U.S. patent application number 13/873583 was filed with the patent office on 2014-01-02 for antibody formulation.
This patent application is currently assigned to NOVARTIS AG. The applicant listed for this patent is Claudia Mueller, Matthias Willmann. Invention is credited to Claudia Mueller, Matthias Willmann.
Application Number | 20140004131 13/873583 |
Document ID | / |
Family ID | 48626495 |
Filed Date | 2014-01-02 |
United States Patent
Application |
20140004131 |
Kind Code |
A1 |
Mueller; Claudia ; et
al. |
January 2, 2014 |
ANTIBODY FORMULATION
Abstract
Anti-CD40 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: |
Mueller; Claudia; (Basel,
CH) ; Willmann; Matthias; (Basel, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mueller; Claudia
Willmann; Matthias |
Basel
Basel |
|
CH
CH |
|
|
Assignee: |
NOVARTIS AG
Basel
CH
|
Family ID: |
48626495 |
Appl. No.: |
13/873583 |
Filed: |
April 30, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61642644 |
May 4, 2012 |
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Current U.S.
Class: |
424/173.1 |
Current CPC
Class: |
A61P 19/02 20180101;
A61K 39/39591 20130101; A61K 39/3955 20130101; C07K 2317/94
20130101; A61K 47/22 20130101; A61K 47/183 20130101; A61P 17/00
20180101; C07K 2317/76 20130101; A61K 9/19 20130101; A61P 37/00
20180101; A61P 29/00 20180101; A61P 37/06 20180101; C07K 16/2878
20130101; A61K 47/26 20130101; A61K 2039/505 20130101; A61K
39/39558 20130101; A61P 37/02 20180101; A61K 47/20 20130101; C07K
2317/21 20130101 |
Class at
Publication: |
424/173.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A lyophilised formulation prepared by lyophilising an aqueous
formulation having a pH of 5.0-7.0 and comprising (i) an anti-CD40
antibody wherein the antibody has a concentration of 20-150 mg/ml,
and wherein said anti-CD40 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) a stabiliser,
(iii) a buffering agent, and (iv) a surfactant.
2. The lyophilised formulation 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-CD40 antibody wherein the antibody has a
concentration of 20-150 mg/ml, and wherein said anti-CD40 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 stabiliser, (iii) histidine
as a buffering agent, and (iv) polysorbate 20 as a surfactant.
3. The lyophilised formulation of claim 2, wherein said formulation
is prepared from an aqueous formulation having a pH of 5.0-7.0 and
comprising (i) an anti-CD40 antibody wherein the antibody has a
concentration of 20-150 mg/ml, and wherein said anti-CD40 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 stabiliser, (iii)
1-60 mM histidine as a buffering agent, and (iv) up to 0.2%
polysorbate 20 as a surfactant.
4. The lyophilised formulation as claimed in claim 1, wherein said
formulation is prepared from an aqueous formulation having a pH of
6.0 and comprising (i) an anti-CD40 antibody wherein the antibody
has a concentration of 50 mg/ml, and wherein said anti-CD40
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 stabiliser, (iii) 10 mM
histidine as a buffering agent, and (iv) 0.02% polysorbate 20 as a
surfactant.
5. The lyophilised formulation as claimed in claim 1, wherein said
formulation is prepared from an aqueous formulation having a pH of
6.0 and comprising (i) an anti-CD40 antibody wherein the antibody
has a concentration of 50 mg/ml, and wherein said anti-CD40
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 stabiliser, (iii) 10 mM
histidine as a buffering agent, (iv) 0.02% polysorbate 20 as a
surfactant, and (v) 17 mM arginine.
6. The lyophilised formulation as claimed in claim 1, wherein said
formulation is prepared from an aqueous formulation having a pH of
6.0 and comprising (i) an anti-CD40 antibody wherein the antibody
has a concentration of 150 mg/ml, and wherein said anti-CD40
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) 270 mM sucrose as a stabiliser, (iii) 30 mM
histidine as a buffering agent, and (iv) 0.06% polysorbate 20 as a
surfactant.
7. An aqueous composition obtained by reconstituting a lyophilised
formulation of claim 1, wherein the reconstitution factor is
between 1:0.5 to 1:6.
8. The aqueous composition of claim 7, wherein the reconstitution
factor is 1:3.
9. An aqueous composition having a pH of 5.0 to 7.0 comprising (i)
an anti-CD40 antibody wherein the antibody has a concentration of
at least 50 mg/ml, and wherein said anti-CD40 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) a stabiliser, (iii) a buffering agent, and (iv) a
surfactant.
10. The aqueous composition of claim 9 comprising (i) an anti-CD40
antibody wherein the antibody has a concentration of at least 50
mg/ml, and wherein said anti-CD40 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 stabiliser, (iii) histidine as a
buffering agent, and (iv) polysorbate 20 as a surfactant.
11. The aqueous composition of claim 10 comprising (i) an anti-CD40
antibody wherein the antibody has a concentration of at least 50
mg/ml, and wherein said anti-CD40 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)
200-300 mM sucrose as a stabiliser, (iii) 25-35 mM histidine as a
buffering agent, and (iv) up to 0.2% polysorbate 20 as a
surfactant
12. The aqueous composition of claim 9, wherein the composition has
a pH of 6.0 and comprises (i) an anti-CD40 antibody wherein the
antibody has a concentration of at least 50 mg/ml, and wherein said
anti-CD40 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) 270 mM sucrose as a stabiliser,
(iii) 30 mM histidine as a buffering agent, and (iv) 0.06%
polysorbate 20 as a surfactant.
13. The aqueous composition of claim 9, wherein the composition has
a pH of 6.0 and comprises (i) an anti-CD40 antibody wherein the
antibody has a concentration of at least 50 mg/ml, and wherein said
anti-CD40 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) 270 mM sucrose as a stabiliser,
(iii) 30 mM histidine as a buffering agent, (iv) 0.06% polysorbate
20 as a surfactant, and (v) 51 mM arginine.
14. The aqueous composition of claim 9, wherein the anti-CD40
antibody has a concentration of 150 mg/ml.
15. The lyophilised formulation of claim 1, wherein the anti-CD40
antibody comprises a V.sub.H domain having the amino acid sequence
of SEQ ID NO: 1 and a V.sub.L domain having the amino acid sequence
of SEQ ID NO: 2.
16. The aqueous composition of claim 9, wherein the anti-CD40
antibody comprises a V.sub.H domain having the amino acid sequence
of SEQ ID NO: 1 and a V.sub.L domain having the amino acid sequence
of SEQ ID NO: 2.
17. The lyophilised formulation of claim 1, wherein the anti-CD40
antibody comprises a heavy chain region and a light chain region
having the amino acid sequence selected from the group consisting
of: a) SEQ ID NO: 9 and SEQ ID NO: 10, respectively; b) SEQ ID NO:
13 and SEQ ID NO: 14, respectively; and c) SEQ ID NO: 17 and SEQ ID
NO: 18, respectively.
18. The aqueous composition of claim 9, wherein the anti-CD40
antibody comprises a heavy chain region and a light chain region
having the amino acid sequence selected from the group consisting
of: a) SEQ ID NO: 9 and SEQ ID NO: 10, respectively; b) SEQ ID NO:
13 and SEQ ID NO: 14, respectively; and c) SEQ ID NO: 17 and SEQ ID
NO: 18, respectively.
19. A delivery device including the aqueous composition of claim
9.
20. A pre-filled syringe including the aqueous composition of claim
9.
21. A method for delivering an anti-CD40 antibody to a mammal,
comprising a step of administering to the mammal an aqueous
composition as claimed claim 9.
22. A method of treating a disease or disorder that is mediated by
CD40, comprising a step of administering the aqueous composition of
claim 9 to a subject.
23. The method of claim 22 wherein the disease or disorder is an
autoimmune disease.
24. The method of claim 23 wherein the autoimmune disease is
rheumatoid arthritis, systemic lupus erythematosus, or Pemphigus
vulgaris.
25. The formulation of claim 1 further comprising and amino
acid.
26. The formulation of claim 25, where in the amino acid is
arginine, methionine or glycine.
27. The aqueous composition of claim 9 further comprising an amino
acid.
28. The aqueous composition of claim 27, wherein the amino acid is
arginine, methionine or glycine.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pharmaceutical
formulation of an antibody against CD40, a process for the
preparation thereof and uses of the formulation.
BACKGROUND
[0002] Despite the availability of several immunosuppressive
treatments for autoimmune diseases, there remains a large unmet
need for more efficacious and safer drugs in a large fraction of
the patient population. For example, despite the reported efficacy
of B cell depleting/inhibiting therapies like Rituximab and
Belimumab in rheumatoid arthritis, systemic lupus erythematosus,
Sjogren's syndrome, and multiple sclerosis, these therapies are
only effective in a portion of diseased individuals, and with
Rituximab, with an accompanying risk of progressive multifocal
leukoencephalopathy. Further, multiple other leukocyte cell types
are often involved in the pathology of these autoimmune diseases
such as macrophages, dendritic cells and T cells, therefore
therapeutic intervention targeting additional cell types or key
immunological pathways that would inhibit their function could
provide benefit. Given the multiple immunologically relevant roles
of CD40-CD154 in the activation and function or these cell types,
it is likely that an anti-CD40 antibody would confer therapeutic
benefit to patients suffering autoimmune diseases outlined above
beyond that currently provided by current therapies. Further, the
central role for CD40-CD154 interactions in intestinal inflammatory
disorders such as Crohn's disease and ulcerative colitis, and
mechanistic links of the CD40 pathway to pathology in more rare
disorders such as autoimmune vasculitis, pemphigus vulgaris, and
idiopathic thrombocytopenic purpura (ITP) also highlights the
potential of anti-CD40 antibodies in these indications.
[0003] The currently available immunosuppressants used after solid
organ transplantation provide excellent short-term efficacy. Acute
rejections within the de novo period are observed in 5%-20% of the
recipients (depending on organ, patient population, and regimen)
and the proportion of grafts lost to acute rejection within the de
novo period is below 5% for any setting. Currently the key unmet
need is the tolerability of immunosuppression with patient and
graft survival in the long term. After renal transplant, 33%
patients die and/or lose their graft within 5 years; the average
age of death of transplant recipient is 58 years. Calcineurin
inhibitors (CNI) remain the mainstay of immunosuppressive therapy
for the vast majority of transplant patients. While nephrotoxicity
and cardiovascular morbidity associated with CNIs is one of the
drivers of chronic allograft nephropathy as well as patient death
with a functioning graft, alternative primary immunosuppression
have not been able to replace CNIs. Overall, there is still room
for improvement in long-term transplant immunosuppression. B-cell
mediated immunological damage of transplanted kidneys may
contribute to poor long-term outcomes and the need for new agents
to target B-cell rejection is increasingly recognised by the
medical community.
