U.S. patent application number 15/895055 was filed with the patent office on 2018-06-28 for antibody formulation.
The applicant listed for this patent is Novartis AG. Invention is credited to Marta Cosenza, Christoph Heusser, Julia Neugebauer, Eveline Schaadt, Stefanie Urlinger, Maximilian Woisetschlaeger.
Application Number | 20180179290 15/895055 |
Document ID | / |
Family ID | 45478272 |
Filed Date | 2018-06-28 |
United States Patent
Application |
20180179290 |
Kind Code |
A1 |
Cosenza; Marta ; et
al. |
June 28, 2018 |
ANTIBODY FORMULATION
Abstract
Anti-BAFFR antibodies are formulated as lyophilisate or liquid
formulation. The lyophilisates can be reconstituted to give a
solution with a high concentration of the antibody active
ingredient for delivery to a patient without high levels of
antibody aggregation. The lyophilisate can be reconstituted with an
aqueous reconstituent to provide an aqueous composition in which
the antibody has a concentration of at least 50 mg/ml. The
lyophilisate or aqueous pharmaceutical composition may include one
or more of a sugar, a buffering agent, a surfactant and/or a free
amino acid.
Inventors: |
Cosenza; Marta; (Basel,
CH) ; Heusser; Christoph; (Oberwil, CH) ;
Neugebauer; Julia; (Munchen, DE) ; Schaadt;
Eveline; (Munchen, DE) ; Urlinger; Stefanie;
(Munchen, DE) ; Woisetschlaeger; Maximilian;
(Oberwill, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG |
Basel |
|
CH |
|
|
Family ID: |
45478272 |
Appl. No.: |
15/895055 |
Filed: |
February 13, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15254351 |
Sep 1, 2016 |
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15895055 |
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13991984 |
Jan 15, 2014 |
9458240 |
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PCT/EP2011/072248 |
Dec 8, 2011 |
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15254351 |
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61421650 |
Dec 10, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/183 20130101;
A61K 47/26 20130101; A61K 9/19 20130101; A61K 9/08 20130101; A61K
2039/505 20130101; C07K 2317/56 20130101; A61K 47/10 20130101; A61K
9/0019 20130101; C07K 2317/565 20130101; C07K 2317/73 20130101;
C07K 16/2878 20130101; A61K 39/39591 20130101; C07K 2317/21
20130101; A61K 39/3955 20130101; A61K 39/39533 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/18 20170101 A61K047/18; A61K 9/08 20060101
A61K009/08; A61K 9/19 20060101 A61K009/19; A61K 39/395 20060101
A61K039/395; A61K 47/10 20170101 A61K047/10; A61K 9/00 20060101
A61K009/00; A61K 47/26 20060101 A61K047/26 |
Claims
1. A method of treating a disease or disorder that is mediated by
BAFF receptor or that can be treated by killing or depleting B
cells, comprising administering to a subject, a lyophilized
formulation comprising (i) an anti-BAFFR antibody wherein the
antibody has a concentration of 20-120 mg/ml, and wherein said
anti-BAFFR antibody includes heavy chain CDR1, CDR2 and CDR3 of SEO
ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2 and CDR3
of SEQ ID NOs: 6, 7 and 8, (ii) a stabilizer (iii)a buffering
agent, (iv) a surfactant, and optionally (v) an amino acid.
2. The method of claim 1, wherein said disease or disorder that is
mediated by BAFF receptor or that can be treated by killing or
depleting B cells is selected from the group consisting of
autoimmune disease, B cell neoplasm, lymphoma, leukemia, myeloma,
rheumatoid arthritis, systemic lupus erythematosus, and Pemphigus
vulgaris.
3. A method for treating a disease or disorder that is mediated by
BAFF receptor or that can be treated by killing or depleting B
cells, comprising administering to a subject the aqueous
pharmaceutical composition of claim 1.
4. The method of claim 3, wherein said disease or disorder that is
mediated by BAFF receptor or that can be treated by killing or
depleting B cells is selected from the group consisting of
autoimmune disease, B cell neoplasm, lymphoma, leukemia, myeloma,
rheumatoid arthritis, systemic lupus erythematosus, and Pemphigus
vulgaris.
5. The method of claim 1, wherein said formulation is prepared from
an aqueous formulation having a pH of 5.0-7.0 and comprising (i) an
anti-BAFFR antibody wherein the antibody has a concentration of
20-120 mg/ml, and wherein said anti-BAFFR antibody includes heavy
chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively,
and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs; 6, 7 and 8, (ii)
sucrose or trehalose as a stabilizer, (iii) histidine as a
buffering agent, (iv) polysorbate 80 as a surfactant, and
optionally (v) an amino acid selected from arginine and
glycine.
6. The method of claim 1, wherein said formulation is prepared from
an aqueous formulation having a pH of 5.0-7.0 and comprising (i) an
anti-BAFFR antibody wherein the antibody has a concentration of
20-120 mg/ml, and wherein said anti-BAFFR antibody includes heavy
chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively,
and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs:: 6, 7 and 8,
(ii) 3-300 mM sucrose or trehalose as a stabilizer, (iii) 1-60 mM
histidine as a buffering agent. (iv) up to 0.2% polysorbate 80 as a
surfactant, and optionally (v) 2-80 mM arginine or glycine.
7. The method of claim 1, wherein said formulation is prepared from
an aqueous formulation having a pH of 6.5 and comprising (i) an
anti-BAFFR antibody wherein the antibody has a concentration of 50
mg/ml, and wherein said anti-BAFFR antibody includes heavy chain
CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and
light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8, (ii) 90
mM sucrose as a stabilizer, (iii) 7 mM histidine as a buffering
agent, and (iv) 0.02% polysorbate 80 as a surfactant.
8. The method of claim 1, wherein said formulation is prepared from
an aqueous formulation having a pH of 6.5 and comprising (i) an
anti-BAFFR antibody wherein the antibody has a concentration of 50
mg/ml, and wherein said anti-BAFFR antibody includes heavy chain
CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and
light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8, (ii) 90
mM sucrose as a stabilizer, (iii) 7 mM histidine as a buffering
agent, (iv) 0.02% polysorbate 80 as a surfactant, and (v) 20 mM
glycine-HCl.
9. The method of claim 1, wherein said formulation is prepared from
an aqueous formulation having a pH of 6.5 and comprising (i) an
anti-BAFFR antibody wherein the antibody has a concentration of 50
mg/ml, and wherein said anti-BAFFR antibody includes heavy chain
CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and
light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8, (ii) 90
mM sucrose as a stabilizer, (iii) 7 mM histidine as a buffering
agent, (iv) 0.02% polysorbate 80 as a surfactant, and (v) 17 mM
arginine-HCl.
10. The method of claim 1, wherein said formulation is prepared
from an aqueous formulation having a pH of 6.5 and comprising (i)
an anti-BAFFR antibody wherein the antibody has a concentration of
66.6 mg/ml, and wherein said anti-BAFFR antibody includes heavy
chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively,
and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 8, 7 and 8, (ii)
80 mM sucrose as a stabilizer, (iii) 7 mM histidine as a buffering
agent, (iv) 0.02% polysorbate 80 as a surfactant, and (v) 17 mM
arginine-HCl.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pharmaceutical
formulation of m antibody against BAFFR (BAFF receptor), a process
for the preparation thereof and uses of the formulation.
BACKGROUND
[0002] The BAFFR:BAFF pair is critically involved in the maturation
of transitional B-cells, for survival and activation of mature
B-cells, and for isotype class switching in response to T
cell-dependent antigens. BAFF and its receptor BAFFR are also
important for survival and growth of malignant B-cells. Further,
BAFFR normally is not expressed on pre-B cells, but was recently
shown to be expressed on human ALL (B-lineage acute lymphoblastic
leukemia) cells (Parameswaran 2010, Cancer Res. 70(11) 4346-4358).
The removal of autoreactive B cells and the blockade of
inappropriate survival/activation mediated by excess BAFF levels in
patents suffering from autoimmune disorders or cancer represents a
well-validated therapeutic goal. Thus, an anti-BAFFR antibody, in
particular an antibody capable of antibody-dependent cell-mediated
cytotoxicity (ADCC) and blockade of ligand binding to BAFFR may
offer an effective therapeutic agent in autoimmune diseases and B
cell neoplasms.
[0003] Antibodies against BAFFR are known from e.g. WO 2010/007082
and include antibodies which are characterized by comprising a
V.sub.H domain with the amino acid sequence of SEQ ID NO: 1 and a
V.sub.L domain with the amino acid sequence of SEQ ID NO: 2. The
antibody MOR6654 in one such antibody (IgG1 kappa). It has the
heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain
amino acid sequence of SEQ ID NO: 10. This antibody may be
expressed from SEQ ID NOs: 14 and 15, preferably in a host cell
which lacks fucosyl-transferase, for example in a mammalian cell
line with an inactive FUT8(-/-) gene, to give a functional
non-fucosylated anti-BAFFR antibody with enhanced ADCC. This
antibody is referred to hereafter as MOR6654B. Alternative ways to
produce non-fucosylated antibodies are known in the art.
[0004] Formulations with high concentration of antibody may have
short shelf lives and the formulated antibodies may loose
biological activity resulting from chemical and physical
instabilities during the storage. Among those, aggregation,
degradation and oxidation are known to be the most common causes of
antibody degradation. In particular, aggregation can potentially
lead to increased immune response in patients, leading to safety
concerns. Thus it must be minimized or prevented.
[0005] It is an object of the invention to provide further and
improved formulations of anti-BAFFR antibodies, and in particular
formulations with high concentration of anti-BAFFR antibodies and
low levels of antibody aggregation.
DISCLOSURE OF THE INVENTION
[0006] Therapeutic antibodies are typically formulated either in
aqueous form ready for parenteral administration or as
lyophilisates for reconstitution with a suitable diluent prior to
administration. According to the invention, an anti-BAFFR antibody
may be formulated either as a lyophilisate, or as an aqueous
composition, for example in pre-filled syringes. Suitable
formulation can provide an aqueous pharmaceutical composition or a
lyophilisate which can be reconstituted to give a solution with a
high concentration of the antibody active ingredient and a low
level of antibody aggregation for delivery to a patient. High
concentrations of antibody are useful as they reduce the amount of
material which must be delivered to a patient. Reduced dosing
volumes minimize the time taken to deliver a fixed dose to the
patient. The aqueous compositions of the invention with high
concentration of anti-BAFFR antibodies are particularly suitable
for subcutaneous administration.
[0007] Thus the invention provides an aqueous composition, suitable
to parenteral administration in a subject, e.g., for subcutaneous
administration, comprising an anti-BAFFR antibody.
[0008] The following specific embodiments of the invention are
described as numbered hereafter: [0009] 1. An aqueous
pharmaceutical composition, suitable for subcutaneous
administration in a subject, comprising an anti-BAFFR antibody in
which the antibody has a concentration of at least 50 mg/ml, and
wherein said anti-BAFFR antibody incudes: (i) one or more heavy
chain CDRs selected from the group consisting of SEQ ID NOs: 3, 4
and 5; and/or (ii) one or more light chain CDRs selected from the
group consisting of SEQ ID NOs; 6, 7 and 8. [0010] 2. The aqueous
pharmaceutical composition, suitable for subcutaneous
administration in a subject, according to Embodiment 1, wherein
said anti-BAFFR antibody includes heavy chain CDR1, CDR2 and CDR3
of SEQ ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2
and CDR3 of SEQ ID NOs: 6, 7 and 8. [0011] 3. The aqueous
pharmaceutical composition of Embodiment 1 or 2, wherein the
anti-BAFFR antibody comprises a V.sub.H domain with amino acid SEQ
ID NO: 1 and a V.sub.L domain with amino acid SEQ ID NO: 2. [0012]
4. The aqueous pharmaceutical composition of Embodiment 1, 2 or 3,
wherein the anti-BAFFR antibody comprises a heavy chain region of
SEQ ID NO: 9 and a light chain region of SEQ ID NO: 10. [0013] 5.
