for 5 weeks. Data obtained is displayed in FIGS. 23
and 24.
[0815] All formulations tested but one contained less than 5%
aggregate following storage at 5.degree. C. The MT103 formulation
comprising 200 mM arginine at pH 8.0 contained over 50% aggregate
material following storage at 5.degree. C. Formulations stored at
25.degree. C. displayed a different stability profile. MT103
precipitated out of solution in all pH 3.0 formulations stored at
25.degree. C. All other MT103 formulations except for the
formulation comprising 200 mM arginine at pH 8.0 remained in
solution with less than 5% aggregate formed. The 200 mM arginine/pH
8.0 formulation contained over 40% of MT103 in aggregate form
following storage at 25.degree. C.
6.2.2. Threshold Level of Stabilizing Excipients
[0816] Threshold concentration of lysine and polysorbate 80 was
determined by comparing the stability of MT-103 formulations that
were identical to each other except for the concentration of lysine
or polysorbate 80, respectively. The base formulation consisted of
55 .mu.g/ml MT103, 10 mM citrate, 10 mM TrisHCl, 200 mM Lysine HCl,
and 0.1% polysorbate 80 at a pH of 6.0. Excipient concentrations
tested are 0, 25, 50, 100, and 200 mM lysine HCl and 0, 0.01, 0.05,
0.1% of polysorbate 80. Stability of a formulation was
characterized by measuring the total non-aggregated MT103 protein
recoverable following extended storage, freeze/thaw cycles, or
shear stress.
[0817] FIG. 24 displays the stability of MT103 liquid formulations
comprising various concentrations of lysine HCl following extended
storage at 40.degree. C. The amount of MT103 protein recoverable
from formulations comprising 25, 50, 100, or 200 mM lysine HCl is
substantially identical over the 65 days time period of the
experiment. There was no change in The protein recovery from
formulations stored at 5.degree. C. or 25.degree. C. did not change
over the 65 day time period of the experiment (data not shown).
[0818] FIG. 25 and FIGS. 26A and 26B display the stability of MT103
formulations comprising various concentrations of lysine HCl or
polysorbate 80 following exposure to i) three freeze/thaw cycles,
ii) mechanical stress on a rocking shaker, or iii) shear stress on
a vortex mixer. The stability of formulations is assessed either by
measuring the total MT103 protein recoverable from a formulation
exposed to stress (FIG. 25) or by measuring the total amount of
aggregates in a formulation exposed to stress (FIGS. 26A and 26B).
Changes in lysine concentration did not significantly alter the
amount of MT103 recoverable or the aggregate formation following
stress. Formulations comprising 0.01, 0.05, or 0.1% polysorbate 80
also displayed identical stability profiles as characterized by the
above described measurements. The formulation comprising 0%
polysorbate (0% PS-80) had a reduced stability profile. The amount
of MT103 recoverable from the unstressed 0% PS-80 formulation was
already significantly reduced; freeze/thaw, mechanical or shear
stress further reduced the amount of recoverable MT103 protein. A
significant amount of MT103 protein precipitated out of the 0%
PS-80 formulation. Because of the precipitation, the level of HPSEC
measured aggregate levels were slightly reduced in unstressed or
freeze/thaw stressed formulations and were not detectable in the 0%
PS-80 formulation following shaking or vortex treatment.
6.2.3. Threshold Level of Trehalose
[0819] Trehalose incorporated into the MT103 formulations described
herein may function as a bulking agent during lyophilization. The
amount of trehalose necessary for the formation of a lyophilized
cake was ascertained by lyophilizing buffer only (no MT103 protein)
formulations comprising various amounts of trehalose. The buffer
only formulations tested are described in Table 10. All buffers
tested produced adequate bulk when subjected to lyophilization. The
cake characteristics and reconstitution times were comparable for
all buffers tested.