[0004] Antibodies against CD40 are known in the art. Chir12.12 is a
fully humanised, non-agonist anti-CD40 antibody (IgG1, kappa) that
blocks CD154 (also known as CD40 ligand; CD40L)-mediated leukocyte
activation and can mediate antibody-dependent cellular cytotoxicity
(ADCC) of human leukocytes and B cell lymphomas in vitro (see WO
2006/073443). WO 2005/044306 describes anti-CD40 antagonist
antibodies, including Chir12.12 for use in particular in the
treatment of autoimmune and inflammatory disorders. Further
Chir12.12 is effective in delaying kidney allograft rejection when
dosed as a monotherapy in Macaca fascicularis (Cynomolgus monkeys)
[Li et al. (2008) Transplantation; 86 (1):10-15]. However,
Chir12.12 can also mediate depletion of peripheral B cells in non
human primates (NHPs).
[0005] Anti-CD40 mAbs with silenced ADCC activity are predicted to
have an improved safety profile relative to the parental anti-CD40
antibodies, and in particular may be more suitable for
non-oncologic indications, such as autoimmune diseases and use in a
transplant setting. The applicant has developed three silent
anti-CD40 antibodies based on the Chir12.12 antibody. These
antibodies, hereinafter designated mAb1, mAb2 and mAb3 are
characterised by certain amino acid mutations in the Fc region
which silence ADCC activity.
[0006] mAb1 comprises an N297A mutation in the antibody heavy chain
amino acid sequence. The antibody comprises the 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, respectively.
mAb1 comprises 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. mAb1 comprises the full length heavy chain amino acid
sequence of SEQ ID NO: 9 and the full length light chain amino acid
sequence of SEQ ID NO: 10.
[0007] mAb2 comprises a D265A mutation in the antibody heavy chain
amino acid sequence. The antibody comprises the 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, respectively.
mAb2 comprises 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. mAb2 comprises the full length heavy chain amino acid
sequence of SEQ ID NO: 13 and the full length light chain amino
acid sequence of SEQ ID NO: 14.
[0008] mAb3 comprises a L234A, L235A mutation (LALA) in the
antibody heavy chain amino acid sequence. The antibody comprises
the 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, respectively. mAb3 comprises 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. mAb3 comprises the full length
heavy chain amino acid sequence of SEQ ID NO: 17 and the full
length light chain amino acid sequence of SEQ ID NO: 18.
[0009] Formulations with high concentration of antibody may have
short shelf lives and the formulated antibodies may lose biological
activity resulting from chemical and physical instabilities during
the storage. Among those, aggregation, deamidation 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
minimised or prevented.
[0010] It is an object of the invention to provide further and
improved formulations of anti-CD40 antibodies, and in particular
formulations with high concentration of anti-CD40 antibodies and
low levels of antibody aggregation.
DISCLOSURE OF THE INVENTION
[0011] 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-CD40 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 minimise the time taken to deliver a fixed dose to the
patient. The aqueous compositions of the invention with high
concentration of anti-CD40 antibodies are particularly suitable for
subcutaneous administration.
[0012] Thus the invention provides an aqueous pharmaceutical
composition, suitable for parenteral administration in a subject,
e.g., for subcutaneous administration, comprising an anti-CD40
antibody.
[0013] The following specific embodiments of the invention are
described as numbered hereafter: [0014] 1. An aqueous
pharmaceutical composition, suitable for subcutaneous
administration in a subject, comprising an anti-CD40 antibody in
which the antibody has a concentration of at least 50 mg/ml, and
wherein said anti-CD40 antibody includes: (i) one or more heavy
chain CDRs selected from the group consisting of SEQ ID NOs: 3, 4
and 5; and/or (i) one or more light chain CDRs selected from the
group consisting of SEQ ID NOs: 6, 7 and 8. [0015] 2. The aqueous
pharmaceutical composition, suitable for subcutaneous
administration in a subject, according to Embodiment 1, wherein
said anti-CD40 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. [0016] 3. The aqueous
pharmaceutical composition of Embodiment 1 or 2, wherein the
anti-CD40 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. [0017]
4. The aqueous pharmaceutical composition of Embodiment 1, 2 or 3,
wherein the anti-CD40 antibody comprises a heavy chain region of
SEQ ID NO: 9 and a light chain region of SEQ ID NO: 10, or a heavy
chain region of SEQ ID NO: 13 and a light chain region of SEQ ID
NO: 14, or a heavy chain region of SEQ ID NO: 17 and a light chain
region of SEQ ID NO: 18. [0018] 5. The aqueous pharmaceutical
composition of any one of Embodiments 1 to 4, wherein less than 5%
of the anti-CD40 antibody is aggregated or degraded. [0019] 6. The
aqueous pharmaceutical composition of any one of Embodiments 1 to
5, comprising one or more of the following components selected
among the group consisting of: a stabiliser, a buffering agent; and
a surfactant. [0020] 7. The aqueous pharmaceutical composition of
Embodiment 6, wherein the stabiliser is a sugar. [0021] 8. The
aqueous pharmaceutical composition of Embodiment 6 or 7,
comprising: a sugar, a buffering agent, and a surfactant. [0022] 9.
The aqueous pharmaceutical composition of Embodiment 6 or 7,
further comprising a free amino acid. [0023] 10. The aqueous
pharmaceutical composition of Embodiment 7 to 9, comprising sucrose
as a sugar. [0024] 11. The aqueous pharmaceutical composition of
Embodiment 10, comprising 200-300 mM sucrose. [0025] 12. The
aqueous pharmaceutical composition of Embodiments 6-11, comprising
a histidine buffer as the buffering agent. [0026] 13. The aqueous
pharmaceutical composition of Embodiment 12, comprising 25-35 mM
histidine buffer. [0027] 14. The aqueous pharmaceutical composition
of Embodiments 6 to 13, comprising polysorbate 20 as a surfactant.
[0028] 15. The aqueous pharmaceutical composition of Embodiment 14,
comprising 0.01 to 0.2% polysorbate 20. [0029] 16. The aqueous
pharmaceutical composition of Embodiment 9, further comprising
arginine and/or methionine as free amino acid. [0030] 17. The
aqueous pharmaceutical composition of Embodiment 16, comprising
40-80 mM arginine. [0031] 18. The aqueous pharmaceutical
composition of any preceding Embodiment, comprising sucrose, a
histidine buffer, polysorbate 20 and arginine. [0032] 19. The
aqueous pharmaceutical composition of any preceding Embodiment,
comprising sucrose, a histidine buffer, polysorbate 20 and
methionine. [0033] 20. The aqueous pharmaceutical composition of
any preceding Embodiment, comprising sucrose, a histidine buffer,
polysorbate 20, arginine and methionine. [0034] 21. A lyophilisate
suitable for preparing the aqueous pharmaceutical composition of
any preceding Embodiments. [0035] 22. A lyophilisate according to
Embodiment 21, comprising sucrose, a histidine buffer, and
polysorbate 20. [0036] 23. A method for preparing a lyophilisate,
comprising the steps of: (i) preparing an aqueous solution
comprising an anti-CD40 antibody, a sugar, a buffering agent, a
surfactant and optionally a free amino acid; and (ii) lyophilising
the aqueous solution. [0037] 24. A delivery device including the
aqueous pharmaceutical composition of any one of Embodiments 1-20.
[0038] 25. A pre-filled syringe including the aqueous
pharmaceutical composition of any one of Embodiments 1-20. [0039]
26. A method for delivering an anti-CD40 monoclonal antibody to a
mammal, comprising a step of administering to the patient a
pharmaceutical composition of any one of Embodiments 1-20. [0040]
27. The composition of any one of Embodiments 1-20, for use in
treating a disease or disorder that is mediated by CD40. [0041] 28.
The composition of Embodiment 27, for the treatment of autoimmune
diseases. [0042] 29. The composition of Embodiment 28, for the
treatment of rheumatoid arthritis, systemic lupus erythematosus, or
Pemphigus vulgaris. [0043] 30. The aqueous pharmaceutical
composition of any one of Embodiments 1-20 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.
[0044] The invention also provides an aqueous pharmaceutical
composition comprising: an anti-CD40 monoclonal antibody as
described above, for example mAb1, mAb2 or mAb3, especially mAb1; a
stabiliser; a buffering agent; and a surfactant. The composition
preferably also includes a free amino acid.
[0045] The invention also provides a lyophilisate comprising: an
anti-CD40 monoclonal antibody as described above, for example mAb1,
mAb2 or mAb3, especially mAb1; a sugar; a buffering agent; and a
surfactant. The lyophilisate preferably also includes a free amino
acid.
[0046] The invention also provides a lyophilisate comprising an
anti-CD40 monoclonal antibody as described above, for example mAb1,
mAb2 or mAb3, especially mAb1; 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 in an aqueous solution.
[0047] The invention also provides an aqueous pharmaceutical
composition comprising high concentration of an anti-CD40
monoclonal antibody as described above, for example mAb1, mAb2 or
mAb3, especially mAb1; wherein less than 5%, 4%, 2% or 1% of the
anti-CD40 antibody is aggregated or degraded.
[0048] The invention also provides a process for preparing a
lyophilisate, comprising steps of: (i) preparing an aqueous
solution comprising an anti-CD40 monoclonal antibody, a sugar, a
buffering agent, a surfactant, and optionally a free amino acid;
and (ii) lyophilising the aqueous solution.
[0049] 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 comprises an anti-CD40 monoclonal antibody, a sugar, a
buffering agent, a surfactant, and optionally a free amino
acid.