The aqueous pharmaceutical composition of any one of Embodiments 1
to 4, wherein less than 5% of the anti-BAFFR anybody is aggregated
or degraded. [0014] 6. The aqueous pharmaceutics composition of any
one of Embodiments 1 to 5, comprising one or more of the following
components selected among the group consisting of: a stabilizer, a
buffering agent; and a surfactant. [0015] 7. The aqueous
pharmaceutics composition of Embodiment 8, wherein the stabilizer
is a sugar. [0016] 8. The aqueous pharmaceutical composition of
Embodiment 6 or 7 comprising: a sugar, a buffering agent, and a
surfactant. [0017] 9. The aqueous pharmaceutical composition of
Embodiment 6 or 7, further comprising a free amino acid. [0018] 10.
The aqueous pharmaceutics composition of Embodiment 7 to 9,
comprising sucrose as a sugar. [0019] 11. The aqueous
pharmaceutical composition of Embodiment 10, comprising 200-300 mM
sucrose. [0020] 12. The aqueous pharmaceutical composition of
Embodiments 6-11, comprising a histidine buffer as the buffering
agent. [0021] 13. The aqueous pharmaceutical composition of
Embodiment 12, comprising 25-35 mM histidine buffer. [0022] 14. The
aqueous pharmaceutical composition of Embodiments 6 to 13,
comprising polysorbate 80 as a surfactant. [0023] 15. The aqueous
pharmaceutical composition of Embodiment 14, comprising 0.01 to
0.1% polysorbate 80. [0024] 16. The aqueous pharmaceutical
composition of Embodiment 9, further comprising arginine as free
amino acid. [0025] 17. The aqueous pharmaceutical composition of
Embodiment 16, comprising 40-80 mM arginine. [0026] 18. The aqueous
pharmaceutical composition of any preceding Embodiment, comprising
sucrose, a histidine buffer, polysorbate 80 and arginine. [0027]
19. A lyophilisate suitable for preparing the aqueous
pharmaceutical composition of any preceding Embodiments. [0028] 20.
A lyophilisate according to Embodiment 19, comprising sucrose, a
histidine buffer, polysorbate 80 and arginine. [0029] 21. A method
for preparing a lyophilisate, comprising the steps of: (i)
preparing an aqueous solution comprising an anti-BAFFR antibody, a
sugar, a buffering agent, a surfactant and optionally a free amino
acid; and (ii) lyophilizing the aqueous solution. [0030] 22. A
delivery device including the aqueous pharmaceutical composition of
any one of Embodiments 1-18. [0031] 23. A pre-filled syringe
including the aqueous pharmaceutical composition of any one of
Embodiments 1-18. [0032] 24. A method for an anti-BAFFR monoclonal
antibody to a mammal, compromising a step of administering to the
patient a pharmaceutical composition of any one of Embodiments
1-18. [0033] 25. The composition of any one of Embodiments 1-18,
for use in treating a disease or disorder that is mediated by BAFF
receptor or that can be treated by killing or depleting B cells.
[0034] 26. The composition of Embodiment 25, for the treatment of
autoimmune diseases. [0035] 27. The composition of Embodiment 25,
for the treatment of B cell neoplasms, such as lymphoma, leukemia
or myeloma. [0036] 28. The composition of Embodiment 26, for the
treatment of rheumatoid arthritis, systemic lupus erythematosus, or
Pemphigus vulgaris. [0037] 29. The aqueous pharmaceutical
composition of any one of Embodiments 1-18 in which the antibody
has a concentration of at least at least 50 mg/ml, at least 100
mg/ml, at least 150 mg/ml, at least 200 mg/ml, at least 250 mg/ml,
or at least 300 mg/ml.
[0038] The invention also provides an aqueous pharmaceutical
composition comprising: an anti- BAFFR monoclonal antibody as
described above, for example MOR6654, especially MOR6654B; a
stabilizer; a buffering agent; and a surfactant. The composition
preferably also includes a free amino acid.
[0039] The invention also provides a lyophilisate comprising: an
anti-BAFFR monoclonal antibody as described above, for example
MOR6654, especially, MOR6654B; a sugar; a buffering agent; and a
surfactant. The lyophilisate preferably also includes a free amino
acid.
[0040] The invention also provides a lyophilisates comprising an
anti-BAFFR monoclonal antibody as described above, for example
MOR6654, especially, MOR6654B, wherein the lyophilisate can be
reconstituted with an aqueous reconstituent to provide an aqueous
composition in which the antibody has a concentration of at least
50 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml,
after reconstitution is an aqueous solution.
[0041] The invention also provides an aqueous pharmaceutical
composition comprising high concentration of an anti-BAFFR
monoclonal antibody as described above, for example MOR6654,
especially, MOR6654B, wherein less than 5%, 4%, 3%, 2% or 1% of the
anti-BAFFR antibody is aggregated or degraded.
[0042] The invention also provides a process for preparing a
lyophilisate, comprising steps of: (i) preparing an aqueous
solution comprising an anti-BAFFR monoclonal antibody, a sugar, a
buffering agent, a surfactant, and optionally a free amino acid;
and (ii) lyophilizing the aqueous solution.
[0043] The invention also provides a process for preparing a
pharmaceutical composition, comprising a step of mixing a
lyophilisate with an aqueous reconstituent, wherein the
lyophilisate compromises an anti-BAFFR monoclonal antibody, sugar,
a buffering agent, a surfactant, and optionally a free amino
acid.
[0044] More specifically the invention provides a lyophilized
formulation prepared by lyophilizing an aqueous formulation having
a pH of 5.0-7.0 and comprising [0045] (i) an anti-BAFFR antibody
wherein the antibody has a concentration of 20-120 mg/ml, and
wherein said anti-BAFFR antibody includes heavy chain CDR1, CDR2
and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and light chain
CDR1, CDR2 and CDR3 of SEQ ID NOs 6, 7 and 8. [0046] (ii) a
stabilizer, [0047] (iii) a buffering agent, [0048] (iv) a
surfactant, and optionally [0049] (v) an amino acid.
[0050] In one embodiment said lyophilized formulation is prepared
from an aqueous formulation having a pH of 5.0-7.0 and comprising
[0051] (i) an anti-BAFFR antibody wherein the antibody has a
concentration of 20-120 mg/ml, and wherein said anti-BAFFR anybody
includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5
respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6,
7 and 8. [0052] (ii) sucrose or trehalose a stabilizer, [0053]
(iii) histidine as a buffering agent, [0054] (iv) polysorbate 80 as
a surfactant, and optionally [0055] (v) an amino add selected from
arginine and glycine.
[0056] In one embodiment said lyophilized formulation is prepared
from an aqueous formulation having a pH of 5.0-7.0 and comprising
[0057] (i) an anti-BAFFR antibody wherein the antibody has a
concentration of 20-120 mg/ml and wherein said anti-BAFFR antibody
includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5
respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6,
7 and 8. [0058] (ii) 3-300 mM sucrose or trehalose as a stabilizer,
[0059] (iii) 1-60 mM histidine as a buffering agent, [0060] (iv) up
to 0.2% polysorbate 80 as a surfactant, optionally [0061] (v) 2-80
mM arginine or glycine.
[0062] In one embodiment said lyophilized formulation us prepared
from an aqueous formulation having a pH of 6.5 and comprising
[0063] (i) an anti-BAFFR antibody wherein the antibody has a
concentration of 50 mg/ml and wherein said anti-BAFFR antibody
includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5
respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6,
7 and 8, [0064] (ii) 90 mM sucrose as a stabilizer, [0065] (iii) 7
mM histidine as a buffering agent, and [0066] (iv) 0.02%
polysorbate 80 as a surfactant.
[0067] In one embodiment said lyophilized formulation is prepared
from an aqueous formulation having a pH of 6.5 and comprising
[0068] (i) an anti-BAFFR antibody wherein the antibody has a
concentration of 50 mg/ml, and wherein said anti-BAFFR antibody
includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO 3, 4 and 5
respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6,
7 and 8. [0069] (ii) 90 mM sucrose as a stabilizer. [0070] (iii) 7
mM histidine as a buffering agent, [0071] (iv) 0.02% polysorbate 80
as a surfactant, and [0072] (v) 20 mM glycine-HCl.
[0073] In one embodiment said lyophilized formulation is prepared
from an aqueous formulation having a pH of 6.5 and compromising
[0074] (i) an anti-BAFFR antibody wherein the antibody has a
concentration of 50 mg/ml, and wherein said anti-BAFFR antibody
includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5
respectively, and light chair CDR1, CDR2 and CDR3 of SEQ ID NOs: 6,
7 and 8, [0075] (ii) 90 mM sucrose a stabilizer, [0076] (iii) 7 mM
histidine as a buffering agent, [0077] (iv) 0.02% polysorbate 80 as
a surfactant, and [0078] (v) 17 mM arginine-HCl.
[0079] In one embodiment said lyophilized formulation is prepared
from an aqueous formulation having a pH of 6.5 and comprising
[0080] (i) an anti-BAFFR antibody wherein the antibody has a
concentration of 66.6 mg/ml, and wherein said anti-BAFFR antibody
includes heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5
respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6,
7 and 8, [0081] (ii) 90 mM sucrose as a stabilizer, [0082] (iii) 7
mM histidine as a buffering agent, [0083] (iv) 0.02% polysorbate 80
as a surfactant, and [0084] (v) 17 mM arginine-HCl.
[0085] The invention also provides an aqueous pharmaceutical
composition obtained by reconstituting a lyophilized formulation as
described above, wherein the reconstitution factor is between 1:0.5
to 1:6. A reconstitution factor of 1:3 is useful.
[0086] The invention also provides an aqueous pharmaceutical
composition having a pH of 5.0 to 7.0 comprising [0087] (i) an
anti-BAFFR antibody wherein the antibody has a concentration of at
least 50 mg/ml, and wherein said anti-BAFFR antibody includes heavy
chain CDR1, CDR2 and CDR3 of SEQ ID NOs 3,4, and 5 respectively,
and light chain CDR1, CDR 2 and CDR3 of SEQ ID NOs: 6, 7 and 8,
[0088] (ii) a stabilizer. [0089] (iii) a buffering agent, [0090]
(iv) a surfactant, and optionally [0091] (v) an amino acid,
[0092] In one embodiment the aqueous pharmaceutical composition
having a pH of 5.0 to 7.0 comprises [0093] (i) an anti-BAFFR
antibody wherein the antibody has a concentration of at least 50
mg/ml, and wherein said anti-BAFFR antibody includes heavy chain
CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and
light chain CDR1, CDR2 and CDR3 Of SEQ ID NOs: 6, 7 and 8, [0094]
(ii) sucrose or trehalose as a stabilizer, [0095] (iii) histidine
as a buffering agent, [0096] (iv) polysorbate 80 as a surfactant,
and optionally [0097] (v) an amino acid selected from arginine and
glycine.
[0098] In one embodiment the aqueous pharmaceutical composition
having a pH of 5.0 is 7.0 comprises [0099] (i) an anti-BAFFR
antibody wherein the antibody has a concentration of at least 50
mg/ml, and wherein said anti-BAFFR antibody includes heavy chain
CDR1, CDR2 and CDR3 of SEQ ID NOs 3, 4 and 5 respectively, and
light chain CDR1, CDR2 and CDR3 of SEQ ID NOs: 6, 7 and 8, [0100]
(ii) 200-300 mM sucrose as a stabilizer, [0101] (iii) 25-35 mM
histidine a buffering agent, [0102] (iv) up to 0.2% polysorbate 80
as a surfactant, and optionally [0103] (v) 10-80 mM arginine or
glycine.