TABLE-US-00011 TABLE 10 Buffer only formulations lyophilized (P80 =
polysorbate 80). B1 25 mM Citrate, 25 mM lysine HCl, 6% trehalose,
0.02% P80 at pH 6.0 B2 10 mM Citrate, 50 mM lysine HCl, 5.5%
Trehalose, 0.02% P80 at pH 6.0 B3 10 mM Citrate, 25 mM lysine HCl,
7.5% Trehalose, 0.05% P80 at pH 6.0 B4 25 mM Citrate, 8% Trehalose,
0.02% P80 at pH 6.0 B5 50 mM Citrate, 6% Trehalose, 0.02% P80 at pH
6.0
6.2.4. Short Term Stability of MT103 Formulations
[0820] Short term stability of formulations 25/25, 10/50, and
25/200 (FIG. 27A) was determined. Stability of the lyophilized
formulations was ascertained by reconstituting lyophilized cakes
following storage at 5.degree. C., 25.degree. C., and 40.degree. C.
and measuring aggregate concentration in the reconstituted liquid.
Lyophilized cakes were prepared in individual vials filled with 0.6
ml of 25/25, 10/50, and 25/200 formulation comprising 175 .mu.g/ml
MT103 using the L1 lyophilization cycle described herein. Aggregate
content of the reconstituted liquid was measured by SEC and is
displayed as a function of lyophilized cake storage time in FIGS.
27B and 27C. The level of aggregates found in reconstituted liquid
formulations 10/50 and 25/25 comprising 175 .mu.g/ml MT103 remained
substantially the same during the 60 days time span of the
experiment at all three temperatures examined (5.degree. C.,
25.degree. C., and 40.degree. C.). Aggregate levels in both
reconstituted formulation were below 2%.
[0821] Stability of pre-lyophilized liquid formulations 25/25,
10/50, and 25/200 comprising 160 .mu.g/ml MT103 (FIG. 27A) was also
determined. Small aliquots of each formulations were stored in a
glass (25/25, 10/50) or polypropylene (25/200) vial at 5.degree. C.
or 25.degree. C. for extended periods of time. Stability of the
formulations were assessed by measuring the level of aggregate
formation using SEC. The % aggregate levels as a function of time
are displayed in FIGS. 28A and 28B. Aggregate levels in 10/50 were
approximately 3% and 4.5% following .about.60 days of storage at
5.degree. C. and 25.degree. C., respectively. Aggregate levels in
25/25 were approximately 2% and 4.5% following .about.60 days of
storage at 5.degree. C. and 25.degree. C., respectively. Aggregate
levels in 25/200 were approximately 0.5% and 3% following .about.60
days of storage at 5.degree. C. and 25.degree. C., respectively.
Aggregate levels in formulation 25/200 rose to 1% by day .about.180
of storage at 5.degree. C., and to 3.5% by day .about.90 of storage
at 25.degree. C.
6.3. Development of a Lyophilized MT-103 Formulation for
Subcutaneous Delivery
[0822] A lyophilized formulation for subcutaneous delivery was
developed by optimizing excipient concentrations to achieve
isotonicity while maintaining protein stability. MT103
concentration was set at 160 .mu.g/ml for the pre-lyophilized
liquid bulk. Lyophilization of candidate formulations was done by
using the optimized 96 hrs lyophilization cycle described
above.
[0823] The stability and lyophilized cake characteristics of the
following formulations were evaluated in an initial set of
experiments: [0824] a) 25 mM citrate, 25 mM lysine HCl, 6%
trehalose, 0.02% polysorbate 80 at pH 6.0; [0825] b) 10 mM citrate,
25 mM lysine HCl, 7.5% trehalose, 0.05% polysorbate 80 at pH 6.0;
[0826] c) 10 mM citrate, 50 mM lysine HCl, 5.5% trehalose, 0.02%
polysorbate 80 at pH 6.0; [0827] d) 25 mM citrate, 8% trehalose,
0.02% polysorbate 80 at pH 6.0; and [0828] e) 50 mM citrate, 6%
trehalose, 0.02% polysorbate 80 at pH 6.0.