[0050] More specifically the invention provides a lyophilised
formulation prepared by lyophilising an aqueous formulation having
a pH of 5.0-7.0 and comprising [0051] (i) an anti-CD40 antibody
wherein the antibody has a concentration of 20-150 mg/ml, and
wherein said anti-CD40 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, [0052] (ii) a stabiliser,
[0053] (iii) a buffering agent, [0054] (iv) a surfactant, and
optionally [0055] (v) an amino acid. [0056] In one embodiment said
formulation is prepared from an aqueous formulation having a pH of
5.0-7.0 and comprising [0057] (i) an anti-CD40 antibody wherein the
antibody has a concentration of 20-150 mg/ml, and wherein said
anti-CD40 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) sucrose or trehalose as a
stabiliser, [0059] (iii) histidine as a buffering agent, [0060]
(iv) polysorbate 20 as a surfactant, and optionally [0061] (v) an
amino acid selected from arginine, methionine and glycine. [0062]
In one embodiment said lyophilised formulation is prepared from an
aqueous formulation having a pH of 5.0-7.0 and comprising [0063]
(i) an anti-CD40 antibody wherein the antibody has a concentration
of 20-150 mg/ml, and wherein said anti-CD40 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) 3-300 mM sucrose or trehalose as a stabiliser, [0065]
(iii) 1-60 mM histidine as a buffering agent, [0066] (iv) up to
0.2% polysorbate 20 as a surfactant, and optionally [0067] (v) 2-80
mM arginine, methionine or glycine. [0068] In one embodiment said
lyophilised formulation is prepared from an aqueous formulation
having a pH of 6.0 and comprising [0069] (i) an anti-CD40 antibody
wherein the antibody has a concentration of 50 mg/ml, and wherein
said anti-CD40 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, [0070] (ii) 90 mM sucrose as a
stabiliser, [0071] (iii) 10 mM histidine as a buffering agent,
[0072] (iv) 0.02% polysorbate 20 as a surfactant. [0073] In one
embodiment said lyophilised formulation is prepared from an aqueous
formulation having a pH of 6.0 and comprising [0074] (i) an
anti-CD40 antibody wherein the antibody has a concentration of 50
mg/ml, and wherein said anti-CD40 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, [0075]
(ii) 90 mM sucrose as a stabiliser, [0076] (iii) 10 mM histidine as
a buffering agent, [0077] (iv) 0.02% polysorbate 20 as a
surfactant, and [0078] (v) 17 mM arginine. [0079] In one embodiment
said lyophilised formulation is prepared from an aqueous
formulation having a pH of 6.0 and comprising [0080] (i) an
anti-CD40 antibody wherein the antibody has a concentration of 150
mg/ml, and wherein said anti-CD40 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) 270 mM sucrose as a stabiliser, [0082] (iii) 30 mM histidine
as a buffering agent, and [0083] (iv) 0.06% polysorbate 20 as a
surfactant. [0084] The invention also provides an aqueous
pharmaceutical composition obtained by reconstituting a lyophilised
formulation as described above, wherein the reconstitution factor
is between 1:0.5 to 1:6. [0085] In one embodiment the
reconstitution factor is 1:3. [0086] The invention also provides an
aqueous pharmaceutical composition having a pH of 5.0 to 7.0
comprising [0087] (i) an anti-CD40 antibody wherein the antibody
has a concentration of at least 50 mg/ml, and wherein said
anti-CD40 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, [0088] (ii) a stabiliser, [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-CD40 antibody wherein the antibody has a
concentration of at least 50 mg/ml, and wherein said anti-CD40
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 stabiliser,
[0095] (iii) histidine as a buffering agent, [0096] (iv)
polysorbate 20 as a surfactant, and optionally [0097] (v) an amino
acid selected from arginine, methionine or glycine. [0098] In one
embodiment the aqueous pharmaceutical composition having a pH of
5.0 to 7.0 comprises [0099] (i) an anti-CD40 antibody wherein the
antibody has a concentration of at least 50 mg/ml, and wherein said
anti-CD40 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
stabiliser, [0101] (iii) 25-35 mM histidine as a buffering agent,
[0102] (iv) up to 0.2% polysorbate 20 as a surfactant, and
optionally [0103] (v) 10-80 mM arginine, methionine or glycine.
[0104] In one embodiment the aqueous pharmaceutical composition has
a pH of 6.0 and comprises [0105] (i) an anti-CD40 antibody wherein
the antibody has a concentration of at least 50 mg/ml, and wherein
said anti-CD40 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
stabiliser, [0107] (iii) 30 mM histidine as a buffering agent, and
[0108] (iv) 0.06% polysorbate 20 as a surfactant. [0109] In one
embodiment the aqueous pharmaceutical composition has a pH of 6.0
and comprises [0110] (i) an anti-CD40 antibody wherein the antibody
has a concentration of at least 50 mg/ml, and wherein said
anti-CD40 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, [0111] (ii) 270 mM sucrose as a
stabiliser, [0112] (iii) 30 mM histidine as a buffering agent,
[0113] (iv) 0.06% polysorbate 20 as a surfactant, and [0114] (v) 51
mM arginine. [0115] In one embodiment the aqueous pharmaceutical
composition of the invention has an anti-CD40 antibody
concentration of 150 mg/ml. [0116] In one embodiment the
lyophilised formulation or the aqueous pharmaceutical composition
of the invention comprises a VH domain having the amino acid
sequence of SEQ ID NO: 1 and a VL domain having the amino acid
sequence of SEQ ID NO: 2. [0117] In one embodiment the lyophilised
formulation or the aqueous pharmaceutical composition of the
invention comprises an anti-CD40 antibody comprising a heavy chain
region of SEQ ID NO: 9 and a light chain region of SEQ ID NO: 10.
[0118] In one embodiment the lyophilised formulation or the aqueous
pharmaceutical composition of the invention comprises the anti-CD40
antibody Chir12.12 having an N297A mutation. [0119] In one
embodiment the lyophilised formulation or the aqueous
pharmaceutical composition of the invention comprises an anti-CD40
antibody comprising a heavy chain region of SEQ ID NO: 13 and a
light chain region of SEQ ID NO: 14. [0120] In one embodiment the
lyophilised formulation or the aqueous pharmaceutical composition
of the invention comprises the anti-CD40 antibody Chir12.12 having
a D265A mutation. [0121] In one embodiment the lyophilised
formulation or the aqueous pharmaceutical composition of the
invention comprises an anti-CD40 antibody comprising a heavy chain
region of SEQ ID NO: 17 and a light chain region of SEQ ID NO: 18.
[0122] In one embodiment the lyophilised formulation or the aqueous
pharmaceutical composition of the invention comprises the anti-CD40
antibody Chir12.12 having an L234A L235A mutation. [0123] The
invention also comprises a delivery device including the aqueous
pharmaceutical composition of the invention. [0124] The invention
also comprises a pre-filled syringe including the aqueous
pharmaceutical composition of the invention. [0125] The invention
also comprises a method for delivering an anti-CD40 antibody to a
mammal, comprising a step of administering to the patient an
aqueous pharmaceutical composition of the invention. [0126] The
invention also comprises a lyophilised formulation or an aqueous
pharmaceutical composition according to the invention for use in
treating a disease or disorder that is mediated by CD40. [0127] The
invention also comprises a lyophilised formulation or aqueous
pharmaceutical composition according to the invention for use in
the treatment of autoimmune diseases. [0128] The invention also
provides a lyophilised formulation or aqueous pharmaceutical
composition according to the invention Multiple Sclerosis, Systemic
Lupus Erythematosus, Sjogren's syndrome, Rheumatoid Arthritis,
transplant rejection; graft-versus-host disease, Pemphigus
vulgaris. Aqueous Pharmaceutical Compositions with High
Concentration of Anti-CD40 Antibodies
[0129] The invention relies, at least partly, in the formulation
properties of antibodies such as mAb1, which retain remarkable
stability and bioactive properties when formulated in a high
concentration either as a liquid (aqueous) or lyophilisate
composition.
[0130] 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 reconstituted
shortly before use. As used herein, the term "aqueous
pharmaceutical composition" refers to the liquid formulation or
reconstituted lyophilised 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.
[0131] 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.
[0132] The use of antibodies as the active ingredient of
pharmaceuticals is now widespread, including the 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.
[0133] 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. [0134] 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-CD40 antibody being at least
about 50 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250 mg/ml, or 300
mg/ml.
[0135] In one aspect, the invention relates to an aqueous
pharmaceutical composition with high concentration of anti-CD40
antibodies.
[0136] 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. 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 factors, including various cells of the immune system
(e.g., effector cells) and the first component (Clq) of the
classical complement system.
[0137] 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 CD40). 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).sub.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).
[0138] 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.
[0139] 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 CD40 is substantially free of antibodies
that specifically bind antigens other than CD40. An isolated
antibody that specifically binds CD40 may, however, have
cross-reactivity to other antigens, such as CD40 molecules from
other species. Moreover, an isolated antibody may be substantially
free of other cellular material and/or chemicals.
[0140] 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.
[0141] 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 296, 57-86).
[0142] The structures and locations of immunoglobulin variable
domains, e.g., CDRs, may be defined using well known numbering
schemes, e.g., the Kabat numbering scheme, the Chothia numbering
scheme, a combination of Kabat and Chothia (AbM), etc. (see, e.g.,
Sequences of Proteins of Immunological Interest, U.S. Department of
Health and Human Services (1991), eds. Kabat et al.; Al Lazikani et
al. (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.
[0143] 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 been grafted onto human framework
sequences.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] The phrases "an antibody recognising an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody which binds specifically to an
antigen".
[0148] As used herein, an antibody that "specifically binds to CD40
polypeptide" or an "anti-CD40 antibody" refers to an antibody that
binds to human CD40 polypeptide of SEQ ID NO: 21 with a K.sub.D of
100 nM or less, 10 nM or less, 1 nM or less. An antibody that
"cross-reacts with an antigen other than CD40" refers to an
antibody that binds that antigen with a K.sub.D of
0.5.times.10.sup.-8 M 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.-9 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.
[0149] In one embodiment, a high concentration of an anti-CD40
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 is at
least 250 mg/ml. In one embodiment, a high concentration is at
least 300 mg/ml.
[0150] In one embodiment, the aqueous pharmaceutical composition of
the invention comprises between 50 mg/ml and 300 mg/ml of an
anti-CD40 antibody, for example, mAb1.
[0151] In one embodiment, the aqueous pharmaceutical composition of
the invention comprises between 75 mg/ml and 250 mg/ml of an
anti-CD40 antibody, for example, mAb1.
[0152] In one embodiment, the aqueous pharmaceutical composition of
the invention comprises between 100 mg/ml and 250 mg/ml of an
anti-CD40 antibody, for example, mAb1.