[0104] In one embodiment the aqueous pharmaceutical composition has
a pH of 6.5 and comprises [0105] (i) an anti-BAFFR antibody wherein
the antibody has a concentration of at least 50 mg/ml, and wherein
said anti-BAFFR antibody includes heavy chain CDR1, CDR2 and CDR3
of SEQ ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2
and CDR3 of SEQ ID NOs: 6, 7 and 8, [0106] (ii) 270 mM sucrose as a
stabilizer, [0107] (iii) 21 mM histidine as a buffering agent and
[0108] (iv) 0.06% polysorbate 80 as a surfactant.
[0109] In one embodiment the aqueous pharmaceutical composition has
a pH of 6.5 and comprises [0110] (i) an anti-BAFFR antibody wherein
the antibody has a concentration of at least 50 mg/ml, and wherein
said anti-BAFFR antibody includes heavy chain CDR1, CDR2 and CDR3
of SEO ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2
and CDR3 of SEQ ID NOs: 6, 7 and 8, [0111] (ii) 270 mM sucrose as a
stabilizer, [0112] (iii) 21 mM histidine as a buffering agent,
[0113] (iv) 0.06% polysorbate 80 as a surfactant, and [0114] (v) 60
mM glycine.
[0115] In one embodiment the aqueous pharmaceutical composition has
a pH of 6.5 and comprises [0116] (i) an anti-BAFFR antibody wherein
the antibody has a concentration of at least 50 mg/ml, and wherein
said anti-BAFFR antibody includes heavy chain CDR1, CDR2 and CDR3
of SEQ ID NOs 3, 4 and 5 respectively, and light chain CDR1, CDR2
and CDR3 of SEQ ID NOs: 6, 7 and 8, [0117] (ii) 270 mM sucrose as a
stabilizer, [0118] (iii) 21 mM histidine as a buffering agent
[0119] (iv) 0.06% polysorbate 80 as a surfactant, and [0120] (v) 51
mM arginine.
[0121] In one embodiment the aqueous pharmaceutical composition of
the invention has a BAFFR antibody concentration o 150 mg/ml.
[0122] In one embodiment the lyophilized formulation or the aqueous
pharmaceutical composition of the invention comprises an anti-BAFFR
antibody comprising a V.sub.H domain with amino acid SEQ ID NO: 1
and a V.sub.L domain with amino acid SEQ ID NO: 2.
[0123] In one embodiment the lyophilized formulation or the aqueous
pharmaceutical composition of the invention comprises an anti-BAFFR
antibody comprising a heavy chain region of SEQ ID NO: 9 and a
light chain region of SEQ ID NO: 10.
[0124] In one embodiment the lyophilized formulation of the aqueous
pharmaceutical composition of the invention comprises the
anti-BAFFR antibody MOR66554 or MOR6654B.
[0125] The invention also comprises a delivery device including the
aqueous pharmaceutical composition of the invention.
[0126] The invention also comprises a pre-filled syringe including
the aqueous pharmaceutical composition of the invention.
[0127] The invention also comprises a method for delivering an
anti-BAFFR antibody to a mammal, comprising a step administering to
the patient an aqueous pharmaceutical composition of the
invention.
[0128] The invention also comprises a lyophilized formulation or an
aqueous pharmaceutical composition according to the invention for
use in treating a disease or disorder that is mediated by BAFF
receptor or that can be treated by killing or depleting B
cells.
[0129] The invention also comprises a lyophilized formulation or
aqueous pharmaceutical composition according to the invention for
use in the treatment of autoimmune diseases.
[0130] The invention also comprises a lyophilized formulation or
aqueous pharmaceutical composition according to the invention for
use in the treatment of B cell neoplasms, such as lymphoma,
leukemia or myeloma.
[0131] The invention also provides a lyophilized formulation or
aqueous pharmaceutical composition according to the invention for
use in the treatment of rheumatoid arthritis, systemic lupus
erythematosus, or Pemphigus vulgaris.
Aqueous Pharmaceutical Compositions with High Concentration of
Anti-BAFFR Antibodies
[0132] The invention relies, at least partly, in the formulation
properties of antibodies such as MOR6654 and MOR6654B, which retain
remarkable stability and Bioactive properties when formulated in a
high concentration either as a liquid (aqueous) or lyophilisate
composition.
[0133] As used herein, an "aqueous" pharmaceutical composition is a
composition suitable for pharmaceutical use, wherein the aqueous
carrier is distilled water. A composition suitable for
pharmaceutical use may be sterile, homogeneous and/or isotonic.
Aqueous pharmaceutical compositions may be prepared either directly
in an aqueous form, for example in pre-filled syringe ready for use
(the "liquid formulations") or as lyophilisate to be reconstructed
shortly before use. As used herein, the term "aqueous
pharmaceutical composition" refers to the liquid formulation or
reconstituted lyophilized formulation. In certain embodiments, the
aqueous pharmaceutical compositions of the invention are suitable
for parenteral administration to a human subject. In a specific
embodiment, the aqueous pharmaceutical compositions of the
invention are suitable for subcutaneous administration.
[0134] As used herein, the phrase "parenteral administration" means
mode of administration other than enteral and topical
administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrastemal injection and infusion.
[0135] The use of antibody as the active ingredient of
pharmaceuticals is now widespread, including file products
HERCEPTIN.TM. (trastuzumab), RITUXAN.TM. (rituximab), SYNAGIS.TM.
(palivizumab), etc. Techniques for purification of therapeutic
antibodies to a pharmaceutical grade are well known in the art.
[0136] The composition will usually be non-pyrogenic e.g.
containing <1 EU (endotoxin unit, a standard measure) per dose,
and preferably <0.1 EU per dose. The composition is preferably
gluten-free.
[0137] In specific embodiments, the aqueous pharmaceutical
compositions of the invention exhibit low to undetectable levels of
antibody aggregation or degradation, with very little to no loss of
the biological activities during manufacture, preparation,
transportation and long periods of storage, the concentration of
the anti-BAFFR antibody being at least about 50 mg/ml, 100 mg/ml,
150 mg/ml, 200 mg/ml, 250 mg/ml, or 300 mg/ml.
[0138] In one aspect, the invention relates to an aqueous
pharmaceutical composition with high concentration of anti-BAFFR
antibodies.
[0139] It is known in the art that such high concentration aqueous
pharmaceutical compositions can be diluted prior to injection, for
example, if lower antibody concentrations are required for specific
therapeutic interventions or when treating patients of lower body
weight including children. Suitable concentrations can be 25 mg/ml
or 10 mg/ml. Alternatively, the original formulation may be
produced with such a lower concentration.
[0140] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chains thereof. A naturally occurring
"antibody" is a glycoprotein comprising at least two heavy (H)
chains and two light (L) chains inter-connected by disulfide bonds.
Each heavy chain is comprised of a heavy chain variable region
(abbreviated herein as V.sub.H) and a heavy chain constant region.
The heavy chain constant region is comprised of three or four
domains, depending on the isotype, C.sub.H1, C.sub.H2, C.sub.H3 and
C.sub.H4. Each light chain is comprised of a light chain variable
region (abbreviated herein as V.sub.L) and a light chain constant
region. The light chain constant region is comprised of one domain,
C.sub.L. The V.sub.H and V.sub.L regions can be further subdivided
into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR) Each V.sub.H and V.sub.L
is composed of three CDRs and four FRs arranged from amino-terminus
to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4. The variable regions of the heavy and light chains
contain a binding domain that interacts with an antigen. The
constant regions of the antibodies may mediate the binding of the
immunoglobulin to host tissues or factor, including various cells
of the immune system (e.g., effector cells) and the first component
(Clq) of the classical complement system.
[0141] The term "antigen-binding portion" of an antibody (or simply
"antigen portion"), as used herein, refers to full length or one or
more fragments of an antibody that retain the ability to
specifically bind to an antigen (e.g., a portion of BAFFR). It has
been shown that the antigen-binding function of an antibody can be
performed by fragments of a full-length antibody. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1
domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; a Fd
fragment consisting of the V.sub.H and C.sub.H1 domains, a Fv
fragment consisting of the V.sub.L and V.sub.H domains of a single
arm of an antibody; a dAb fragment (Ward et al. 1989 Nature
341:544-546), which consists of a V.sub.H domain; and an isolated
complementarity determining region (CDR).
[0142] Furthermore, although the two domains of the Fv fragment,
V.sub.L and V.sub.H, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the
V.sub.L and V.sub.H regions pair to form monovalent molecules
(known as single chain Fv (scFv): see e.g., Bird et al., 1988
Science 242:423-426: and Huston et al., 1988 Proc. Natl. Acad. Sci.
85:5879-5883). Such single chain antibodies are also intended to be
encompassed within the term "antigen-binding region" of an
antibody. These antibody fragments are obtained using conventional
techniques known to those of skill in the art, and the fragments
are screened for utility in the same manner as are intact
antibodies.
[0143] An "isolated antibody", as used herein, refers to an
antibody that is substantially free of other antibodies having
different antigenic specificities, e.g., an isolated antibody that
specifically binds human BAFFR is substantially free of antibodies
that specifically bind antigens other than BAFFR. An isolated
antibody that specifically binds BAFFR may, however, have
cross-reactivity to other antigens, such as BAFFR molecules from
other species. Moreover, an isolated antibody may be substantially
free of other cellular material and/or chemicals.
[0144] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope.
[0145] The term "human antibody", as used herein, includes
antibodies having variable regions in which both the framework and
CDR regions are derived from sequences of human origin.
Furthermore, if the antibody contains a constant region, the
constant region also is derived from such human sequences, e.g.,
human germline sequences, or mutated versions of human germline
sequences or antibody containing consensus framework sequences
derived from human framework sequences analysis, for example, as
described in Knappik, et al. (2000. J Mol Biol 57-86).
[0146] The structures and locations of immunoglobulin variable
domains, e.g., CDRs, may be defined using well knows numbering
schemes, e.g., the Kabat numbering scheme, the Chothia numbering
scheme, a combination of Kabat and Chothia is (AbM), etc. (see,
e.g., Sequences of Proteins of Immunological Interest, U.S.
Department of Health and Human Services (1991). eds. Kabat et al.,
A Lazikani et at. (1997) J. Mol. Bio. 273:927 948). Throughout this
specification, the complementarity determining region ("CDR") is
defined according to the Kabat definition with the exception of
CDRH1 which is the stretch of amino acids defined by a combination
of both Kabat and Chothia definitions for this CDR.
[0147] The human antibodies of the invention may include amino acid
residues not encoded by human sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo). However, the term "human antibody", as used
herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have beers grafted onto human framework
sequences.
[0148] The term "human monoclonal antibody" refers to antibodies
displaying a single binding specificity which have variable regions
in which both the framework and CDR regions are derived from human
sequences.
[0149] The term "recombinant human antibody", as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as antibodies isolated from
an animal (e.g., a mouse) that is transgenic or transchromosomal
for human immunoglobulin genes or a hybridoma prepared therefrom,
antibodies isolated from a host cell transformed to express the
human antibody, e.g., from a transfectoma, antibodies isolated from
a recombinant, combinatorial human antibody library, and antibodies
prepared, expressed, created or isolated by any other means that
involve splicing of all or a portion of a human immunoglobulin
gene, sequences to other DNA sequences. Such recombinant human
antibodies have variable regions in which the framework and CDR
regions are derived from human germline immunoglobulin sequences.