[0829] Stability of the liquid formulations were assessed by
measuring the % protein aggregate level following storage at 5 or
25.degree. C. Liquid formulations used for the stability
experiments comprised 160 .mu.g/ml MT103 protein. Experimental
results are shown in FIGS. 27A-27C.
[0830] The cake characteristics and reconstitution times were
comparable for all formulations tested.
[0831] Excipient concentrations and formulation pH were optimized
using a 3 level, 4 factor Box-Behnken experimental design. Response
variable was the change in % aggregate levels in the formulation
following four weeks of storage at 25.degree. C. The factors
optimized and the levels tested for each factor are listed in Table
11. The stability of 25 different formulations, including the
center point formulation, was determined in three blocks following
the standard Box-Behnken experimental design. The stability of the
center point formulation was measured in duplicates in each
experimental block to improve the statistical significance of the
results. Data analyses predicted that an optimal isotonic
formulation would comprise 30 mM citrate, 75 mM lysine HCl, and
6.5% trehalose, and has a pH of 7.0.
TABLE-US-00012 TABLE 11 Excipient concentrations used in
optimization experiment. Levels High Medium Low Factors pH 5.0 6.0
7.0 Lysine HCl 30 mM 50 mM 70 mM Citrate 10 mM 30 mM 50 mM
Trehalose 5% 6.5% 8%
6.4. In Vivo Bioavailability of MT103
[0832] Candidate formulations of MT103 drug product were evaluated
in a mouse model for serum bioavailability following subcutaneous
route of administration. A formulation was identified that
consistently yielded the highest level of bioavailability in the
mouse model, and was evaluated in cynomolgus monkeys.
Bioavailability following subcutaneous bolus injection in
cynomolgus monkeys was comparable to that observed in mice. These
animals represent pharmacologically non-relevant models for
evaluating off-target activity of MT103 (MEDI-538) based on its
lack of specific binding to T and B cells in these species.
6.4.1. Methods
[0833] ELISA for the detection of MT103 in mouse serum: A specific
MT103-capture ELISA was employed to determine the concentration of
MT103 in serum samples of CD-1 mice. The ELISA assay utilized two
unique and non-competing anti-MT103 monoclonal Abs (F3 and 6E12) to
capture the analyte, which was subsequently detected with a
ruthinylated antibody against the hexahistidine (6.times.-his) tag
of the molecule. For the final assay method, F3 and 6E12 were both
spotted in 5 .mu.L on the same well of a Meso Scale Discovery (MSD)
Hi Bind plate using phosphate buffered saline (PBS). Plates were
dried overnight and non-specific sites were blocked with 1% BSA+PBS
for 1 hr at room temperature (RT) with shaking. A MT103 standard
curve was prepared with serial 1:2 dilutions from 800 ng/mL to 0.39
ng/mL in a pool of normal CD-1 mouse serum. Plates were washed 3
times with PBS+0.5% Tween-20 and standard curve calibrators,
quality control (QC) samples and mouse samples were added to the
blocked wells neat or with dilution (up to 1:250) at 25 .mu.L per
well and incubated for 1 hour at RT, with rotation (approximately
600 rpm). For detection of captured MT103, a rabbit
anti-6.times.-His polyclonal Ab was conjugated to MSD Sulfo-Tag
(ST) and 25 .mu.L per well of the conjugated reagent was added
subsequently to the plates in assay buffer (PBS+1% casein). Plates
were incubated for one additional hour at RT, with rotation. Plates
were washed 3 times with PBS+0.05% Tween-20 and developed using
2.times.MSD Read Buffer (150 .mu.L per well). Plates were
immediately read on the MSD Sector 6000 reader and data analysis
was conducted using MSD Workbench software. Concentrations of MT103
present in the mouse serum samples were estimated by non-linear
regression analysis from a 4-parameter fit model of the MT103
standard data set. The lower limit of quantitation for this assay
was determined to be 0.4 ng of MT103 per mL of mouse serum.