[0153] In one embodiment, the aqueous pharmaceutical composition of
the invention comprises between 100 mg/ml and 200 mg/ml of an
anti-CD40 antibody, for example, mAb1.
[0154] In one embodiment, the aqueous pharmaceutical composition of
the invention comprises 150 mg/ml of an anti-CD40 antibody, for
example, mAb1.
[0155] 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-CD40 antibody, for example, mAb1.
[0156] Furthermore, the aqueous pharmaceutical compositions are
stable such that, even after storage for 4 weeks at 2-8.degree. C.,
less than 5%, 4%.sub., 3%.sub., 2%.sub., 1%, 0.05% or 0.01% of the
total anti-CD40 antibody is aggregated as measured by SEC-HPLC.
[0157] The aqueous pharmaceutical compositions may include, in
addition to the anti-CD40 antibody, further components such as one
or more of the following: (i) a stabiliser; (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-CD40 antibody.
[0158] Suitable stabilisers for use with the invention can act,
e.g., as viscosity enhancing agents, bulking agents, solubilising
agents, and/or the like. The stabiliser 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,
D-mannose, sorbose and the like; disaccharides, 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 stabiliser they include
salts such as NaCl or amino acid components such as
arginine-HCl.
[0159] 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.
[0160] 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 5.5 and 7.5,
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 6.0.
[0161] 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.
[0162] 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, polyoxyethylene sorbitan monooleate,
polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan
monopalmitate, polyoxyethylene 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),
polyoxyethylene polyoxypropylene alkyl 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.
polyoxyethylene 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.16 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, glycerophospholipid, 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 acid
esters e.g. polysorbate 20, 40, 60 or 80. Polysorbate 80 (Tween 80)
is particularly useful.
[0163] Suitable free amino acids for use with the invention
include, but are not limited to, arginine, lysine, histidine,
methionine, ornithine, isoleucine, leucine, alanine, glycine,
glutamic acid or aspartic acid. The inclusion of a basic amino acid
is preferred ie. 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.
[0164] When present, components (i) to (iv) will be at a
concentration sufficient to maintain the anti-CD40 antibody in a
form which is active and soluble after either
(i) lyophilisation and storage and reconstitution (for
lyophilisates), or (ii) conditioning in dosing units and storage
(for liquid formulations).
[0165] 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. 50-380 mM, 100-350 mM, 200-300 mM. A concentration of 270 mM
sucrose is useful.
[0166] 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 60 mM e.g. 10-50 mM, 20-40 mM, 25-35 mM. A
concentration of 30 mM histidine buffer is useful.
[0167] 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 20 is useful. In some embodiments polysorbate 80 may be
used.
[0168] A free amino acid 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 2 and 100 mM e.g. 10-80 mM, 20-70 mM, 30-60 mM, 40-60 mM. A
concentration of 51 mM arginine (e.g. arginine-HCl) or 60 mM
methionine or glycine (e.g. glycine-HCl) is useful.
[0169] A formulation containing histidine buffer, sucrose and
polysorbate 20 has been shown to be suitable for lyophilisation of
antibody mAb1 at a concentration of at least 150 mg/ml after
reconstitution.
[0170] In one embodiment the aqueous pharmaceutical composition
consists of 150 mg/ml mAb1, 30 mM histidine, 270 mM sucrose and
0.06% polysorbate 20.
[0171] In one embodiment the aqueous pharmaceutical composition
consists of 150 mg/ml mAb1, 30 mM histidine, 270 mM sucrose, 0.06%
polysorbate 20 and 51 mM arginine-HCl.
[0172] In one embodiment the aqueous pharmaceutical composition
consists of 150 mg/ml mAb1, 30 mM histidine, 270 mM sucrose, 0.06%
polysorbate 20 and 60 mM glycine-HCl.
[0173] In one embodiment the aqueous pharmaceutical composition
consists of 200 mg/ml mAb1, 30 mM histidine, 270 mM sucrose, and
0.06% polysorbate 20.
[0174] In one embodiment the aqueous pharmaceutical composition
consists of 200 mg/ml mAb1, 30 mM histidine, 270 mM sucrose, 0.06%
polysorbate 20 and 51 mM arginine-HCl.
[0175] In one embodiment the aqueous pharmaceutical composition
consists of 75 mg/ml mAb1, 30 mM histidine, 270 mM sucrose, and
0.06% polysorbate 20.
[0176] In one embodiment the aqueous pharmaceutical composition
consists of 75 mg/ml mAb1, 30 mM histidine, 270 mM sucrose, 0.06%
polysorbate 20 and 51 mM arginine-HCl.
[0177] Other contemplated excipients, which may be utilised 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).
[0178] The aqueous pharmaceutical compositions of the invention may
include further active ingredients in addition to the anti-CD40
antibody. Further pharmacological agents may include, for instance,
chemotherapeutic compounds.
Lyophilisates
[0179] 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.TM. to 21 mg/ml, SYNAGIS.TM.
and RAPTIVA.TM. to 100 mg/ml, and XOLAIR.TM. to 125 mg/ml.
Pre-Lyophilisates, Lyophilisates and Aqueous Reconstitution
[0180] 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.
[0181] 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
lyophilisation:volume of formulation before) 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-CD40 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.
[0182] Typical reconstituents for lyophilised 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's buffering agent) or it may
instead include no buffering agent (e.g. WFI (water for injection),
or physiological saline).
[0183] When present, components (i) to (iv) will be at a
pre-lyophilisation concentration sufficient to maintain the
anti-CD40 antibody in a form which is active and soluble after
storage (under normal conditions) and reconstitution. The
components will also be present after reconstitution.
[0184] 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 10 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 amino acid, such as arginine, methionine or
glycine, may be present before lyophilisation at a concentration of
between 2 and 80 mM e.g. 3-60 mM, 3-50 mM, 6-30 mM, 10-25 mM, 15-20
mM. A concentration of 17 mM arginine-HCl or 20 mM glycine-HCl or
60 mM methionine is useful. The anti-CD40 antibody is present
before lyophilisation 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.
[0185] The pre-lyophilisate of the invention has a pH between 5.0
and 8.0, between 5.0 and 7.0, between 5.5 and 6.5. In a specific
embodiment, the pre-lyophilisate of the invention has a pH of about
6.0.
[0186] In one embodiment the pre-lyophilisate of the invention has
a molar ratio of sucrose:antibody of 90:1 and a molar ratio of
histidine:antibody of 10:1.
[0187] In one embodiment the pre-lyophilisate of the invention has
a molar ratio of sucrose:antibody of 90:1, a molar ratio of
histidine:antibody of 10:1, and a molar ratio of
arginine-HCl:antibody of 17:1.
[0188] In one embodiment the pre-lyophilisate of the invention has
a molar ratio of sucrose:antibody of 90:1, a molar ratio of
histidine:antibody of 10:1, and a molar ratio of
glycine-HCl:antibody of 60:1.
[0189] A formulation containing histidine buffer, sucrose,
polysorbate 20 and, optionally arginine, methionine or glycine has
been shown to be suitable for lyophilisation of antibody mAb1.
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
[0190] The aqueous pharmaceutical compositions of the invention
comprising anti-CD40 antibodies can be used to treat, ameliorate or
prevent CD40-related autoimmune disorders, CD40-related
inflammatory disorders and/or to prevent or reduce the risk of
graft rejection in transplantation. For the purposes of the present
invention, the term "inflammatory disorders" includes "autoimmune
disorders".
[0191] As used herein, the term "autoimmunity" is generally
understood to encompass inflammatory immune-mediated processes
involving "self" antigens. In autoimmune diseases, self antigen(s)
trigger host immune responses.
[0192] Pharmaceutical compositions comprising anti-CD40 antibodies
are particularly useful to treat Multiple Sclerosis, Systemic Lupus
Erythematosus, Sjogren's syndrome, Rheumatoid Arthritis, transplant
rejection; graft-versus-host disease, Pemphigus vulgaris; and B
cell neoplasms such as acute lymphoblastic leukemia (ALL) and
B-cell chronic lymphocytic leukemia (CLL).
[0193] Also, the present invention includes treatment of
inflammation associated with tissue transplant rejection.
"Transplant rejection" or "graft rejection" refers to any
host-mounted immune response against a graft including but not
limited to HLA antigens, blood group antigens, and the like.
[0194] The invention can also be used to treat graft versus host
disease, such as that associated with bone marrow transplantation,
for example. In such graft versus host disease, the donor bone
marrow includes lymphocytes and cells that mature into lymphocytes.
The donor's lymphocytes recognise the recipient's antigens as
non-self and mount an inflammatory immune response. Hence, as used
herein, "graft versus host disease" or "graft versus host reaction"
refers to any T cell mediated immune response in which donor
lymphocytes react to the host's antigens.
[0195] The antagonist anti-CD40 antibodies or proteins described
herein, for example mAb1, mAb2 or mAb3, can be used in accordance
with the methods of the invention to treat autoimmune and/or
inflammatory disorders including, but not limited to, systemic
lupus erythematosus (SLE), discoid lupus, lupus nephritis,
sarcoidosis, inflammatory arthritis, including juvenile arthritis,
rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome,
ankylosing spondylitis, and gouty arthritis, rejection of an organ
or tissue transplant, hyperacute, acute, or chronic rejection
and/or graft versus host disease, multiple sclerosis, hyper IgE
syndrome, polyarteritis nodosa, primary biliary cirrhosis,
inflammatory bowel disease, Crohn's disease, celiac's disease
(gluten-sensitive enteropathy), primary Sjogren's syndrome (pSS),
autoimmune hepatitis, pernicious anemia, autoimmune hemolytic
anemia, psoriasis, scleroderma, myasthenia gravis, autoimmune
thrombocytopenic purpura, autoimmune thyroiditis, Grave's disease,
Hasimoto's thyroiditis, immune complex disease, chronic fatigue,
immune dysfunction syndrome (CFIDS), polymyositis and
dermatomyositis, cryoglobulinemia, thrombolysis, cardiomyopathy,
pemphigus vulgaris, pulmonary interstitial fibrosis, Type I and
Type II diabetes mellitus, type 1, 2, 3, and 4 delayed-type
hypersensitivity, allergy or allergic disorders,
unwanted/unintended immune responses to therapeutic proteins (see
for example, U.S. Patent Application No. US 2002/0119151 and Koren,
et al. (2002) Curr. Pharm. Biotechnol. 3: 349-60), asthma,
Churg-Strauss syndrome (allergic granulomatosis), atopic
dermatitis, allergic and irritant contact dermatitis, urtecaria,
IgE-mediated allergy, atherosclerosis, ANCA-associated
Vasculitides, vasculitis, idiopathic inflammatory myopathies,
hemolytic disease, Alzheimer's disease, chronic inflammatory
demyelinating polyneuropathy, and the like.