In certain embodiments, however, such recombinant human antibodies
can be subjected to in vitro mutagenesis (or, when an animal
transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the V.sub.H and
V.sub.L regions of the recombinant antibodies are sequences that,
while derived from and related to human germline V.sub.H and
V.sub.L sequences, may not naturally exist within the human
antibody germline repertoire in vivo.
[0150] As used herein, "isotype" refers to the antibody class
(e.g., IgM, IgA, IgD, IgE and IgG such as IgG1, IgG2, IgG3 or IgG4)
that is provided by the heavy chain constant region genes.
[0151] The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody which binds specifically to an
antigen".
[0152] As used herein, an antibody that "specifically binds to
BAFFR polypeptide" or an "anti-BAFFR antibody" refers to an
antibody that binds to human BAFFR polypeptide of SEQ ID NO: 13
with a K.sub.D of 100 nM or less, 10 nM or less, 1 nM or less. An
antibody that "cross-resets with an antigen other than BAFFR"
refers to an antibody that binds that antigen with a K.sub.D of
0.5.times.10.sup.-8 nM or less, 5.times.10.sup.-9 M or less, or
2.times.10.sup.-9 M or less. An antibody that "does not cross-react
with a particular antigen" is intended to refer to an antibody that
binds to that antigen, with a K.sub.D of 1.5.times.10.sup.-8 M or
greater, or a K.sub.D of 5-10.times.10.sup.-8 M or
1.times.10.sup.-7 M or greater. In certain embodiments, such
antibodies that do not cross-react with the antigen exhibit
essentially undetectable binding against these proteins in standard
binding assays.
[0153] In one embodiment, a high concentration of an anti-BAFFR
antibody in the aqueous pharmaceutical composition of the invention
is at least 50 mg/ml. In one embodiment, a high concentration is at
least 100 mg/ml. In one embodiment, a high concentration is at
least 150 mg/ml. In one embodiment, a high concentration is at
least 200 mg/ml. In one embodiment, a high concentration at least
250 mg/ml, in one embodiment, a high concentration is at least 300
mg/ml.
[0154] In one embodiment, the aqueous pharmaceutical composition of
the invention comprises between 50 mg/ml and 300 mg/ml of an
anti-BAFFR antibody, for example, MOR6654, especially MOR6654B.
[0155] In one embodiment, the aqueous pharmaceutical composition of
the invention comprises between 75 mg/ml and 250 mg/ml of an
anti-BAFFR antibody, for example, MOR6654. especially MOR6654B.
[0156] In one embodiment, the aqueous pharmaceutics composition of
the invention comprises between 100 mg/ml and 250 mg/ml of an
anti-BAFFR antibody, for example, MOR6654, especially MOR6654B.
[0157] In one embodiment, the aqueous pharmaceutical composition of
the invention comprises between 100 mg/ml and 200 mg/ml of an
anti-BAFFR antibody, for example, MOR6654, especially MOR6654B.
[0158] In one embodiment, the aqueous pharmaceutical composition of
the invention comprises 150 mg/ml of an anti-BAFFR antibody, for
example, MOR6654, especially MOR6654B.
[0159] In one embodiment, the aqueous pharmaceutical composition of
the invention comprises about 50 mg/ml, about 60 mg/ml, about 70
mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110
mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150
mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190
mg/ml, about 200 mg/ml, about 210 mg/ml, about 220 mg/ml, about 230
mg/ml, about 240 mg/ml, about 250 mg/ml or about 300 mg/ml of an
anti-BAFFR antibody, for example, MOR6654, especially MOR6654B.
[0160] Furthermore, the aqueous pharmaceutical compositions are
stable such that even after storage for 4 weeks at 2-8.degree. C.,
less than 5%, 4%, 3%, 2%, 1%, 0.05% or 0.01% of the total
anti-BAFFR antibody is aggregated as measured by SEC-HPLC.
[0161] The aqueous pharmaceutical compositions may include, in
addition to the anti-BAFFR antibody, further components such as one
or more of the following: (i) a stabilizer; (ii) a buffering agent,
(iii) a surfactant; and (iv) a free amino acid inclusion of each of
such additional components can give compositions with low
aggregation of the anti-BAFFR antibody.
[0162] Suitable stabilizer for use with the invention can act,
e.g., as viscosity enhancing agents, bulking agents, solubilizing
agents, and/or the like. The stabilizer can be ionic or non ionic
(e.g. sugars). As sugars they include, but are not limited to,
monosaccharides, e.g., fructose, maltose, galactose, glucose,
O-mannose, sorbose and the like; disacchandes, e.g. lactose,
sucrose, trehalose, cellobiose, and the like; polysaccharides, e.g.
raffinose, melezitose, maltodextrins, dextrans, starches, and the
like; and alditols, such as mannitol, xylitol, maltitol, lactitol,
xylitol sorbitol (glucitol) and the like. For example, the sugar
may be sucrose, trehalose, raffinose, maltose, sorbitol or
mannitol. The sugar may be a sugar alcohol or an amino sugar.
Sucrose is particularly useful. As ionic stabilizer they include
salts such as NaCl or amino acid components such as
arginine-HCl.
[0163] Suitable buffering agents for use with the invention
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 phtalic acid; Tris,
thomethamine hydrochloride, or phosphate buffer. In addition, amino
acid components can also be used as buffering agent. Such amino
acid component includes without limitation glycine and histidine. A
histidine buffer is particularly useful.
[0164] The aqueous pharmaceutical compositions include such
buffering agent or pH adjusting agent to provide improved pH
control. In one embodiment an aqueous pharmaceutical composition of
the invention has a pH between 5.0 and 8.0, between 5.0 and 7.0,
between 6.0 and 8.0, or between 6.0 and 7.0. In a specific
embodiment, an aqueous pharmaceutical composition of the invention
has a pH of about 6.5.
[0165] As used herein, the term "surfactant" herein refers to
organic substances having amphipathic structures; i.e., 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 dispersing agents
for various pharmaceutical compositions and preparations of
biological materials.
[0166] Suitable surfactants for use with the invention include, but
are not limited to, non-ionic surfactants, ionic surfactants and
zwitterionic surfactants. Typical surfactants for use with the
invention include, but are not limited to, sorbitan fatty acid
esters (e.g. sorbitan monocaprylate, sorbitan monolaurate, sorbitan
monopalmitate), sorbitan trioleate, glycerine fatty acid esters
(e.g. glycerine monocaprylate, glycerine monomyristate, glycerine
monostearate), polyglycerine fatty acid esters (e.g. decaglyceryl
monostearate, decaglyceryl distearate, decaglyceryl monolinoleate),
polyoxyethylene sorbitan fatty acid esters (e.g. polyoxyethylene
sorbitan monolaurate, polyoxythylene sorbitan monocleate,
polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan
monopalmitate, polyoxyethyene sorbitan trioleate, polyoxyethylene
sorbitan tristearate), polyoxyethylene sorbitol fatty acid esters
(e.g. polyoxyethylene sorbitol tetrastearate, polyoxyethylene
sorbitol tetraoleate), polyoxyethylene glycerine fatty acid esters
(e.g. polyoxyethylene glyceryl monostearate), polyethylene glycol
fatty acid esters (e.g. polyethylene glycol distearate),
polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl ether),
polyoxyethlene polyoxypropylene alky ethers (e.g. polyoxyethylene
polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl
ether, polyoxyethylene polyoxypropylene cetyl ether),
polyoxyethylene alkylphenyl ethers (e.g. polyoxyethylene
nonylphenyl ether), polyoxyethylene hydrogenated castor oils (e.g.
polyoxythylene castor oil, polyoxyethylene hydrogenated castor
oil), polyoxyethylene beeswax derivatives (e.g. polyoxyethylene
sorbitol beeswax), polyoxyethylene lanolin derivatives (e.g.
polyoxyethylene lanolin) and polyoxyethylene fatty acid amides
(e.g. polyoxyethylene stearic acid amide); C.sub.10-C.sub.18 alkyl
sulfates (e.g. sodium cetyl sulfate, sodium lauryl sulfate, sodium
oleyl sulfate), polyoxyethylene C.sub.10-C.sub.18 alkyl ether
sulfate with an average of 2 to 4 moles of ethylene oxide units
added (e.g. sodium polyoxyethylene lauryl sulfate), and
C.sub.1-C.sub.18 alkyl sulfosuccinate ester salts (e.g. sodium
lauryl sulfosuccinate ester); and natural surfactants such as
lecithin, glycerophosopholipid, sphingophospholipids (e.g.
sphingomyelin), and sucrose esters of C.sub.12-C.sub.18 fatty
acids. A composition may include one or more of these surfactants.
Preferred surfactants are polyoxyethylene sorbitan fatty esters
e.g. polysorbate 20, 40, 60 or 80. Polysorbate 80 (Tween 80) is
particularly useful.
[0167] Suitable free amino acids for use with the invention
include, but are not limited to, arginine, lysine, histidine,
ornithine, isoleucine, leucine, alanine, glycine glutamic acid or
aspartic acid. The inclusion of a basic amino acid is preferred
i.e. arginine, lysine and/or histidine. If a composition includes
histidine then this may act both as a buffering agent and a free
amino acid, but when a histidine buffer is used it is typical to
include a non-histidine free amino acid e.g. to include histidine
buffer and lysine. An amino acid may be present in its D- and/or
L-form, but the L-form is typical. The amino acid may be present as
any suitable salt e.g. a hydrochloride salt, such as
arginine-HCl.
[0168] When present components (i) to (iv) will be at a
concentration sufficient to maintain the anti-BAFFR antibody in a
form which is active and soluble after either
[0169] (i) lyophilisation and storage and reconstitution (for
lyophilisates), or
[0170] (ii) conditioning in dosing units and storage (for liquid
formulations).
[0171] Thus a sugar may be present in the aqueous pharmaceutical
composition of the invention, e.g. after reconstitution of a
lyophilisate in water, at a concentration of between 3 and 400 mM
e.g. 50-300 mM, 200-300 mM, 250-300 mM. A concentration of 270 mM
sucrose is useful.
[0172] A buffering agent may be present in the aqueous
pharmaceutical composition of the invention, e.g. after
reconstitution of a lyophilisate in water, at a concentration of
between 1 and 600 mM e.g. 10-40 mM, 15-30 mm. 15-25 mM. A
concentration of 21 mM histidine buffer is useful.
[0173] A surfactant may be present in the aqueous pharmaceutical
composition of the invention, e.g. after reconstitution of a
lyophilisate in water, at a concentration of up to 0.2% (by volume)
e.g. 0.01-0.1%, 0.03-0.08%, 0.04-0.08%. A concentration of 0.06%
polysorbate 80 or polysorbate 20 is useful.
[0174] A free amino acid may be present in the aqueous
pharmaceutical composition of the invention, e.g. after
reconstitution of a lyophilisate water, at a concentration of
between 2 and 100 mM e.g. 10-80 mM, 20-70 mM, 30-60 mM, 40-60 mM. A
concentration of 51 mM arginine-HCl or 60 mM glycine-HCl is
useful.
[0175] A formulation containing histidine buffer, sucrose and
polysorbate 80 has been shown to be suitable for lyophilisation of
antibody MOR6654B at a concentration of at least 150 mg/ml after
reconstitution.
[0176] In one embodiment the aqueous pharmaceutical composition
consists of 150 mg/ml MOR6654 or MOR6654B, 21 mM histidine, 270 mM
sucrose and 0.06% polysorbate 80.
[0177] In one embodiment the aqueous pharmaceutical composition
consists of 150 mg/ml MOR6654 or MOR6654B, 21 mM histidine, 270 mM
sucrose, 0.06% polysorbate 80 and 60 mM glycine-HCl.