[0834] ELISA for the detection of MT103 in cynomolgus monkey serum:
Samples were tested using an electrochemiluminescent (ECL) assay to
quantitate the levels of MT103 in cynomolgus serum. Meso Scale
Discovery (MSD) 96 well plates were coated with two monoclonal
antibodies (mAb) specific for MT103 (F3 and 6E12). F3 and 6E12 were
diluted to 50 .mu.g/mL and 90 .mu.g/mL, respectively, in Dulbecco's
Phosphate Buffered Saline (D-PBS) containing 0.015% Tween-20 and 5
.mu.l of this solution was spotted on the surface of the MSD plate.
Plates were dried overnight and non-specific sites were blocked
with 1% BSA+D-PBS for 1 hr at room temperature (RT). A MT103
standard curve was prepared with serial 1:2.5 dilutions from 1200
ng/mL to 0.126 ng/mL in normal cynomolgus serum (NCS). Plates were
washed 4 times with D-PBS+0.1% Tween-20 and standard curve
calibrators, quality control (QC) samples and study samples were
added to the blocked wells without dilution at 25 .mu.L per well
and incubated for 1 hour at RT, with rotation (approximately 600
rpm). For detection of captured MT103, a rabbit anti-hexa-histidine
polyclonal Ab was conjugated to MSD Sulfo-Tag (ST) and 25 .mu.L per
well of the conjugated reagent at 1.5 .mu.g/mL was added
subsequently to the plates in assay buffer (D-PBS+1% casein).
Plates were incubated for one additional hour at RT, with rotation.
Plates were washed 4 times with D-PBS+0.1% Tween-20 and developed
using 2.times.MSD Read Buffer (150 .mu.L per well). Plates were
immediately read on the SECTOR Imager 6000 instrument (MSD) to
measure the ECL signal and data was analyzed using the Discovery
Workbench Software, version 2.0 (MSD). The lower limit and upper
limits of quantitation for this assay were determined to be 200
ng/mL and 1.5 ng/mL, respectively.
6.4.2. Results
6.4.2.1. Bioavailability of MT103 Following a Single-Dose
Intravenous (IV) or Subcutaneous (SC) Bolus Administration of Four
Different MT103 Formulations in CD-1 Mice.
[0835] In a single-dose IV and SC pharmacokinetic study, serum
levels of MT103 were determined in samples collected during a
single day. CD-1 mice (n=4/group) were treated with a single bolus
IV or SC infusion of 4 different formulations of MT103 at two dose
levels (0.75 and 2.5 mg/kg). Formulations utilized in this study
are detailed in Table 12. Serum samples were collected at 6
different time points over a time interval of 24 hours after
administration. The concentrations of MT103 in serum samples were
determined using a specific ELISA method with a LLOQ of 0.4 ng/mL.
The plot of the mean serum concentrations versus time profile of
MT103 is presented in FIGS. 29A and 29B. Absolute bioavailability
was estimated by taking the ratio of the mean area under the curve
(AUC) for the SC treatment groups and the mean AUC of the IV
treatment groups. The bioavailability was 17%, 33%, 35%, and 24%
for groups of mice treated with 0.75 mg/kg of Formulation X, Y, Z
and W, respectively, and 21%, 26%, 30%, and 37% for groups of mice
treated with 2.5 mg/kg of Formulation X, Y, Z, and W, respectively.
Formulation Z was chosen for subsequent studies.