[0196] Genetic ablation or pharmacological inhibition of the
CD40-CD154 pathway has previously demonstrated therapeutic benefit
in either the clinic or in preclinical models of SLE, pSS, ITP, MS,
Crohn's disease, Pemphigus vulgaris, autoimmune vasculitis and RA
(Law C L, Grewal I S. (2009). Adv. Exp. Med. Biol. 2009; 647:8-36);
the medical need of which is detailed below.
[0197] In preferred embodiments the anti-CD40 antibodies or
proteins of the invention are useful in treating: (i) systemic
lupus erythematosus (lupus nephritis), preferably in providing
effective steroid-sparing therapies for induction and maintenance
of remission, and prevention of end-stage renal disease; (ii)
primary Sjogren's syndrome, preferably in prevention of salivary
and lacrimary gland destruction, and induction and maintenance of
remission of extraglandular manifestations; (ii) autoimmune
thrombocytopenic purpura, preferably treatment of patients
refractory to standard of care; (iv) ANCA-associated Vasculitides,
preferably inducing and maintaining remission in patients
refractory to corticosteroids, and steroid-sparing treatment; (v)
Pemphigus Vulgaris, preferably in induction and maintenance of
remission in patients refractory to corticosteroids, and
steroid-sparing treatment; (vi) Multiple Sclerosis, preferably in
providing more effective treatments for prevention of relapses and
disability progression, and achieving disease-free status; and
(vii) Crohn's disease, preferably in providing more effective
therapies for maintenance of remission, and treatment of patients
refractory to anti-TNF.
[0198] In some other embodiments, the anti-CD40 antibodies or
proteins of the invention are useful in treating pulmonary
inflammation including but not limited to lung graft rejection,
asthma, sarcoidosis, emphysema, cystic fibrosis, idiopathic
pulmonary fibrosis, chronic bronchitis, allergic rhinitis and
allergic diseases of the lung such as hypersensitivity pneumonitis,
eosinophilic pneumonia, bronchiolitis obliterans due to bone marrow
and/or lung transplantation or other causes, graft
atherosclerosis/graft phlebosclerosis, as well as pulmonary
fibrosis resulting from collagen, vascular, and autoimmune diseases
such as rheumatoid arthritis, scleroderma and lupus
erythematosus.
[0199] "Treatment" is herein defined as the application or
administration of an anti-CD40 antibody or protein according to the
invention, for example, mAb1, mAb2 or mAb3 antibody, to a subject,
or application or administration a pharmaceutical composition
comprising said anti-CD40 antibody or protein of the invention to
an isolated tissue or cell line from a subject, where the subject
has an autoimmune disease and/or inflammatory disease, a symptom
associated with an autoimmune disease and/or inflammatory disease,
or a predisposition toward development of an autoimmune disease
and/or inflammatory disease, where the purpose is to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the autoimmune disease and/or inflammatory disease, any associated
symptoms of the autoimmune disease and/or inflammatory disease, or
the predisposition toward the development of the autoimmune disease
and/or inflammatory disease.
[0200] By "treatment" is also intended the application or
administration of a pharmaceutical composition comprising an
anti-CD40 antibodies or protein of the invention, for example,
mAb1, mAb2 or mAb3 antibody, to a subject, or application or
administration of a pharmaceutical composition comprising said
anti-CD40 antibody or protein of the invention to an isolated
tissue or cell line from a subject, where the subject has an
autoimmune disease and/or inflammatory disease, a symptom
associated with an autoimmune disease and/or inflammatory disease,
or a predisposition toward development of an autoimmune disease
and/or inflammatory disease, where the purpose is to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the autoimmune disease and/or inflammatory disease, any associated
symptoms of the autoimmune disease and/or inflammatory disease, or
the predisposition toward the development of the autoimmune disease
and/or inflammatory disease.
[0201] By "anti-inflammatory activity" is intended a reduction or
prevention of inflammation. Therapy with at least one anti-CD40
antibody or protein according to the invention causes a
physiological response that is beneficial with respect to treatment
of an autoimmune disease and/or inflammatory disease, where the
disease involves cells expressing the CD40 antigen. It is
recognised that the methods of the invention may be useful in
preventing phenotypic change in cells such as proliferation,
activation, and the like.
Patient Administration
[0202] A pharmaceutical composition of the invention can be
administered to a patient.
[0203] 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 effective amount of the
anti-CD40 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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
then 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
humanisation. Equinised, bovinised, caninised and felinised
antibodies are known in the art. The antibody will bind to CD40
from the target species, but it may also cross-react with CD40 from
other species.
[0208] Dosage can be by a single dose schedule or a multiple dose
schedule.
[0209] Ingredients for forming compositions of the invention (e.g.
lyophilisates and reconstituents) may be supplied in
hermetically-sealed containers.
The Anti-CD40 Antibody
[0210] The invention concerns the formulation of anti-CD40
antibodies and more specifically the antibodies designated mAb1,
mAb2, and mAb3, especially mAb1.
[0211] One suitable antibody that can be comprised in the
pharmaceutical compositions of the invention is the human
recombinant antibody mAb1, structurally characterised as further
described below. The V.sub.H amino acid sequence of such isolated
anti-CD40 antibody is shown in SEQ ID NO: 1. The V.sub.L amino acid
sequence of such isolated anti-CD40 antibody is shown in SEQ ID NO:
2. The full length heavy chain amino acid sequence of such isolated
anti-CD40 antibody is shown in SEQ ID NO: 9. The full-length light
chain amino acid sequence of such isolated anti-CD40 antibody is
shown in SEQ ID NO: 10. Another example of heavy and light chain
amino acid sequences of such isolated anti-CD40 antibodies are
those encoded by the nucleotide sequences of SEQ ID NO: 11 and SEQ
ID NO: 12 respectively.
[0212] Other anti-CD40 antibodies that can be used for preparing
the pharmaceutical compositions of the invention include anti-CD40
antibodies with amino acids that have been mutated by amino acid
deletion, insertion or substitution, yet have no more than 1, 2, 3,
4 or 5 amino acid deletions, insertions or substitutions 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.
[0213] Conservative amino acid 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-CD40 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 CD40.
[0214] Antibodies may typically be glycosylated. N-linked glycans
attached to the C.sub.H2 domain of a heavy chain, for instance, can
influence Clq and FcR binding, and aglycosylated antibodies (for
example comprising an N297A mutation) 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.
[0215] Another modification of the anti-CD40 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).
[0216] As used herein, the term "polyethylene glycol" is intended
to encompass any of the forms of PEG that have been used to
derivatise other proteins, such as mono (C1-C10) alkoxy- or
aryloxy-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 0 154 316 by Nishimura et al. and EP 0 401 384
by Ishikawa et al.
[0217] Any other natural or non-natural post-translational
modification of anti-CD40 antibodies (e.g. mAb1) is further
contemplated as specific embodiments of anti-CD40 antibodies that
could be used for preparing the pharmaceutical compositions of the
invention.
[0218] Antibodies can be prepared in a form free from products with
which they would naturally be associated. Contaminant components of
an antibody's natural environment include materials such as
enzymes, hormones, or other host cell proteins.
[0219] The various features and embodiments of the present
invention, referred to in individual sections and embodiments above
apply, as appropriate, to other sections and embodiments, mutatis
mutandis. Consequently features specified in one section or
embodiment may be combined with features specified in other
sections or embodiments, as appropriate.
EXAMPLES
Preparing Anti-CD40 Antibodies
[0220] CHIR-12.12, and mAb1 (N297A CHIR-12.12), mAb2 (D265A
CHIR-12.12), and mAb3 (CHIR-12.12 LALA) bind specifically to CD40.
Tables 1 and 2 below summarise the sequence characteristics of
these antibodies. These antibodies 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.