[0178] In one embodiment the aqueous pharmaceutical composition
consists of 150 mg/ml MOR6654 or MOR6654B, 21 mM histidine, 270 mM
sucrose, 0.06% polysorbate 80 and 51 mM arginine-HCl.
[0179] In one embodiment the aqueous pharmaceutical composition
consists of 200 mg/ml MOR6654B, 21 mM histidine, 270 my sucrose,
0.06% polysorbate 80 and 51 mM arsinine-HCl.
[0180] In one embodiment the aqueous pharmaceutical composition
consists of 75 mg/ml MOR6654 or MOR6654B, 21 mM histidine, 270 mM
sucrose, 0.06% polysorbate 80 and 51 mM arginine-HCl.
[0181] Other contemplated excipients, which may be utilized in the
aqueous pharmaceutical compositions 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), recombinant human albumin,
gelatin, casein, salt-forming counterions such 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 "The Handbook of
Pharmaceutical Excipients, 4.sup.th edition, Rowe et al., Eds.,
American Pharmaceuticals Association (2003); and Remington: the
Science and Practice of Pharmacy, 21.sup.th edition, Gennaro, Ed.,
Lippincott Williams & Wilkins (2005).
[0182] The aqueous pharmaceutical compositions of the invention may
include further active ingredients in addition to the anti-BAFFR
antibody. Further pharmacological agents may include, for instance,
chemotherapeutic compounds.
Lyophilisates
[0183] Techniques for lyophilisation of antibodies are well known
in the art e.g. see John F. Carpenter and Michael J. Pikal, 1997
(Pharm. Res. 14, 969-975); Xialin (Charlie) Tang and Michael J
Pikal, 2004 (Pharm Res. 21, 191-200). For example, the monoclonal
antibody products SYNAGIS.TM., REMICADE.TM., RAPTIVA.TM.,
SIMULECT.TM., XOLAIR.TM. and HERCEPTIN.TM. are supplied as
lyophilisates. These antibodies are reconstituted to various final
concentrations e.g, SIMULECT.TM. is reconstituted to a
concentration of 4 mg/ml antibody, REMICADE.TM. is reconstituted to
a concentration of 10 mg/ml, HERCEPTIN to 21 mg/ml. SYNAGIS and
RAPTIVA.TM. to 100 mg/ml, and XOLAIR.TM. to 125 mg/ml.
Pre-lyophilisates, Lyophilisates and Aqueous Reconstitution
[0184] Before a lyophilisate can be administered to a patient it
should be reconstituted with an aqueous reconstituent. This step
permits antibody and other components in the lyophilisate to
re-dissolve to give a solution which is suitable for injection to a
patient.
[0185] The volume of aqueous material used for reconstitution
dictates the concentration of the antibody in a resulting
pharmaceutical composition. Reconstitution with a smaller volume of
reconstituent than the pre-lyophilisation volume provides a
composition which is more concentrated than before lyophilisation.
The reconstitution factor (volume of formulation after
lyophilization volume of formulation before lyophilization) may be
from 1:0.5 to 1:6. A reconstitution factor of 1:3 is useful. As
mentioned above, lyophilisates of the invention can be
reconstituted to give aqueous compositions with an anti-BAFFR
antibody concentration of at least 50 mg/ml, 100 mg/ml, 150 mg/ml,
200 mg/ml, 250 mg/ml or 300 mg/ml, and the volume of reconstituent
will be selected accordingly, if required, the reconstituted
formulation can be diluted prior to administration to a patient as
appropriate to deliver the intended dose.
[0186] Typical reconstituents for lyophilized antibodies include
sterile water or buffer, optionally containing a preservative. If
the lyophilisate includes a buffering agent then the reconstituent
may include further buffering agent (which may be the same as or
different from the lyophilisate buffering agent) or it may instead
include no buffering agent (e.g. WFI (water for injection), or
physiological saline).
[0187] When present, components (i) to (iv) will be at a
pre-lyophilisation concentration sufficient to maintain the
anti-BAFFR antibody in a form which is active and soluble after
storage (under normal conditions) and reconstitution. The
components will also be present after reconstitution.
[0188] Thus a sugar, such as sucrose or trehalose, may be present
before lyophilisation at a concentration of between 3 and 300 mM
e.g. 15-200 mM 30-150 mM, 80-100 mM. A concentration of 90 mM
sucrose is useful. A buffering agent, such as histidine, may be
present before lyophilisation at a concentration of between 1 and
60 mM e.g. 3-30 mM, 5-20 mM, 5-15 mM. A concentration of 7 mM
histidine buffer is useful. A surfactant, such as polysorbate 80 or
polysorbate 20 may be present before lyophilisation at a
concentration of up to 0.2% (by volume) e.g. 0.01-0.1%, 0,01-0,08%,
0.01-0.04%. A concentration of 0.02% polysorbate 80 or polysorbate
20 is useful. A free ammo acid, such as arginine or glycine, may be
present before lyophilization at a concentration of between 2 and
80 mM e.g. 3-50 mM, 6-30 mM, 10-25 mM, 15-20 mM. A concentration of
17 mM arginine-HCI or 20 mM glycine-HCl is useful. The anti-BAFFR
antibody is present before lyophilization at a concentration of
between 20 mg/ml and 120 mg/ml, e.g. 20 mg/ml, 30 mg/ml, 40 mg/ml,
50 mg/ml, 60 mg/ml. 66.6 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100
mg/ml, 110 mg/ml, or 120 mg/ml. A concentration of 50 mg/ml is
useful.
[0189] The pre-lyophilisate of the invention has a pH between 5.0
and 8.0, between 5.0 and 7.0, between 6.0 and 8.0, or between 6.0
and 7.0. In a specific embodiment, the pre- lyophilisate of the
invention has a pH of about 6.5.
[0190] In one embodiment the pre-lyophilisate of the invention has
a molar ratio of sucrose:antibody of 270:1 and a molar ratio of
histidine:antibody of 21:1.
[0191] In one embodiment the pre-lyophilisate of the invention has
a molar ratio of sucrose:antibody of 270:1, a molar ratio of
histidine:antibody of 21.1, and a molar ratio of
arginine-HCl:antibody of 51:1.
[0192] In one embodiment the pre-lyophilisate of the invention has
a molar ratio of sucrose:antibody of 270:1, a molar ratio of
histidine-antibody of 21:1, and a molar ratio of
glycine-HCl:antibody of 60:1.
[0193] In one embodiment the pre-lyophilisate of the invention has
a molar ratio of sucrose:antibody of 203:1, a molar ratio of
histidine:antibody of 16:1, and a molar ratio of
arginine-HCl:antibody of 38:1.
[0194] A formulation containing histidine buffer, sucrose,
polysorbate 80 and, optionally arginine or glycine has been shown
to be suitable for lyophilisation of antibody MOR6654B. After
reconstitution the components of the lyophilisate may be present at
a concentration of the aqueous pharmaceutical compositions as
described hereinbefore.
Target Diseases and Disorders
[0195] The aqueous pharmaceutical compositions of the invention
comprising anti-BAFFR antibodies can be used to treat, ameliorate
or prevent a variety of diseases or disorders. Pharmaceutical
compositions comprising anti-BAFFR antibodies are particularly
useful to treat BAFFR related disorders such as autoimmune
disorders, e.g., systemic lupus erythematosus, Pemphigus vulgaris,
rheumatoid arthritis, multiple sclerosis and B cell neoplasms such
as acute lymphoblastic leukemia (ALL) and B-cell chronic
lymphocytic leukemia (CLL).
[0196] As used herein, "a BAFFR-related disorder" includes
conditions associated with or characterized by aberrant BLyS levels
and/or diseases or conditions that can be treated by depleting or
killing B cells. These includes, without limitations, inflammatory
conditions, autoimmune diseases, severe infections, and organ or
tissue transplant rejection. These further include B-cell
neoplasms.
[0197] For example, the aqueous pharmaceutical compositions of the
invention comprising anti-BAFFR antibodies may be used for the
treatment, amelioration or prevention of recipients of heart, lung,
combined heart-lung, liver, kidney, pancreatic, skin or corneal
transplants, including allograft rejection or xenograft rejection,
and for the prevention of graft-versus-host disease, such as
following bone marrow transplant, and organ transplant associated
arteriosclerosis. Further, the aqueous pharmaceutical compositions
of the invention are useful in solid organ transplantation and in
antibody-mediated acute and chronic transplant rejection.
[0198] The aqueous pharmaceutical compositions of the invention
comprising anti-BAFFR antibodies are useful for the treatment,
prevention, or amelioration of autoimmune disease and of
inflammatory conditions, in particular inflammatory conditions with
an etiology including an autoimmune component such as arthritis
(for example rheumatoid arthritis, arthritis chronica progrediente
and arthritis deformans) and rheumatic diseases, including
inflammatory conditions and rheumatic diseases involving bone loss,
inflammatory pain, spondyloarthropathies including ankylosing
spondylitis, Reiter syndrome, reactive arthritis, psoriatic
arthritis, and enterophethics arthritis, hypersensitivity
(including both airways hypersensitivity and dermal
hypersensitivity) and allergies. Specific auto-immune diseases for
which antibodies of the invention may be employed include
autoimmune haematological disorders (including e.g. hemolytic
anaemia, aplastic anaemia, pure red cell anaemia and idiopathic
thrombocytopenia), acquired hemophilia A, cold agglutinin disease,
cryoglobulinemia, thrombotic thrombocytopenic purpura, Sjogren's
syndrome, systemic lupus, erythematosus, inflammatory muscle
disorders, polychondritis, scleroderma, vasculitis such as
cryoglobulinemia, large vessel vasculitides such as giant cell
arteritis, polymyalgia rheumatics, necrotizing vasculitides,
including anti-neutrophil cytoplasmic antibody- associate
vasculitis, Takayasu's arteritis, polyarteritis nodosa,
Henoch-Schonlein purpura, and Churg-Strauss syndrome. IgM mediated
neuropathy, seronegative spondarthritis, opsocionus myoclonus
syndrome, Wegener granulomatosis, dermatomyositis, anti-neutrophil
cytoplasmatic autoantibody (ANCA) vasculitis, chronic active
hepatitis, myasthenia gravis, psoriasis. Steven-Johnson syndrome,
pemphigus vulgaris, pemphigus foliacius, idiopathic sprue,
autoimmune inflammatory bowel disease including e.g. ulcerative
colitis, Crohn's disease and irritable Bowel Syndrome), endocrine
ophthalmopathy, Graves' disease, sarcoidosis, multiple sclerosis,
neuromyelitis optica, primary biliary cirrhosis, juvenile diabetes
(diabetes mellitus type I), uveitis (anterior intermediate and
posterior as well as panuveitis), keratoconjunctives sicca and
vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic
arthritis and glomerulonephritis (with and without nephrotic
syndrome, e.g. including idiopathic nephrotic syndrome or minimal
change nephropathy), acute nephritic lupus, tumors, inflammatory
disease of skin and cornea, myositis, loosening of bone implants,
metabolic disorders, such as atherosclerosis, diabetes, and
dislipidemia.
[0199] The aqueous pharmaceutical compositions of the invention may
also be useful in preventing, ameliorating or treating B-cell
neoplasms. Examples of such diseases and conditions include, but
are not limited to, B-cell Non-Hodgkins lymphomas, such as small
lymphocytic lymphoma, lymphoplasmacytoid lymphoma, mantle cell
lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue
lymphoma, diffuse large cell lymphoma, and Burkitt's lymphoma;
acute lymphoblastic leukemia (ALL), precursor B-lymphoblastic
leukemia; B-cell chronic lymphocytic leukemia (CLL), and multiple
myeloma. Other B-cell neoplasms are encompassed within the scope of
the invention.