TABLE-US-00013 TABLE 12 Formulations of MT103 Poly- Citrate, Lysine
sorbate Estimated pH HCL Trehalose 80 Osmolarity 7.0 (mM) (mM) (%
w/v) (% v/v) (mOs) Formulation X 25 200 15 0.1 1100 Formulation Y
25 200 400 Formulation Z 25 25 6 0.02 520 Formulation W 10 50 5.5
0.02 400
[0836] In CD-1 mice, SC administered MEDI-538 (MT103) was 20-30%
bioavailable and exhibited serum concentrations that were
dose-proportional, reached a peak at 4 hrs, and had a similar
terminal half-life as IV administered drug (FIG. 32). After
injection of 0.25, 0.75, or 2.5 mg/kg of MEDI-538, serum samples
were collected at 5 min, 1, 2, 4, 8, 15, 24, and 44 hrs. Serum
concentration of MEDI-538 were measured by ELISA method and
reported here as a the arithmetic mean for each sample at each time
interval after injection. A two-phase exponential decay, non-linear
regression model was used for IV treated mice. Dose optimization
studies showed that varying the dose concentration or volume for a
given dose level did not effect the concentration of MEDI-538 in
the serum after SC administration.
TABLE-US-00014 TABLE 13 Pharmacokinetic parameters of MEDI-538 in
CD-1 mice. N listed for each dataset. C.sub.max T.sub.max t.sub.1/2
AUC.sub.obs AUC.sub.inf CL Bioavail- Route (dose) N (ng/ml) (hr)
(hr) (ng hr/ml) (ng hr/ml) (ml/hr/kg) ibility IV (2.5 mg/kg) 7
23,100 0.0083 4.7 17,700 17,600 140 1.00 IV (0.75 mg/kg) 5 8,090
0.083 6.2 4,670 4,760 160 1.00 IV (0.25 mg/kg) 6 1,030 0.083 5.7
809 827 300 1.00 SC (2.5 mg/kg) 4 437 4 4.4 4,430 4,440 560 0.25 SC
(0.75 mg/kg) 4 151 4 4.5 1,330 1,380 550 0.28 SC (0.25 mg/kg) 4 106
4 4.6 543 554 450 0.67 Cmax, peak serum concentration; Tmax, time
to peak serum concentration; t.sub.1/2, half-life of serum
concentration; AUCobs, are under the serum concentration-time curve
up to the last measureable sampling time; AUCinf, are under the
serum-concentration-time curve to infinity; CL, total body plasma
clearance estimated as Dose/AUCinf after intravenous
administration; Bioavailability, absolute bioavailability estimated
as AUCinf (SC)/AUCinf (IV).
6.4.2.2. Bioavailability of MT103 Following a Single-Dose
Intravenous (IV) or Subcutaneous (SC) Bolus Administration of the
Lead Formulation of MT103 in CD-1 Mice.
[0837] In a single-dose IV and SC pharmacokinetic study, serum
levels of MT103 were determined in samples collected during a
single day. CD-1 mice (n=5/group) were treated with a single bolus
IV or SC infusion of MT103 in the lead formulation (Formulation Z)
at two dose levels (1.6 and 5.0 mg/kg). Serum samples were
collected at 6 different time points over a time interval of 24
hours after administration. The concentrations of MT103 in serum
samples were determined using a specific ELISA method with a LLOQ
of 0.4 ng/mL. The plot of the mean serum concentrations versus time
profile of MT103 is presented in FIGS. 30A and 30B. Absolute
bioavailability was 28% and 29% for groups of mice treated with 1.6
mg/kg and 5.0 mg/kg, respectively, and was comparable to the
bioavailability observed following a single-dose IV and SC
pharmacokinetic study in cynomolgus monkeys. Thus, the results of
the cynomolgus monkeys supported the results observed in the mouse
model.
6.4.2.3. Bioavailability of MT103 Following a Single-Dose
Intravenous (IV) or Subcutaneous (SC) Bolus Administration of MT103
in Cynomolgus Monkey.
[0838] A study was conducted to compare the bioavailability of
MT103 when delivered by a single intravenous (IV) bolus or
subcutaneous (SC) injection at 0.5 mg/kg to male cynomolgus monkeys
over a one-week period (FIGS. 31A and 31B). The study used a
cross-over design with one week washout period between two phases.