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 CHIR- 12.12, mAb1, mAb2 and mAb3 2 Amino acid
sequence of the variable region (V.sub.L) of the light chain of
CHIR- 12.12, mAb1, mAb2 and mAb3 3 Amino acid sequence of HCDR1 of
CHIR-12.12, mAb1, mAb2 and mAb3 4 Amino acid sequence of HCDR2 of
CHIR-12.12, mAb1, mAb2 and mAb3 5 Amino acid sequence of HCDR3 of
CHIR-12.12, mAb1, mAb2 and mAb3 6 Amino acid sequence of LCDR1 of
CHIR-12.12, mAb1, mAb2 and mAb3 7 Amino acid sequence of LCDR2 of
CHIR-12.12, mAb1, mAb2 and mAb3 8 Amino acid sequence of LCDR3 of
CHIR-12.12, mAb1, mAb2 and mAb3 9 Amino acid sequence of the full
length heavy chain of mAb1 10 Amino acid sequence of the full
length light chain of mAb1 11 Nucleotide sequence encoding full
length heavy chain of mAb1 (SEQ ID NO: 9) 12 Nucleotide sequence
encoding full length light chain of mAb1 (SEQ ID NO: 10) 13 Amino
acid sequence of the full length heavy chain of mAb2 14 Amino acid
sequence of the full length light chain of mAb2 15 Nucleotide
sequence encoding full length heavy chain of mAb2 (SEQ ID NO: 13)
16 Nucleotide sequence encoding full length light chain of mAb2
(SEQ ID NO: 14) 17 Amino acid sequence of the full length heavy
chain of mAb3 18 Amino acid sequence of the full length light chain
of mAb3 19 Nucleotide sequence encoding full length heavy chain of
mAb3 (SEQ ID NO: 17) 20 Nucleotide sequence encoding full length
light chain of mAb3 (SEQ ID NO: 18) 21 Amino acid sequence of human
CD40
TABLE-US-00002 TABLE 2 Sequence listing SEQ ID NO: Amino acid or
Nucleotide Sequence 1
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV
ISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIA
APGPDYWGQGTLVTVSS 2
DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLI
SLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFG PGTKVDIR 3
SYGMH 4 VISYEESNRYHADSVKG 5 DGGIAAPGPDY 6 RSSQSLLYSNGYNYLD 7
LGSNRAS 8 MQARQTPFT 9
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV
ISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIA
APGPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVL T VLHQDWLNGK
EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSREE MTKNQVSLTC LVKGFYPSDI
AVEWESNGQP ENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 10
DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLI
SLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFG
PGTKVDIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 11
CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGG
TCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGG
CATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGC
CGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGG
GCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAG
ATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGG
ACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCT
GGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTG
GCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCC
TGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGG
CGCCCTGACCTCCGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCCTCC
GGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCTAGCTCTCTGG
GCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAG
GTGGACAAGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTC
CCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTT
CCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTG
ACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCA
ATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAG
AGAGGAACAGTACGCCTCCACCTACCGGGTGGTGTCTGTGCTGACCGTG
CTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCA
ACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGG
CCAGCCCCGCGAGCCACAGGTGTACACACTGCCCCCCAGCCGGGAAGA
GATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAAGGCTTCTACC
CCTCCGATATCGCCGTGGAGTGGGAGTCCAACGGACAGCCCGAGAACAA
CTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGT
ACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTT
CTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG
TCCCTGTCCCTGAGCCCCGGCAAG 12
GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCG
AGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAAC
GGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCAC
AGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCG
GTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGG
TGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGA
CCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGT
GGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAG
AGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGG
AGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA
GCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCT
GAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTG
TACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGA
GCTTCAACAGGGGCGAGTGC 13
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV
ISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIA
APGPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 14
DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLI
SLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFG
PGTKVDIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PV TKSFNRGEC
15 CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGG
TCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGG
CATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGC
CGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGG
GCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAG
ATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGG
ACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCT
GGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTG
GCCCCCTCCAGCAAGTCCACCTCTGGCGGCACCGCCGCTCTGGGCTGCC
TGGTGAAAGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACTCTGG
CGCCCTGACCTCCGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCCTCC
GGCCTGTACTCCCTGTCCTCCGTGGTGACCGTGCCCTCTAGCTCTCTGG
GCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAG
GTGGACAAGCGGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTC
CCCCCTGCCCTGCCCCTGAACTGCTGGGCGGACCTTCCGTGTTCCTGTT
CCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCCGAAGTG
ACCTGCGTGGTGGTGGCCGTGTCCCACGAGGACCCTGAAGTGAAGTTCA
ATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAG
AGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCTGTGCTGACCGTG
CTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTCTCCA
ACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAAGGCCAAGGG
CCAGCCCCGCGAGCCACAGGTGTACACACTGCCCCCCAGCCGGGAAGA
GATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAAGGCTTCTACC
CCTCCGATATCGCCGTGGAGTGGGAGTCCAACGGACAGCCCGAGAACAA
CTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGT
ACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTT
CTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAG
TCCCTGTCCCTGAGCCCCGGCAAG 16
GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCG
AGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAAC
GGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCAC
AGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCG
GTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGG
TGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGA
CCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGT
GGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAG
AGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGG
AGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA
GCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCT
GAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTG
TACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGA
GCTTCAACAGGGGCGAGTGC 17
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAV
ISYEESNRYHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIA
APGPDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN
HKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL TVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 18
DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLI
SLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFG
PGTKVDIRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD
NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 19
CAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCCGG
TCCCTGAGACTGTCTTGCGCCGCCTCCGGCTTCACCTTCTCCAGCTACGG
CATGCACTGGGTGCGACAGGCCCCTGGCAAGGGACTGGAATGGGTGGC
CGTGATCTCCTACGAGGAATCCAACAGATACCACGCTGACTCCGTGAAGG
GCCGGTTCACAATCTCCCGGGACAACTCCAAGATCACCCTGTACCTGCAG
ATGAACTCCCTGCGGACCGAGGACACCGCCGTGTACTACTGCGCCAGGG
ACGGAGGAATCGCCGCTCCTGGACCTGATTATTGGGGCCAGGGCACCCT
GGTGACAGTGTCCTCCGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTG
GCCCCTTCCAGCAAGTCTACCTCCGGCGGCACAGCTGCTCTGGGCTGCC
TGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGC
GCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCG
GCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGG
CACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGG
TGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCC
TCCCTGCCCTGCTCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCC
CTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGAC
CTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATT
GGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGG
AGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCT
GCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAAC
AAGGCCCTGCCTGCCCCTATCGAAAAGACAATCTCCAAGGCCAAGGGCC
AGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAAT
GACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTT
CCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTA
CAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACT
CCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTC
CTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCC
CTGTCCCTGTCTCCCGGCAAG 20
GACATCGTGATGACCCAGTCCCCCCTGTCCCTGACCGTGACACCTGGCG
AGCCTGCCTCTATCTCCTGCAGATCCTCCCAGTCCCTGCTGTACTCCAAC
GGCTACAACTACCTGGACTGGTATCTGCAGAAGCCCGGCCAGTCCCCAC
AGGTGCTGATCTCCCTGGGCTCCAACAGAGCCTCTGGCGTGCCCGACCG
GTTCTCCGGCTCTGGCTCTGGCACCGACTTCACACTGAAGATCTCACGGG
TGGAAGCCGAGGACGTGGGCGTGTACTACTGCATGCAGGCCCGGCAGA
CCCCCTTCACCTTCGGCCCTGGCACCAAGGTGGACATCCGGCGTACGGT
GGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAG
AGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGG
AGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACA
GCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCT
GAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTG
TACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGA
GCTTCAACAGGGGCGAGTGC 21
MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDC
TEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTIC
TCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVS
SAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAIL
LVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVT
QEDGKESRISVQERQ
EXAMPLES OF FORMULATIONS
[0221] A high concentration lyophilised or liquid formulation of
mAb1 was desired and so formulation studies were performed. A
lyophilised formulation comprising a sugar, a buffering agent and a
surfactant was stable and could maintain high antibody
concentrations after reconstitution.
[0222] Five formulations (F1, F2, F3, F4 and F5) of mAb1 were
evaluated for stability. F1 was a liquid formulation at 50 mg/mL
mAb1 at pH 6.0. Formulations F2, F3, F4, and F5 had prior to
lyophilisation, 50 mg/vial mAb1. Formulations F2, F3, F4 and F5
had, prior to lyophilisation, 50 mg/ml mAb1 at pH 6.0. Formulations
F2, F3, F4 and F5 had a fill volume of 3.6 ml. The five
formulations included buffer, sugar, surfactant and free amino acid
as listed in Table 3:
TABLE-US-00003 TABLE 3 Examples of formulations Histidine Amino
mAb1 buffer Stabiliser Surfactant acid F1 50 mg/ml 10 mM 190 mM
0.02% polysorbate 40 mM trehalose 20 arginine- HCl F2 50 mg/ml 10
mM 90 mM 0.02% polysorbate 17 mM trehalose 20 arginine- HCl F3 50
mg/ml 10 mM 90 mM 0.02% polysorbate 17 mM sucrose 20 arginine- HCl
F4 50 mg/ml 10 mM 90 mM 0.02% polysorbate -- trehalose 20 F5 50
mg/ml 10 mM 90 mM 0.02% polysorbate -- sucrose 20
[0223] The F2, F3, F4, and F5 lyophilisates were reconstituted with
WFI (1.0 ml) to give a reconstituted volume of 1.2 ml (1/3 the
original aqueous volume). The reconstituted compositions were as
listed in Table 4:
TABLE-US-00004 TABLE 4 Examples of formulations Histidine Amino
mAb1 buffer Stabiliser Surfactant acid F2 150 mg/ml 30 mM 270 mM
0.06% polysorbate 51 mM histidine trehalose 20 arginine- HCl F3 150
mg/ml 30 mM 270 mM 0.06% polysorbate 51 mM histidine sucrose 20
arginine- HCl F4 150 mg/ml 30 mM 270 mM 0.06% polysorbate --
histidine trehalose 20 F5 150 mg/ml 30 mM 270 mM 0.06% polysorbate
-- histidine sucrose 20
[0224] The lyophilisation cycle used is reported in 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 06:00 -37.degree. C. Ambient 6 Chamber Vacuum as required
-37.degree. C. 0.2 mbar.sup.a 7 Primary drying 16:00 -37.degree. C.
to 25.degree. C. 0.2 mbar.sup.a ramp 8 Secondary drying 24:00
25.degree. C. 0.2 mbar.sup.a hold 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 five formulations were tested for stability at various
time points after formulation/reconstitution as listed below.
[0226] Size exclusion--High Pressure Liquid Chromatography
(SEC-HPLC) was used to assess the amount of mAb1 in each
formulation at the time of formulation (T0), 4 weeks (25.degree.
C.), 4 weeks (40.degree. C.), 6 months (2-8.degree. C.), 6 months
(25.degree. C.) and 6 months (40.degree. C.). The amount of
antibody is expressed as a percentage of the starting amount, as
listed in Table 6. The F1 formulation remains close to 50
mg/ml.+-.5 mg/ml (100%.+-.10%), the F2, F3, F4 and F5 formulations
remain close to 150 mg/ml.+-.15 mg/ml (100%.+-.10%).
TABLE-US-00006 TABLE 6 Amount of antibody from SEC-HPLC 4 weeks 4
weeks 6 months 6 months 6 months T0 (25.degree. C.) (40.degree. C.)
(2-8.degree. C.) (25.degree. C.) (40.degree. C.) F1 100% .+-. 10%
100% .+-. 10% 100% .+-. 10% 100% .+-. 10% 100% .+-. 10% 100% .+-.
10% F2 100% .+-. 10% -- 100% .+-. 10% -- 100% .+-. 10% 100% .+-.
10% F3 100% .+-. 10% -- 100% .+-. 10% -- -- 100% .+-. 10% F4 100%
.+-. 10% -- 100% .+-. 10% -- 100% .+-. 10% 100% .+-. 10% F5 100%
.+-. 10% -- 100% .+-. 10% -- 100% .+-. 10% 100% .+-. 10%
[0227] The potency of the mAb1 antibody in each formulation was
measured by ELISA assay at T0, 4 weeks (25.degree. C.), 4 weeks
(40.degree. C.), 6 months (2-8.degree. C.), 6 months (25.degree.
C.) and 6 months (40.degree. C.). The results, expressed as a
percentage of the starting potency (100%) are listed in Table 7.