Patient Administration
[0200] A pharmaceutical composition of the invention can be
administered to a patient. Administration will typically be via a
syringe. Thus the invention provides a delivery device (e.g. a
syringe) including a pharmaceutical composition of the invention
(e.g., pre- filled syringe). Patients will receive an affective
amount of the anti-BAFFR antibody as the principal active
ingredient i.e. an amount that is sufficient to treat, ameliorate,
or prevent the disease or disorder in question. Therapeutic effects
may also include reduction in physical symptoms. The optimum
effective amount and concentration of antibody for any particular
subject will depend upon various factors, including the patient's
age size health and/or gender, the nature and extent of the
condition, the activity of the particular antibody, the rate of its
clearance by the body, and also on any possible further
therapeutic(s) administered in combination with the antibody. The
effective amount delivered for a given situation can be determined
within the judgment of a clinician. For purposes of the present
invention, an effective dose may be from about 0.005 mg/kg to about
50 mg/kg, or about 0.05 mg/kg to about 10 mg/kg. Known
antibody-based pharmaceuticals provide guidance in this respect
e.g. HERCEPTIN.TM. is administered with an initial loading dose of
4 mg/kg body weight and a weekly maintenance dose of 2 mg/kg body
weight; RITUXAN.TM. is administered weekly at 375 mg/m.sup.2;
SYNAGIS.TM. is administered intramuscularly at 15 mg/kg body
weight; etc.
[0201] The invention provides a method for delivering a monoclonal
antibody to a mammal, comprising a step of administering to the
patient a pharmaceutical composition of the invention.
[0202] The invention also provides a method for delivering a
monoclonal antibody to a mammal, comprising steps of: (i)
reconstituting a lyophilisate of the invention to give an aqueous
formulation, and (ii) administering the aqueous formulation to the
patient. Step (ii) ideally takes place within 24 hours of step (i)
e.g. within 12 hours, within 6 hours, within 3 hours, or within 1
hour.
[0203] The invention also provides formulations of the invention
for use as medicaments e.g. for use in delivering an antibody to a
mammal, or for use in treating, preventing or ameliorating one or
more of the diseases and disorders described above.
[0204] The mammal is preferably a human but may also be, for
example, a horse or a cow or a dog or a cat. The antibodies will
ideally be chosen to match the target species e.g. a human antibody
for human administration, an equine antibody for horses, a canine
antibody for dogs, etc. If native host antibodies are not available
than transfer of antibody specificity from one species to another
can be achieved by transfer of CDR residues (and typically, in
addition, one or more framework residues) from a donor antibody
into a recipient framework from the host species e.g. as in
humanization. Equinized, bovinized, caninized and felinized
antibodies are known in the art. The antibody will bind to BAFFR
from the target species, but it may also cross-react with BAFFR
from other species.
[0205] Dosage can be by a single dose schedule or a multiple dose
schedule.
[0206] Ingredients for forming compositions of the invention (e.g.
lyophilisates and reconstituents) may be supplied in
hermetically-seated containers.
The Anti-BAFFR Antibody
[0207] The invention concerns the formulation of anti-BAFFR
antibodies and more specifically MOR6654 and MOR6654B.
[0208] One suitable antibody that can be comprised in the
pharmaceutical compositions of the invention is the human
recombinant antibody MOR6654, structurally characterized as further
described below. The V.sub.H amino acid sequence of such isolated
anti-BAFFR antibody is shown in SEQ ID NO: 1. The V.sub.L amino
acid sequence of such isolated anti-BAFFR antibody is shown in SEQ
ID NO: 2. An example of the full length heavy chain amino acid
sequence of such isolated anti-BAFFR antibody is shown in SEQ ID
NO: 9. An example of the full-length light chain amino acid
sequence of such isolated anti- BAFFR antibody is shown in SEQ ID
NO: 10. Another example of heavy and light chain amino acid
sequences of such isolated anti-BAFFR antibodies are those encoded
by the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO. 12
respectively. Another example of heavy and light chain amino acid
sequences of antibodies are those encoded by corresponding DNA
sequences contained in plasmid pBW510 as deposited by Novartis
Pharma AG, Forum 1. CH-4002 Basel, Switzerland, at DSMZ on Apr. 29,
2009 with accession number DSM22542.
[0209] Other anti-BAFFR antibodies that can be used for preparing
the pharmaceutical compositions of the invention include anti-BAFFR
antibodies, with amino acids that have been mutated by amino add
deletion, insertion or substitution, yet have no more than 1, 2, 3,
4 or 5 amino acid deletion, insertion or substitution in either the
heavy or light chain regions described above. In a specific
embodiment, such amino acid changes appear only within the
framework and/or constant regions and the CDR regions are 100%
identical to the heavy chain CDR1, CDR2 and CDR3 regions of SEQ ID
NO: 3, 4 and 5 and to the light chain CDR1, CDR2 and CDR3 regions
of SEQ ID NO: 6, 7, and 8 respectively. In one more specific
embodiment, the changes that have been made are only conservative
amino acid substitutions outside of the CDR regions.
[0210] Conservative amino add substitutions are ones in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues outside of the CDR regions of an
anti-BAFFR antibody, can be replaced with other amino acid residues
from the same side chain family, and the altered antibody can be
tested for retained function, in particular the same binding
properties to BAFFR.
[0211] Antibodies may typically be glycosylated. N-linked glycans
attached to the C.sub.H2 domain of a heavy chain, for instance, can
influence C1q and FcR binding, and aglycosylated antibodies may
have lower or different affinity for these receptors. The glycan
structure can also affect activity e.g. differences in
complement-mediated cell death may be seen depending on the number
of galactose sugars (0, 1 or 2) at the terminus of a glycan's
biantennary chain. An antibody's glycans preferably do not lead to
a human immunogenic response after administration.
[0212] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, such as a hypofucosylated of
non-fucosylated antibody having reduced amounts of or no fucosyl
residues or an antibody having increased bisecting GlcNac
structures. Such altered glycosylation patterns have been
demonstrated to increase the antibody-dependent cell-mediated
cytotoxicity (ADCC) ability of antibodies. Such carbohydrate
modifications can be accomplished by, for example, expressing the
antibody in a host cell with altered glycosylation machinery. Cells
with an altered glycosylation machinery have been described in the
art and can be used as host cells in which to express recombinant
antibodies of the invention to thereby produce an antibody with
altered glycosylation. For example, EP 1,176,196 by Hang et al.
describes a cell line with a functionally disrupted FUT8 gene,
which encodes a fucosyl transferase, such that antibodies expressed
in such a cell line exhibit hypofucosylation or are devoid of
fucosyl residues. Therefore, in one embodiment, the anti-BAFFR
antibodies that are included in the pharmaceutical compositions of
the invention are produced by recombinant expression in a cell line
which exhibit hypofucosylation or non-fucosylation pattern, for
example, a mammalian cell line with deficient expression of the
FUT8 gene encoding fucosyltransferase.
[0213] As used herein, the term MOR6654 encompasses any type of
glycosyation pattern. In a specific embodiment, the pharmaceutical
compositions comprises an anti-BAFFR antibody consisting of MOR6654
as produced in a cell line which exhibits a hypofucosylation or
non-fucosylation pattern, such as MOR6654B, which exhibit non-
fucosylation pattern (devoid of fuscosyl residues). PCT Publication
WO 03/035835 by Presta describes a variant CHO cell line, LecI3
cells, with reduced ability to attach fucose to Asn(297)-linked
carbohydrates, also resulting in hypofucosylation of antibodies
expressed in that host cell (see also Shields. R. L et al. 2002 J.
Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana
et al. describes cell lines engineered to express
glycoprotein-modifying glycosyl transferases (eg., beta(1.4)-N
acetylglucosaminyltransferase III (GnTIII)) such that antibodies
expressed in the engineered cell lines exhibit increased bisecting
GlcNac structures which results in increased ADCC activity of the
antibodies (see ate Umana et al., 1999 Nat. Biotech. 17:176-180).
Eureka Therapeutics further describes genetically engineered CHO
mammalian cells capable of producing antibodies with altered
mammalian glycosylation pattern devoid of fucosyl residues
(http://www.eurekainc.com/about_us/companyoverview.html).
Alternatively, the anti-BAFFR antibodies can be produced in yeasts
or filamentous fungi engineered for mammalian-like glycosylation
pattern and capable of producing antibodies lacking fucose as
glycosylate pattern (see for example EP1297172B1).
[0214] Another modification of the anti-BAFFR antibodies herein
that is contemplated by the invention is pegylation. An antibody
can be pegylated to, for example, increase the biological (e.g.,
serum) half-life of the antibody. To pegylate an antibody, the
antibody, or fragment thereof, typically may be reacted with
polyethylene glycol (PEG), such as a reactive ester or aldehyde
derivative of PEG, under conditions in which one or more PEG groups
become attached to the antibody or antibody fragment. The
pegylation can be carried out by an acylation reaction or an
alkylation reaction with a reactive PEG molecule (or an analogous
reactive water-soluble polymer).
[0215] As used herein, the term "polyethylene glycol" is intended
to encompass any of the forms of PEG that have been used to
derivative other proteins, such as mono (C1-C10) alkoxy- or
arytoxy-polyethylene glycol or polyethylene glycol-maleimide. In
certain embodiments, the antibody to be pegylated is an
aglycosylated antibody. Methods for pegylating proteins are known
in the art and can be applied to the antibodies of the invention.
See for example, EP 0 154 31 by Nishimura et al. and EP 0 401 364
by Ishikawa et al.
[0216] Any other natural or non-natural post-translational
modification of anti-BAFFR antibodies (e.g. MOR6654) is further
contemplated as specific embodiments of anti-BAFFR antibodies that
could be used for preparing the pharmaceutical compositions of the
invention.
[0217] Antibodies can be prepared in a form free from products with
which they would naturally be associated. Contaminant component of
an antibody's natural environment include materials such as
enzymes, hormones, or other host cell proteins.
EXAMPLES
Preparing Anti-BAFFR Antibodies
[0218] Antibody MOR6654 binds specially to BAFFR and is also
described in reference WO2010/007082. It is a human IgG1 kappa
antibody obtained via phage display. Its heavy and light chains
consist of SEQ ID NOs: 9 and 10. The Tables 1 and 2 below summarize
the sequence characteristics of MOR6654.
[0219] This antibody may be produced in mammalian host cells, such
as, a CHO cell line transfected with expression vectors carrying
heavy and light chain coding sequences under suitable expression
promoters.
[0220] This antibody is preferably produced in a mammalian call
line, e.g. a CHO cell line, wherein the gene encoding
fucosyltransferase (FUT8 gene) has been inactivated. The resulting
antibody is non-fucosylated and designated here as MOR6654B.