Three animals were randomized in each of two groups. During Phase
1, Group 1 animals were dosed intravenously and Group 2 animals
were dosed subcutaneously and vice-versa during Phase 2. MT103,
formulated in 25 mM Citrate, 25 mM Lysine HCL, 6% Trehalose, 0.02%
Polysorbate 80, pH 6.0, was delivered in a 1 mL per kg dose volume
for both routes. All animals appeared healthy throughout the study
with no signs of irritation at the sites of injection. Serum
concentrations of MT103 were determined using a sensitive and
specific ELISA assay based on electrochemiluminescence (ECL)
detection with a lower limit of quantitation (LLOQ) equal to 1.5
ng/ml. PK analysis results showed the average half-life of MT103
was approximately 6.3 and 7.5 hours for intravenous (IV) and
subcutaneous (SC) groups, respectively. The average Cmax was 12393
and 152 .mu.g/mL, and was attained 0.1 and 4.3 hours post-dose for
the IV and SC groups, respectively. The mean observed AUC for the
IV and SC groups was 12723.1 and 2655.28 .mu.ghr/mL, respectively,
whereas the average AUC(0-.infin.) was 12766.0 and 2711.2
.mu.ghr/mL, respectively, for the two groups. The clearance (CL) of
MT103 following IV injection is 39.96 mL/hr/kg and the apparent
clearance (CL/F) following SC injection is 185.63 mL/hr/kg. The
bioavailability of MT103 delivered via a single SC dose was 22%
estimated based on the ratio of AUC(0-.infin.). Bioavailability
data obtained is summarized in FIG. 33.
TABLE-US-00015 TABLE 14 Pharmacokinetic parameters of MEDI-538 in
cynomolgus monkeys. N = 6 (3 M, 3F). C.sub.max T.sub.max t.sub.1/2
AUC.sub.obs AUC.sub.inf CL Bioavail- Route (ng/ml) (hr) (hr) (ng
hr/ml) (ng hr/ml) (ml/hr/kg) ibility IV 12,400 (1370) 0.1 (0.0) 6.3
(1.1) 12,700 (2,100) 12,800 (2,100) .sup. 40 (5.9) 1.00 SC 150 (25)
4.3 (0.8) 7.5 (1.3) 2,660 (220) 2,700 (230) 186 (17) 0.22 (0.033)
Cmax, peak serum concentration; Tmax, time to peak serum
concentration; t.sub.1/2, half-life of serum concentration; AUCobs,
are under the serum concentration-time curve up to the last
measureable sampling time; AUCinf, are under the
serum-concentration-time curve to infinity; CL, total body plasma
clearance estimated as Dose/AUCinf after intravenous
administration; Bioavailability, absolute bioavailability estimated
as AUCinf (SC)/AUCinf (IV). Values in parentheses, standard
deviation of the mean for each parameter.
6.5. Pharmacodynamic Activity, and Anti-Tumor Efficacy of the
CD19/CD3 Specific BiTE.RTM. Antibody MEDI-538 (MT103/Blinatumomab)
Delivered Subcutaneously in Animal Models
[0839] Animal models were utilized to evaluate the bioavailability,
pharmacodynamic effects, and anti-tumor potency of MEDI-538
following subcutaneous (SC) administration.
[0840] Results:
[0841] MEDI-538 provided anti-tumor efficacy following SC
administration in NOD/SCID mice that were engrafted with human B
cell tumor cells mixed with human PBMC and that were capable of
demonstrating human T cell-mediated killing of tumor cells.