The potency of the F1 formulation decreases with progression of
time and increase of temperature; after 6 m at 40.degree. C.
potency was found to be 79%. The lowest value measured was 85% for
F4 (6 m at 25.degree. C.) and 86% for F3 (6 m at 40.degree. C.)
TABLE-US-00007 TABLE 7 Potency of antibody as measured in ELISA 4
weeks 4 weeks 6 months 6 months 6 months T0 (25.degree. C.)
(40.degree. C.) (2-8.degree. C.) (25.degree. C.) (40.degree. C.) F1
91.3 104.0 98.0 91.0 91.0 79.0 F2 93.2 F3 95.7 86.0 F4 96.5 85.0
90.0 F5 97.8
[0228] Stability of the formulations was evaluated by % impurities
as measured by size exclusion--High Pressure Liquid Chromatography
(SEC-HPLC) and Dynamic Light Scattering (DLS). The results are
shown in Tables 8 and 9.
TABLE-US-00008 TABLE 8 Aggregation products results from SEC-HPLC 4
weeks 4 weeks 6 months 6 months 6 months T0 (25.degree. C.)
(40.degree. C.) (2-8.degree. C.) (25.degree. C.) (40.degree. C.) F1
0.26% 0.26% 0.59% 0.38% 0.47% 2.76% F2 0.38% 1.37% 1.40% 3.80% F3
0.29% 0.96% 2.88% F4 0.38% 1.94% 1.97% 5.59% F5 0.33% 1.32% 1.40%
4.01%
TABLE-US-00009 TABLE 9 Small particles; results for PolyDispensity
Index (PDI) from Dynamic Light Scattering 4 weeks 4 weeks 6 months
6 months 6 months T0 (25.degree. C.) (40.degree. C.) (2-8.degree.
C.) (25.degree. C.) (40.degree. C.) F1 52.8% 30.2% 39.3% 41.3%
52.9% 59.9% F2 48.8% 61.0% 44.4% 46.6% F3 50.4% 57.0% 40.8% F4
54.7% 61.8% 61.9% 59.2% F5 47.2% 60.0% 60.5% 51.3%
[0229] The F1 formulation showed an increase of aggregation
products with increasing temperature and time. For the F2 to F5
formulations addition of arginine (F2 and F3) slightly reduced the
level of aggregation; and sucrose (F3 and F5) was superior to
trehalose.
[0230] Aggregation products were also evaluated by measuring
turbidity. The F1 formulation showed an increase of turbidity with
increase in temperature and with increase in time. The F2 to F5
formulations showed slightly increased turbidity values for
formulations containing arginine (F2 and F3).
TABLE-US-00010 TABLE 10 Turbidity measurements (absorbance units) 4
weeks 4 weeks 6 months 6 months 6 months T0 (25.degree. C.)
(40.degree. C.) (2-8.degree. C.) (25.degree. C.) (40.degree. C.) F1
7.38 11.30 12.90 9.7 12.7 17.9 F2 9.36 14.90 12.1 11.3 F3 8.98
14.40 11.0 F4 6.34 10.40 9.2 7.6 F5 6.91 10.70 9.7 8.2
[0231] Stability of the formulations was evaluated by % degradation
products as measured SEC-HPLC. With the F2 to F5 formulations the
addition of arginine slightly reduced degradation. The results are
shown in Table 11.
TABLE-US-00011 TABLE 11 Degradation products results from SEC-HPLC
4 weeks 4 weeks 6 months 6 months 6 months T0 (25.degree. C.)
(40.degree. C.) (2-8.degree. C.) (25.degree. C.) (40.degree. C.) F1
0.11% 1.40% 2.76% 1.04% 2.10% 8.88% F2 0.11% 0.14% 0.12% 0.05% F3
0.15% 0.18% 0.00% 2.97% F4 0.10% 0.19% 0.15% 5.70% F5 0.11% 0.18%
0.14% 4.10%
[0232] The four lyophilised formulations F2, F3, F4, and F5 were
assessed for visual clarity after reconstitution. The results are
presented in Table 12.
TABLE-US-00012 TABLE 12 Visual clarity 4 weeks 4 weeks 6 months 6
months 6 months T0 (25.degree. C.) (40.degree. C.) (2-8.degree. C.)
(25.degree. C.) (40.degree. C.) F2 clear clear clear clear clear
clear without without without without without without particles
particles particles particles particles particles F3 clear clear
clear clear clear clear without without without without without
without particles particles particles particles particles particles
F4 clear clear clear clear clear clear without without without
without without without particles particles particles particles
particles particles F5 clear clear clear clear clear clear without
without without without without without particles particles
particles particles particles particles
[0233] The physico-chemical properties of the formulations were
assessed; the results are presented in Table 13. The viscosity
values are the values of the formulations having 50 mg/mil antibody
prior to lyophilisation. The osmolality values correspond to the
reconstituted compositions having 150 mg/ml antibody.
TABLE-US-00013 TABLE 13 Physico-chemical properties Viscosity
Osmolality mPa s mOsm/kg F1 2.1 318 F2 1.7 580 F3 1.6 556 F4 1.6
461 F5 1.6 443
CONCLUSIONS
[0234] Overall, the formulations tested showed good results. The
bioassays (SEC and ELISA) showed that the formulations are
comparable. Soluble aggregates were slightly reduced in the
presence of arginine and were slightly lower for the sucrose
containing formulations compared to the trehalose containing
formulations. Turbidity was slightly increased in the presence of
arginine; there was no difference between trehalose or sucrose
containing formulations. Degradation was reduced in the presence of
arginine. With regard to physico-chemical properties the pH,
viscosity, residual moisture content, and visual aspects were
comparable for all formulations. Osmolality was >300 mOsm/kg for
all formulations, and was even higher in the presence of
arginine-HCl (F2 and F3). It is desirable to have a formulation
which is isotonic to plasma (290 mOsm/kg), or as close to this
value as possible. The formulations having sucrose (F3 and F5) were
considered preferable to those comprising trehalose. Whilst some
beneficial effects on degradation were seen with arginine (F3), the
lower osmolality of the F5 formulation was considered to be a more
important factor. The F5 formulation was deemed to be the most
optimal formulation.
Sequence CWU 1
1
211120PRTHomo sapiens 1Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr
Glu Glu Ser Asn Arg Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Ile Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Gly Gly Ile Ala Ala Pro Gly Pro Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 2112PRTHomo
sapiens 2Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Thr Val Thr
Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Leu Tyr Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu
Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Val Leu Ile Ser Leu Gly
Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Arg Gln
Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Arg 100 105 110
35PRTHomo sapiens 3Ser Tyr Gly Met His 1 5 417PRTHomo sapiens 4Val
Ile Ser Tyr Glu Glu Ser Asn Arg Tyr His Ala Asp Ser Val Lys 1 5 10
15 Gly 511PRTHomo sapiens 5Asp Gly Gly Ile Ala Ala Pro Gly Pro Asp
Tyr 1 5 10 616PRTHomo sapiens 6Arg Ser Ser Gln Ser Leu Leu Tyr Ser
Asn Gly Tyr Asn Tyr Leu Asp 1 5 10 15 77PRTHomo sapiens 7Leu Gly
Ser Asn Arg Ala Ser 1 5 89PRTHomo sapiens 8Met Gln Ala Arg Gln Thr
Pro Phe Thr 1 5 9450PRTHomo sapiens 9Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Val Ile Ser Tyr Glu Glu Ser Asn Arg Tyr His Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ile Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Gly Gly Ile Ala Ala Pro Gly Pro Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg 290 295 300 Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445 Gly Lys 450 10219PRTHomo sapiens 10Asp Ile Val Met Thr Gln
Ser Pro Leu Ser Leu Thr Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly
Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45
Pro Gln Val Leu Ile Ser Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50
55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys
Met Gln Ala 85 90 95 Arg Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr
Lys Val Asp Ile Arg 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180
185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
111350DNAHomo sapiens 11caggtgcagc tggtggaatc tggcggcgga gtggtgcagc
ctggccggtc cctgagactg 60tcttgcgccg cctccggctt caccttctcc agctacggca
tgcactgggt gcgacaggcc 120cctggcaagg gactggaatg ggtggccgtg
atctcctacg aggaatccaa cagataccac 180gctgactccg tgaagggccg
gttcacaatc tcccgggaca actccaagat caccctgtac 240ctgcagatga
actccctgcg gaccgaggac accgccgtgt actactgcgc cagggacgga
300ggaatcgccg ctcctggacc tgattattgg ggccagggca ccctggtgac
agtgtcctcc 360gctagcacca agggcccctc cgtgttccct ctggccccct
ccagcaagtc cacctctggc 420ggcaccgccg ctctgggctg cctggtgaaa
gactacttcc ccgagcccgt gaccgtgtcc 480tggaactctg gcgccctgac
ctccggcgtg cacacctttc cagccgtgct gcagtcctcc 540ggcctgtact
ccctgtcctc cgtggtgacc gtgccctcta gctctctggg cacccagacc
600tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagcg
ggtggaaccc 660aagtcctgcg acaagaccca cacctgtccc ccctgccctg
cccctgaact gctgggcgga 720ccttccgtgt tcctgttccc cccaaagccc
aaggacaccc tgatgatctc ccggaccccc 780gaagtgacct gcgtggtggt
ggacgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 840tacgtggacg
gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacgcc
900tccacctacc gggtggtgtc tgtgctgacc gtgctgcacc aggactggct
gaacggcaaa 960gagtacaagt gcaaggtctc caacaaggcc ctgcctgccc
ccatcgaaaa gaccatctcc 1020aaggccaagg gccagccccg cgagccacag
gtgtacacac tgccccccag ccgggaagag 1080atgaccaaga accaggtgtc
cctgacctgt ctggtcaaag gcttctaccc ctccgatatc 1140gccgtggagt