TABLE-US-00001 TABLE 1 Brief description of the sequences listed in
the sequence listing of Table 2 SEQ ID NO: Description of the
sequence 1 Amino acid sequence of the variable region (V.sub.H) of
the heavy chain of MOR6654 2 Amino acid sequence of the variable
region (V.sub.L) of the light chain of MOR6654 3 Amino acid
sequence of HCDR1 of MOR6654 4 Amino acid sequence of HCDR2 of
MOR6654 5 Amino acid sequence of HCDR3 of MOR6654 6 Amino acid
sequence of LCDR1 of MOR6654 7 Amino acid sequence of LCDR2 of
MOR6654 8 Amino acid sequence of LCDR3 of MOR6654 9 Amino acid
sequence of the full length heavy chain of MOR6654 10 Amino acid
sequence of the full length light chain of MOR6654 11 Nucleotide
sequence encoding SEQ ID NO: 1 12 Nucleotide sequence encoding SEQ
ID NO: 2 13 Human BAFFR amino acid sequence 14 Full length
nucleotide sequence (including leader sequence and constant part)
of MOR6654 heavy chain; nt 1-57 = leader; nt 58-429 = VH; nt
430-1419 = constant region (hIgG1) 15 Full length nucleotide
sequence (including leader sequence and constant part) of MOR6654
light chain; nt 1-60 = leader; nt 61-384 = VL; nt 385-705 =
constant region (hkappa)
TABLE-US-00002 TABLE 2 Sequence listing SEQ ID NO: Amino acid or
Nucleotide Sequence 1 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWGWIR
QSPGRGLEWLGRIYYRSKWYNSYAVSVKSRITINPDTSKN
QFSLQLNSVTPEDTAVYYCARYDWVPKIGVFDSWGQGTLV TVSS 2
DIVLTQSPATLSLSPGERATLSCRASQFISSSYLSWYQQK
PGQAPRLLIYGSSSRATGVPARFSGSGSGTDFTLTISSLE
PEDFAVYYCQQLYSSPMTFGQGTKVEIK 3 GDSVSSNSAAWG 4 RIYYRSKWYNSYAVSVKS 5
YDWVPKIGVFDS 6 RASQFISSSYLS 7 GSSSRAT 8 QQLYSSPMT 9
QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWGWIR
QSPGRGLEWLGRIYYRSKWYNSYAVSVKSRITINPDTSKN
QFSLQLNSVTPEDTAVYYCARYDWVPKIGVFDSWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL
GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT
TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 10
DIVLTQSPATLSLSPGERATLSCRASQFISSSYLSWYQQK
PGQAPRLLIYGSSSRATGVPARFSGSGSGTDFTLTISSLE
PEDFAVYYCQQLYSSPMTFGQGTKVEIKRTVAAPSVFIFP
PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS
QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGFC 11
CATGCAGCTGCAGCAGAGCGGCCCAGGCCTGGTCAAGCCC
TCTCAGACCTGTCACTGACCTGCGCCATTTCAGGCGACAG
CGTGAGCAGCAACAGCGCCGCCTGGGGCTGGATCAGGCAG
AGCCCCGGTAGGGGCCTGGAATGGCTGGGCAGGATCTACT
ACAGGTCCAGGTGGTACAACAGCTACGCCGTGAGCGTGAA
GAGCAGGATCACCATCAACCCTGACACCAGCAAGAACCAG
TTCTCACTGCAGCTCAACAGCGTGACCCCCGAGGACACCG
CCGTGTACTACTGCGCCAGATACGACTGGGTGCCCAAGAT
CGGCGTGTTCGACAGCTGGGGCAGGGCACCCTGGTGACCG TGTCAAGC 12
GATATGGTGCTGACACAGAGCCCCGCCACCCTGAGCCTGA
GCCCAGGCGAGAGGGCCACCCTGTCCTGCAGGGCCAGCCA
GTTTATCAGCAGCAGCTACCTGTCCTGGTATCAGCAGAAG
CCCGGCCAGGCCCCTAGACTGCTGATCTACGGCAGCTCCT
CTCGGGCCACCGGCGTGCCCGCCAGGTTCAGCGGCAGCGG
CTCCGGCACCGACTTCACCCTGACAATCAGCAGCCTGGAG
CCCGAGGACTTCGCCGTGTACTACTGCCAGCAGCTGTACA
GCTCACCCATGACCTTCGGCCAGGGCACCAAGGTGGAGAT CAAG 13
MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRT
PRPKPAGASSPAPRTALQPQESVGAGAGEAALPLPGLLFG
APALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDGD
KDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHS VPVPATELGSTELVTTKTAGPEQQ
14 ATGGCCTGGGTGTGGACCCTGCCCTTCCTGATGGCCGCTG
CCCAGTCAGTGCAGGCCGAGGTGCAGCTGCAGCAGAGCGG
CCCAGGCCTGGTCAAGCCCTCTCAGACCCTGTCACTGAGC
TGCGCCATTTCAGGCGACAGCGTGAGCAGCAACAGCGCCG
CCTGGGGCTGGATCAGGCAGAGCCCCGGTAGGGGCCTGGA
ATGGCTGGGCAGGATCTACTACAGGTCGAAGTGGTACAAC
AGCTACGCCGTGAGCGTGAAGAGCAGGATCACCATCAACC
CTGAGACCAGCAAGAACGAGTTCTCACTGCAGCTCAACAG
CGTGACCCCCGAGGACACCGCGGTGTACTACTGCGCCAGA
TACGACTGGGTGCCCAAGATCGGCGTGTTCGACAGCTGGG
GCCAGGGCACCCTGGTGACCGTGTCAAGCGCCAGCACCAA
GGGCCCCAGCGTGTTGCCCCTGGCCCCCAGCAGGAAGAGC
ACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGG
ACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGG
AGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTG
CAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAG
TGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAA
CGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGA
GTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCC
CCTGCCCAGCCCCAGAGCTGCTGGGCGGACGCTCCGTGTT
CCTGTTCCCCCCCAAGCCCAAGGACACGCTGATGATCAGC
AGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCC
ACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGG
CGTGGAGGTGGACAACGCCAAGACCAAGCCCAGAGAGGAG
CAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCG
TGCTGCACCAGGACTGGGTGAACGGCAAGGAATAGAAGTG
CAAGGTCTCCAACAAGGCGCTGCCAGCCCGCATCGAAAAG
ACCATCAGGAAGGCCAAGGGCCAGCCACGGGAGCCCCAGG
TGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAA
CCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCC
AGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCG
AGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGA
CGGCAGGTTCTTCCTGTACAGCAAGCTGACCGTGGACAAG
TCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGA
TGCACGAGGGCCTGCACAACCACTACACCCAGAAGAGCCT GAGCCTGTCCCCCGGCAAG 15
ATGAGCGTGCTGACCCAGGTGCTGGCTCTGCTGCTGCTGT
GGCTGACCGGCACGAGATGCGATATCGTGCTGACACAGAG
CCCCGGCACCCTGAGCGTGAGCCCAGGCGAGAGGGCCACC
CTGTCGTGCAGGGCCAGCCAGTTTATCAGCAGCAGCTACC
TGTCCTGGTATCAGCAGAAGCCCGGGCAGGCCCCTAGACT
GCTGATGTACGGCAGGTCCTCTCGGGCCACCGGCGTGCCC
GCCAGGTTCAGCGGCAGCGGCTCCGGCACCGACTTCACCC
TGACAATCAGCAGCCTGGAGCCCGAGGAGTTCGCCGTGTA
CTACTGCCAGCAGCTGTACAGCTCAGCCATGACCTTCGGC
GAGGGCACCAAGGTGGAGATCAAGCGTACGGTGGCCGCTC
CCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAA
GAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTC
TACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACG
GCCTGCAGAGCGGCAACAGCGAGGAGAGCGTCACCGAGCA
GGACAGCAAGGACTCCACCTACAGCGTGAGCAGCACCCTG
ACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACG
CCTGCGAGGTGACCCACCAGGGGGTGTGCAGCGCCGTGAC
GAAGAGGTTCAACAGGGGCGAGTGC
Examples of Formulations
[0221] A high concentration lyophilized or liquid formulation of
MOR6654B was desired and so formulation studies were performed. A
lyophilized formulation comprising a sugar, a buffering agent and a
surfactant was stable and could maintain high anybody
concentrations after reconstitution.
[0222] Three formulations (F1, F2, F3) of MOR6654B at 150 mg/vial
and one formulation at 200 mg/vial (F3b) were evaluated for
stability. Formulations F1, F2 and F3 had, prior to lyophilisation,
50 mg/ml MOR6654B at pH 6.5, and formulation F3b 66.6 mg/ml at pH
6.5. The four formulations had a volume of 3.6 ml and included
buffer, sugar, surfactant and free amino acid as fellows in Table
3:
TABLE-US-00003 TABLE 3 Examples of formulations MOR6654B Buffer
Sugar Surfactant Amino acid F1 50 mg/ml 7 mM 90 mM 0.02% --
histidine sucrose polysorbate 80 F2 50 mg/ml 7 mM 90 mM 0.02% 20 mM
histidine sucrose polysorbate glycine-HCl 80 F3 50 mg/ml 7 mM 90 mM
0.02% 17 mM histidine sucrose polysorbate arginine-HCl 80 F3b 66.6
mg/ml 7 mM 90 mM 0.02% 17 mM histidine sucrose polysorbate
arginine-HCl 80
[0223] The lyophilisates were reconstituted with WFI (1 ml) to give
a reconstituted volume of 1.2 ml (20% overage; 1/3 the original
aqueous volume). The reconstituted compositions were as in Table
4:
TABLE-US-00004 TABLE 4 Examples of formulations MOR6654B Buffer
Sugar Surfactant Amino acid F1 150 mg/ml 21 mM 270 mM 0.06% --
histidine sucrose polysorbate 80 F2 150 mg/ml 21 mM 270 mM 0.06% 60
mM histidine sucrose polysorbate glycine-HCl 80 F3 150 mg/ml 21 mM
270 mM 0.06% 51 mM histidine sucrose polysorbate arginine-HCl 80
F3b 200 mg/ml 21 mM 270 mM 0.06% 51 mM histidine sucrose
polysorbate arginine-HCl 80
[0224] The lyophilisation cycle used is reported to Table 5.
TABLE-US-00005 TABLE 5 The lyophilisation cycle parameters Time
Chamber Step Operation [hh:mm] Shelf temp. pressure 1 Vial loading
As required 20.degree. C. Ambient 2 5.degree. C. cooling 00:30
5.degree. C. Ambient 3 5.degree. C. hold 03:00 5.degree. C. Ambient
4 Freeze ramp 01:24 5.degree. C. to -37.degree. C. Ambient 5 Freeze
hold 04:00 -37.degree. C. Ambient 6 Chamber Vacuum 00:10
-37.degree. C. 0.2 mbar.sup.a 7 Primary drying ramp 16:00
-37.degree. C. to 25.degree. C. 0.2 mbar.sup.a 8 Secondary drying
hold 24:00 25.degree. C. 0.2 mbar.sup.a 11 Vial stoppering
25.degree. C. 850 .+-. 50 mbar .sup.aChamber pressure was
controlled using sterile filtered nitrogen. The pressure was
determined by instruments based on capacitance measurements.
[0225] The four reconstituted formulations were tested for
stability (i) prior to lyophilisation, (ii) after immediate
post-lyophilisation reconstitution, and (iii) after reconstitution
following storage at 2-8.degree. C. or 40.degree. C. for four
weeks. Stability was evaluated by % impurities as measured by size
exclusion-High Pressure Liquid Chromatography (SEC-HPLC), Dynamic
Light Scattering (DLS) and visual inspection (assessed after
overnight storage at 2-8.degree. C.). The results are shown in
Tables 8 to 10 below.