Pharmacodynamic effects were observed following SC administration
of hys103 (anti-human CD19.times.anti-mouse CD3), a hybrid mouse
surrogate form of MEDI-538, to immunocompetent human CD19
transgenic knock-in mice. Hys103 recruits murine T cells for
redirected lysis of human CD19 expressing mouse B cells. Treatment
caused a reduction in B cells and an increase in activated T cells
in the spleen as measured by flow cytometry
[0842] These results demonstrated that SC delivery of MEDI-538
yielded comparable PK characteristics in multiple animal models,
provided sufficient systemic levels for efficacy against tumor
challenge in xenograft mouse models, and depleted B cells in an
immunocompetent mouse model. Taken together, the data support the
SC route of administration as an alternative method for delivery of
MEDI-538.
6.5.1. In Vivo Anti-Tumor Efficacy.
[0843] Experimental design of in vivo mouse tumor models used are
outlined in FIG. 34.
[0844] Cohorts of 6 NOD/SCID mice were inoculated SC with 10.sup.6
Namalwa cells in the absence (without T cells) or presence of
2.times.10.sup.6 human CD3+ T cells isolated from healthy donors.
Five daily doses of MEDI-538 as indicated or a negative control
BiTE.RTM. were administered by subcutaneous injection on the
indicated days following tumor cell/T cell engraftment. Mean values
of tumor growth curves are shown in FIG. 35.
[0845] Cohorts of 6 NOD/SCID mice were inoculated IV with 105 Ramos
cells mixed with 5.times.10.sup.6 human PBMCs isolated from healthy
donors. Five daily doses of MEDI-538 as indicated or a negative
control BiTE.RTM. were administered by subcutaneous injection on
the indicated days following tumor cell/PBMC engraftment. The
number of mice alive versus time after treatment is graphed.
Animals were euthanized when overt signs of paralysis were
observed. Results are shown in FIG. 36.
6.5.2. Pharmacodynamic Effects.
[0846] Pharmacodynamic effects were evaluated using hys103
(anti-human CD19.times.anti-mouse CD3), a hybrid mouse surrogate
form of MEDI-538, and immunocompetent human CD19 (huCD19)
transgenic knock-in mice. The huCD19 transgenic mice was
established by Tom Tedder's laboratory. The expression of huCD19 is
restricted to B cells and can replace the function of murine CD19.
The expression level of huCD19 on the murine transgenic B cells is
comparable to the expression level observed on normal human blood B
cells. Functional characteristics of T cells from the huCD19
transgenic mouse are comparable to that of T cells from the
background mouse strain (C57BL/6).
[0847] CD3+ human T cells or CD3+ mouse T cells from huCD19
transgenic mice (spleen) were combined with DiOC.sub.18(3)-labeled
human CD19+ NALM-6 targets cells in the presence of the indicated
concentrations of MEDI-538 or hys103. After 18 hrs for the human T
cells and 42 hrs for the mouse T cells, specific cell lysis was
determined by means of a flow cytometry-based assay. E:T ratio was
5:1. EC50 is the concentration at half maximal target cell lysis.
Results for replicate experiments are shown in FIGS. 37A and
37B.
[0848] Four huCD19 transgenic mice were administered a single SC
dose of hys103 at various dose levels. Hys103-induced effects on
the splenic and bone marrow CD19+B220+ B cell populations were
monitored by flow cytometry analysis at various time intervals
following treatment, and reported as the percentage of B cells
following treatment normalized to the number of B cells present in
PBS treated mice in FIGS. 38 and 39.
[0849] Four huCD19 transgenic mice were administered a single SC
dose of hys103 at various dose levels. Hys103-induced effects on
the splenic and bone marrow CD3+CD8+ T cell populations were
monitored by flow cytometry analysis at various time intervals
following treatment, and reported as the fold increase of the mean
fluorescence intensity (MFI) of CD69 following hys103 treatment as
compared to the MFI of CD69 on T cells in PBS treated mice
evaluated at the same time interval (FIGS. 40 and 41). Similar
levels of CD69 activation were observed with CD3+CD4+ T cells.