gggagtccaa cggacagccc gagaacaact acaagaccac cccccctgtg
1200ctggactccg acggctcatt cttcctgtac tccaagctga ccgtggacaa
gtcccggtgg 1260cagcagggca acgtgttctc ctgctccgtg atgcacgagg
ccctgcacaa ccactacacc 1320cagaagtccc tgtccctgag ccccggcaag
135012657DNAHomo sapiens 12gacatcgtga tgacccagtc ccccctgtcc
ctgaccgtga cacctggcga gcctgcctct 60atctcctgca gatcctccca gtccctgctg
tactccaacg gctacaacta cctggactgg 120tatctgcaga agcccggcca
gtccccacag gtgctgatct ccctgggctc caacagagcc 180tctggcgtgc
ccgaccggtt ctccggctct ggctctggca ccgacttcac actgaagatc
240tcacgggtgg aagccgagga cgtgggcgtg tactactgca tgcaggcccg
gcagaccccc 300ttcaccttcg gccctggcac caaggtggac atccggcgta
cggtggccgc tcccagcgtg 360ttcatcttcc cccccagcga cgagcagctg
aagagcggca ccgccagcgt ggtgtgcctg 420ctgaacaact tctacccccg
ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 480agcggcaaca
gccaggagag cgtcaccgag caggacagca aggactccac ctacagcctg
540agcagcaccc tgaccctgag caaggccgac tacgagaagc ataaggtgta
cgcctgcgag 600gtgacccacc agggcctgtc cagccccgtg accaagagct
tcaacagggg cgagtgc 65713450PRTHomo sapiens 13Gln Val Gln Leu Val
Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ala Val Ile Ser Tyr Glu Glu Ser Asn Arg Tyr His Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ile Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Gly Ile Ala Ala Pro Gly
Pro Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro
Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Ala Val Ser His Glu 260 265 270 Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295
300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420
425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 445 Gly Lys 450 14219PRTHomo sapiens 14Asp Ile Val Met
Thr Gln Ser Pro Leu Ser Leu Thr Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Val Leu Ile Ser Leu Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90 95 Arg Gln Thr Pro Phe Thr Phe Gly Pro
Gly Thr Lys Val Asp Ile Arg 100 105 110 Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165
170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 151350DNAHomo sapiens 15caggtgcagc tggtggaatc tggcggcgga
gtggtgcagc ctggccggtc cctgagactg 60tcttgcgccg cctccggctt caccttctcc
agctacggca tgcactgggt gcgacaggcc 120cctggcaagg gactggaatg
ggtggccgtg atctcctacg aggaatccaa cagataccac 180gctgactccg
tgaagggccg gttcacaatc tcccgggaca actccaagat caccctgtac
240ctgcagatga actccctgcg gaccgaggac accgccgtgt actactgcgc
cagggacgga 300ggaatcgccg ctcctggacc tgattattgg ggccagggca
ccctggtgac agtgtcctcc 360gctagcacca agggcccctc cgtgttccct
ctggccccct ccagcaagtc cacctctggc 420ggcaccgccg ctctgggctg
cctggtgaaa gactacttcc ccgagcccgt gaccgtgtcc 480tggaactctg
gcgccctgac ctccggcgtg cacacctttc cagccgtgct gcagtcctcc
540ggcctgtact ccctgtcctc cgtggtgacc gtgccctcta gctctctggg
cacccagacc 600tacatctgca acgtgaacca caagccctcc aacaccaagg
tggacaagcg ggtggaaccc 660aagtcctgcg acaagaccca cacctgtccc
ccctgccctg cccctgaact gctgggcgga 720ccttccgtgt tcctgttccc
cccaaagccc aaggacaccc tgatgatctc ccggaccccc 780gaagtgacct
gcgtggtggt ggccgtgtcc cacgaggacc ctgaagtgaa gttcaattgg
840tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc ccagagagga
acagtacaac 900tccacctacc gggtggtgtc tgtgctgacc gtgctgcacc
aggactggct gaacggcaaa 960gagtacaagt gcaaggtctc caacaaggcc
ctgcctgccc ccatcgaaaa gaccatctcc 1020aaggccaagg gccagccccg
cgagccacag gtgtacacac tgccccccag ccgggaagag 1080atgaccaaga
accaggtgtc cctgacctgt ctggtcaaag gcttctaccc ctccgatatc
1140gccgtggagt gggagtccaa cggacagccc gagaacaact acaagaccac
cccccctgtg 1200ctggactccg acggctcatt cttcctgtac tccaagctga
ccgtggacaa gtcccggtgg 1260cagcagggca acgtgttctc ctgctccgtg
atgcacgagg ccctgcacaa ccactacacc 1320cagaagtccc tgtccctgag
ccccggcaag 135016657DNAHomo sapiens 16gacatcgtga tgacccagtc
ccccctgtcc ctgaccgtga cacctggcga gcctgcctct 60atctcctgca gatcctccca
gtccctgctg tactccaacg gctacaacta cctggactgg 120tatctgcaga
agcccggcca gtccccacag gtgctgatct ccctgggctc caacagagcc
180tctggcgtgc ccgaccggtt ctccggctct ggctctggca ccgacttcac
actgaagatc 240tcacgggtgg aagccgagga cgtgggcgtg tactactgca
tgcaggcccg gcagaccccc 300ttcaccttcg gccctggcac caaggtggac
atccggcgta cggtggccgc tcccagcgtg 360ttcatcttcc cccccagcga
cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg 420ctgaacaact
tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag
480agcggcaaca gccaggagag cgtcaccgag caggacagca aggactccac
ctacagcctg 540agcagcaccc tgaccctgag caaggccgac tacgagaagc
ataaggtgta cgcctgcgag 600gtgacccacc agggcctgtc cagccccgtg
accaagagct tcaacagggg cgagtgc 65717450PRTHomo
sapiens 17Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Glu Glu Ser
Asn Arg Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Ile Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Asp Gly Gly Ile Ala Ala Pro Gly Pro Asp Tyr Trp Gly Gln 100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 225 230 235
240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360
365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450
18219PRTHomo sapiens 18Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Thr Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Val Leu Ile
Ser Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90
95 Arg Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Arg
100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 191350DNAHomo
sapiens 19caggtgcagc tggtggaatc tggcggcgga gtggtgcagc ctggccggtc
cctgagactg 60tcttgcgccg cctccggctt caccttctcc agctacggca tgcactgggt
gcgacaggcc 120cctggcaagg gactggaatg ggtggccgtg atctcctacg
aggaatccaa cagataccac 180gctgactccg tgaagggccg gttcacaatc
tcccgggaca actccaagat caccctgtac 240ctgcagatga actccctgcg
gaccgaggac accgccgtgt actactgcgc cagggacgga 300ggaatcgccg
ctcctggacc tgattattgg ggccagggca ccctggtgac agtgtcctcc
360gctagcacca agggcccctc cgtgttccct ctggcccctt ccagcaagtc
tacctccggc 420ggcacagctg ctctgggctg cctggtcaag gactacttcc
ctgagcctgt gacagtgtcc 480tggaactctg gcgccctgac ctctggcgtg
cacaccttcc ctgccgtgct gcagtcctcc 540ggcctgtact ccctgtcctc
cgtggtcaca gtgccttcaa gcagcctggg cacccagacc 600tatatctgca
acgtgaacca caagccttcc aacaccaagg tggacaagcg ggtggagcct
660aagtcctgcg acaagaccca cacctgtcct ccctgccctg ctcctgaagc
tgctggcggc 720ccttctgtgt tcctgttccc tccaaagccc aaggacaccc
tgatgatctc ccggacccct 780gaagtgacct gcgtggtggt ggacgtgtcc
cacgaggatc ctgaagtgaa gttcaattgg 840tacgtggacg gcgtggaggt
gcacaacgcc aagaccaagc ctcgggagga acagtacaac 900tccacctacc
gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaaa
960gagtacaagt gcaaagtctc caacaaggcc ctgcctgccc ctatcgaaaa
gacaatctcc 1020aaggccaagg gccagcctag ggaaccccag gtgtacaccc
tgccacccag ccgggaggaa 1080atgaccaaga accaggtgtc cctgacctgt
ctggtcaagg gcttctaccc ttccgatatc 1140gccgtggagt gggagtctaa
cggccagcct gagaacaact acaagaccac ccctcctgtg 1200ctggactccg
acggctcctt cttcctgtac tccaaactga ccgtggacaa gtcccggtgg
1260cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa
ccactacacc 1320cagaagtccc tgtccctgtc tcccggcaag 135020657DNAHomo
sapiens 20gacatcgtga tgacccagtc ccccctgtcc ctgaccgtga cacctggcga
gcctgcctct 60atctcctgca gatcctccca gtccctgctg tactccaacg gctacaacta
cctggactgg 120tatctgcaga agcccggcca gtccccacag gtgctgatct
ccctgggctc caacagagcc 180tctggcgtgc ccgaccggtt ctccggctct
ggctctggca ccgacttcac actgaagatc 240tcacgggtgg aagccgagga
cgtgggcgtg tactactgca tgcaggcccg gcagaccccc 300ttcaccttcg
gccctggcac caaggtggac atccggcgta cggtggccgc tcccagcgtg
360ttcatcttcc cccccagcga cgagcagctg aagagcggca ccgccagcgt
ggtgtgcctg 420ctgaacaact tctacccccg ggaggccaag gtgcagtgga
aggtggacaa cgccctgcag 480agcggcaaca gccaggagag cgtcaccgag
caggacagca aggactccac ctacagcctg 540agcagcaccc tgaccctgag
caaggccgac tacgagaagc ataaggtgta cgcctgcgag 600gtgacccacc
agggcctgtc cagccccgtg accaagagct tcaacagggg cgagtgc 65721277PRTHomo
sapiens 21Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu
Leu Thr 1 5 10 15 Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu
Lys Gln Tyr Leu 20 25 30 Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln
Pro Gly Gln Lys Leu Val 35 40 45 Ser Asp Cys Thr Glu Phe Thr Glu
Thr Glu Cys Leu Pro Cys Gly Glu 50 55 60 Ser Glu Phe Leu Asp Thr
Trp Asn Arg Glu Thr His Cys His Gln His 65 70 75 80 Lys Tyr Cys Asp
Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr 85 90 95 Ser Glu
Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr 100 105 110
Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly 115
120 125 Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys
Glu 130 135 140 Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala
Phe Glu Lys 145 150 155 160 Cys His Pro Trp Thr Ser Cys Glu Thr Lys
Asp Leu Val Val Gln Gln 165 170 175 Ala Gly Thr Asn Lys Thr Asp Val
Val Cys Gly Pro Gln Asp Arg Leu 180 185 190 Arg Ala Leu Val Val Ile
Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile 195 200 205 Leu Leu Val Leu
Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn 210 215 220 Lys Ala
Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp 225 230 235
240 Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His
245 250 255 Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg
Ile Ser 260 265 270 Val Gln Glu Arg Gln 275
* * * * *