TABLE-US-00006 TABLE 6 Aggregation products results from SEC-HPLC
Pre- Post- 2-8.degree. C. for 4 40.degree. C. for 4 lyophilisation
lyophilisation weeks weeks F1 <0.1% <0.1% <0.1% 0.24% F2
<0.1% <0.1% <0.1% 0.18% F3 <0.1% <0.1% <0.1%
0.14% F3b <0.1% <0.1% <0.1% 0.23%
TABLE-US-00007 TABLE 7 Degradation products results from SEC-HPLC
Pre- Post- 2-8.degree. C. for 4 40.degree. C. for 4 lyophilisation
lyophilisation weeks weeks F1 0.15% 0.11% 0.16% 0.16% F2 0.13%
0.14% 0.14% 0.12% F3 0.12% 0.15% 0.13% 0.10% F3b 0.11% 0.12% 0.15%
0.11%
TABLE-US-00008 TABLE 8 Small particles; results for PolyDispensity
Index (PDI) from Dynamic Light Scattering Pre- Post- 2-8.degree. C.
for 4 40.degree. C. for 4 lyophilisation lyophilisation weeks weeks
F1 12.5% 27.5% 23.8% 23.8% F2 12.1% 43.9% 23.5% 23.5% F3 7.4% 19.4%
17.3% 16.1% F3b 7.7% 22.8% 18.3% 17.8%
TABLE-US-00009 TABLE 9 Small particles; results for particles
radius (r.nm) from Dynamic Light Scattering (diluted samples) Pre-
Post- 2-8.degree. C. for 4 40.degree. C. for 4 lyophilisation
lyophilisation weeks weeks F1 4.1 nm 2.9 nm 3.6 nm 4.0 nm F2 4.2 nm
3.0 nm 4.0 nm 4.4 nm F3 5.6 nm 5.5 nm 5.5 nm 5.7 nm F3b 5.7 nm 5.7
nm 5.9 nm 5.7 nm
TABLE-US-00010 TABLE 10 Visual clarity Pre- Post- 2-8.degree. C.
for 4 40.degree. C. for 4 lyophilisation lyophilisation weeks weeks
F1 Clear, Clear, Clear, Clear, no visible no visible no visible no
visible particles particles particles particles F2 Clear, Clear,
Clear, Clear, no visible no visible no visible no visible particles
particles particles particles F3 Clear, Clear, Clear, Clear, no
visible no visible no visible no visible particles particles
particles particles F3b Clear, Clear, Clear, Clear, no visible no
visible no visible no visible particles particles particles
particles
[0226] Overall, all the formulation tested showed good results. F3
showed the lowest aggregation of MOR8S54S before and after
reconstitution, measured by Dynamic tight Scattering (DLS, see
Table 8). Based on these results a stability study was performed
with formulation F3. The reconstituted F3 formulation was prepared
as described above but in a 10 L scale. Table 11 shows the
stability results obtained with F3 as measured by SEC-HPLC.
TABLE-US-00011 TABLE 11 Stability of F3 as measured by SEC-HPLC
Storage Conditions Sum of Sum of Purity/Impurities [%] Purity
aggregates fragments Initial analysis 99.6 0.36 <0.10 5.degree.
C. 3 months 99.5 0.44 <0.10 6 months 99.5 0.45 <0.10
25.degree. C./60% RH 3 months 99.3 0.67 <0.10 6 months 99.1 0.76
<0.10 40.degree. C./75% RH 3 months 98.6 1.4 <0.10 6 months
98.1 1.7 0.10
Sequence CWU 1
1
151124PRTHomo sapiens 1Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu
Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser
Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Gly Trp Ile
Arg Gln Ser Pro Gly Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Ile
Tyr Tyr Arg Ser Lys Trp Tyr Asn Ser Tyr Ala 50 55 60 Val Ser Val
Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln
Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90
95 Tyr Tyr Cys Ala Arg Tyr Asp Trp Val Pro Lys Ile Gly Val Phe Asp
100 105 110 Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
2108PRTHomo sapiens 2Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Phe Ile Ser Ser Ser 20 25 30 Tyr Leu Ser Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ser Ser
Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Leu Tyr Ser Ser Pro 85 90
95 Met Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
312PRTHomo sapiens 3Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Gly
1 5 10 418PRTHomo sapiens 4Arg Ile Tyr Tyr Arg Ser Lys Trp Tyr Asn
Ser Tyr Ala Val Ser Val 1 5 10 15 Lys Ser 512PRTHomo sapiens 5Tyr
Asp Trp Val Pro Lys Ile Gly Val Phe Asp Ser 1 5 10 612PRTHomo
sapiens 6Arg Ala Ser Gln Phe Ile Ser Ser Ser Tyr Leu Ser 1 5 10
77PRTHomo sapiens 7Gly Ser Ser Ser Arg Ala Thr 1 5 89PRTHomo
sapiens 8Gln Gln Leu Tyr Ser Ser Pro Met Thr 1 5 9454PRTHomo
sapiens 9Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro
Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser
Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Gly Trp Ile Arg Gln Ser
Pro Gly Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Ile Tyr Tyr Arg
Ser Lys Trp Tyr Asn Ser Tyr Ala 50 55 60 Val Ser Val Lys Ser Arg
Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu
Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr
Cys Ala Arg Tyr Asp Trp Val Pro Lys Ile Gly Val Phe Asp 100 105 110
Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115
120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly 130 135 140 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro 145 150 155 160 Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr 165 170 175 Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val 180 185 190 Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 195 200 205 Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro 210 215 220 Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 225 230 235
240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp 260 265 270 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly 275 280 285 Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp 305 310 315 320 Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 325 330 335 Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 340 345 350 Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 355 360
365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 385 390 395 400 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu 435 440 445 Ser Leu Ser Pro Gly
Lys 450 10215PRTHomo sapiens 10Asp Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Phe Ile Ser Ser Ser 20 25 30 Tyr Leu Ser Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly
Ser Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70
75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Leu Tyr Ser Ser
Pro 85 90 95 Met Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195
200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 11372DNAHomo sapiens
11caggtgcagc tgcagcagag cggcccaggc ctggtcaagc cctctcagac cctgtcactg
60acctgcgcca tttcaggcga cagcgtgagc agcaacagcg ccgcctgggg ctggatcagg
120cagagccccg gtaggggcct ggaatggctg ggcaggatct actacaggtc
caagtggtac 180aacagctacg ccgtgagcgt gaagagcagg atcaccatca
accctgacac cagcaagaac 240cagttctcac tgcagctcaa cagcgtgacc
cccgaggaca ccgccgtgta ctactgcgcc 300agatacgact gggtgcccaa
gatcggcgtg ttcgacagct ggggccaggg caccctggtg 360accgtgtcaa gc
37212324DNAHomo sapiens 12gatatcgtgc tgacacagag ccccgccacc
ctgagcctga gcccaggcga gagggccacc 60ctgtcctgca gggccagcca gtttatcagc
agcagctacc tgtcctggta tcagcagaag 120cccggccagg cccctagact
gctgatctac ggcagctcct ctcgggccac cggcgtgccc 180gccaggttca
gcggcagcgg ctccggcacc gacttcaccc tgacaatcag cagcctggag
240cccgaggact tcgccgtgta ctactgccag cagctgtaca gctcacccat
gaccttcggc 300cagggcacca aggtggagat caag 32413184PRTHomo sapiens
13Met Arg Arg Gly Pro Arg Ser Leu Arg Gly Arg Asp Ala Pro Ala Pro 1
5 10 15 Thr Pro Cys Val Pro Ala Glu Cys Phe Asp Leu Leu Val Arg His
Cys 20 25 30 Val Ala Cys Gly Leu Leu Arg Thr Pro Arg Pro Lys Pro
Ala Gly Ala 35 40 45 Ser Ser Pro Ala Pro Arg Thr Ala Leu Gln Pro
Gln Glu Ser Val Gly 50 55 60 Ala Gly Ala Gly Glu Ala Ala Leu Pro
Leu Pro Gly Leu Leu Phe Gly 65 70 75 80 Ala Pro Ala Leu Leu Gly Leu
Ala Leu Val Leu Ala Leu Val Leu Val 85 90 95 Gly Leu Val Ser Trp
Arg Arg Arg Gln Arg Arg Leu Arg Gly Ala Ser 100 105 110 Ser Ala Glu
Ala Pro Asp Gly Asp Lys Asp Ala Pro Glu Pro Leu Asp 115 120 125 Lys
Val Ile Ile Leu Ser Pro Gly Ile Ser Asp Ala Thr Ala Pro Ala 130 135
140 Trp Pro Pro Pro Gly Glu Asp Pro Gly Thr Thr Pro Pro Gly His Ser
145 150 155 160 Val Pro Val Pro Ala Thr Glu Leu Gly Ser Thr Glu Leu
Val Thr Thr 165 170 175 Lys Thr Ala Gly Pro Glu Gln Gln 180
141419DNAHomo sapiens 14atggcctggg tgtggaccct gcccttcctg atggccgctg
cccagtcagt gcaggcccag 60gtgcagctgc agcagagcgg cccaggcctg gtcaagccct
ctcagaccct gtcactgacc 120tgcgccattt caggcgacag cgtgagcagc
aacagcgccg cctggggctg gatcaggcag 180agccccggta ggggcctgga
atggctgggc aggatctact acaggtccaa gtggtacaac 240agctacgccg
tgagcgtgaa gagcaggatc accatcaacc ctgacaccag caagaaccag
300ttctcactgc agctcaacag cgtgaccccc gaggacaccg ccgtgtacta
ctgcgccaga 360tacgactggg tgcccaagat cggcgtgttc gacagctggg
gccagggcac cctggtgacc 420gtgtcaagcg ccagcaccaa gggccccagc
gtgttccccc tggcccccag cagcaagagc 480accagcggcg gcacagccgc
cctgggctgc ctggtgaagg actacttccc cgagcccgtg 540accgtgtcct
ggaacagcgg agccctgacc tccggcgtgc acaccttccc cgccgtgctg
600cagagcagcg gcctgtacag cctgtccagc gtggtgacag tgcccagcag
cagcctgggc 660acccagacct acatctgcaa cgtgaaccac aagcccagca
acaccaaggt ggacaagaga 720gtggagccca agagctgcga caagacccac
acctgccccc cctgcccagc cccagagctg 780ctgggcggac cctccgtgtt
cctgttcccc cccaagccca aggacaccct gatgatcagc 840aggacccccg
aggtgacctg cgtggtggtg gacgtgagcc acgaggaccc agaggtgaag
900ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc
cagagaggag 960cagtacaaca gcacctacag ggtggtgtcc gtgctgaccg
tgctgcacca ggactggctg 1020aacggcaagg aatacaagtg caaggtctcc
aacaaggccc tgccagcccc catcgaaaag 1080accatcagca aggccaaggg
ccagccacgg gagccccagg tgtacaccct gcccccctcc 1140cgggaggaga
tgaccaagaa ccaggtgtcc ctgacctgtc tggtgaaggg cttctacccc
1200agcgacatcg ccgtggagtg ggagagcaac ggccagcccg agaacaacta
caagaccacc 1260cccccagtgc tggacagcga cggcagcttc ttcctgtaca
gcaagctgac cgtggacaag 1320tccaggtggc agcagggcaa cgtgttcagc
tgcagcgtga tgcacgaggc cctgcacaac 1380cactacaccc agaagagcct
gagcctgtcc cccggcaag 141915705DNAHomo sapiens 15atgagcgtgc
tgacccaggt gctggctctg ctgctgctgt ggctgaccgg caccagatgc 60gatatcgtgc
tgacacagag ccccgccacc ctgagcctga gcccaggcga gagggccacc
120ctgtcctgca gggccagcca gtttatcagc agcagctacc tgtcctggta
tcagcagaag 180cccggccagg cccctagact gctgatctac ggcagctcct
ctcgggccac cggcgtgccc 240gccaggttca gcggcagcgg ctccggcacc
gacttcaccc tgacaatcag cagcctggag 300cccgaggact tcgccgtgta
ctactgccag cagctgtaca gctcacccat gaccttcggc 360cagggcacca
aggtggagat caagcgtacg gtggccgctc ccagcgtgtt catcttcccc
420cccagcgacg agcagctgaa gagcggcacc gccagcgtgg tgtgcctgct
gaacaacttc 480tacccccggg aggccaaggt gcagtggaag gtggacaacg
ccctgcagag cggcaacagc 540caggagagcg tcaccgagca ggacagcaag
gactccacct acagcctgag cagcaccctg 600accctgagca aggccgacta
cgagaagcat aaggtgtacg cctgcgaggt gacccaccag 660ggcctgtcca
gccccgtgac caagagcttc aacaggggcg agtgc 705
* * * * *
References