[0850] A study was conducted to compare the pharmacokinetic profile
of hys103 (anti-human CD19.times.anti-mouse CD3), a hybrid mouse
surrogate form of MEDI-538, following a single intravenous (IV)
bolus or subcutaneous (SC) injection to huCD19 transgenic mice.
Experimental animals received a single dose of 0.375 mg/kg, 0.75
mg/kg, 1.5 mg/kg or 3 mg/kg of hys103, formulated in 25 mM Citrate,
25 mM Lysine HCL, 6% Trehalose, 0.02% Polysorbate 80, pH 6.0, by
both routes of administration. Serum level of hys103 was monitored
by ELISA over a 24 hr period following administration. A
representative sample of the data is shown in FIGS. 42A and 42B.
Serum half-life of hys103 was approximately 8 hrs. Serum
bioavailability of hys103 was approximately 18% following
subcutaneous administration.
[0851] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0852] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference. In addition, U.S.
Provisional Application No. 60/990,772, filed Nov. 28, 2007,
61/039,985, filed Mar. 27, 2008, and 61/040,384, filed Mar. 28,
2008 are hereby incorporated by reference in their entirety for all
purposes.
[0853] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
Sequence CWU 1
1
51111PRTartificialrecombinant polypeptide 1Asp Ile Gln Leu Thr Gln
Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr
Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp
Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile
His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln
Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110 2124PRTartificialrecombinant
polypeptide 2Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg
Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp
Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr
Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu
Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala
Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105
110 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
3119PRTartificialrecombinant polypeptide 3Asp Ile Lys Leu Gln Gln
Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met
Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met
His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50
55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp
Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115
4106PRTArtificialrecombinant polypeptide 4Asp Ile Gln Leu Thr Gln
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr
Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp
Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45
Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser 50
55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala
Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn
Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
105 5531PRTArtificialrecombinant polypeptide 5Met Gly Trp Ser Cys
Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser
Asp Tyr Lys Asp Asp Asp Asp Lys Asp Ile Gln Leu Thr 20 25 30 Gln
Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile 35 40
45 Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu
50 55 60 Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro Lys Leu Leu
Ile Tyr 65 70 75 80 Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro Arg
Phe Ser Gly Ser 85 90 95 Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile
His Pro Val Glu Lys Val 100 105 110 Asp Ala Ala Thr Tyr His Cys Gln
Gln Ser Thr Glu Asp Pro Trp Thr 115 120 125 Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser 145 150 155 160 Gly
Ala Glu Leu Val Arg Pro Gly Ser Ser Val Lys Ile Ser Cys Lys 165 170
175 Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gln
180 185 190 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln Ile Trp Pro
Gly Asp 195 200 205 Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys
Ala Thr Leu Thr 210 215 220 Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met
Gln Leu Ser Ser Leu Ala 225 230 235 240 Ser Glu Asp Ser Ala Val Tyr
Phe Cys Ala Arg Arg Glu Thr Thr Thr 245 250 255 Val Gly Arg Tyr Tyr
Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr 260 265 270 Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln 275 280 285 Ser
Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys 290 295
300 Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys
305 310 315 320 Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile
Asn Pro Ser 325 330 335 Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys
Asp Lys Ala Thr Leu 340 345 350 Thr Thr Asp Lys Ser Ser Ser Thr Ala
Tyr Met Gln Leu Ser Ser Leu 355 360 365 Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp 370 375 380 His Tyr Cys Leu Asp
Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 385 390 395 400 Ser Val
Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 405 410 415
Gly Val Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala 420
425 430 Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser
Val 435 440 445 Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser
Pro Lys Arg 450 455 460 Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly
Val Pro Tyr Arg Phe 465 470 475 480 Ser Gly Ser Gly Ser Gly Thr Ser
Tyr Ser Leu Thr Ile Ser Ser Met 485 490 495 Glu Ala Glu Asp Ala Ala
Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn 500 505 510 Pro Leu Thr Phe
Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His 515 520 525 His His
His 530
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