U.S. patent application number 17/101966 was filed with the patent office on 2021-07-22 for anti-pdl1 antibody formulations.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Sreedhara ALAVATTAM, Ying YANG.
Application Number | 20210221892 17/101966 |
Document ID | / |
Family ID | 1000005489798 |
Filed Date | 2021-07-22 |
United States Patent
Application |
20210221892 |
Kind Code |
A1 |
YANG; Ying ; et al. |
July 22, 2021 |
ANTI-PDL1 ANTIBODY FORMULATIONS
Abstract
The invention provides stable aqueous pharmaceutical
formulations comprising an anti-PDL1 antibody. The invention also
provides methods for making such formulations and methods of using
such formulations.
Inventors: |
YANG; Ying; (South San
Francisco, CA) ; ALAVATTAM; Sreedhara; (South San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
1000005489798 |
Appl. No.: |
17/101966 |
Filed: |
November 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15081785 |
Mar 25, 2016 |
10875922 |
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17101966 |
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PCT/US2014/057821 |
Sep 26, 2014 |
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15081785 |
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61883953 |
Sep 27, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/52 20130101;
C07K 2317/41 20130101; C07K 2317/76 20130101; A61K 39/39591
20130101; A61K 2039/505 20130101; C07K 16/2827 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395 |
Claims
1-39. (canceled)
40: A method of treating cancer in a subject comprising
administering an effective amount of a stable aqueous
pharmaceutical formulation comprising an anti-programmed death
ligand 1 (anti-PD-L1) monoclonal antibody in a concentration of 60
mg/ml, histidine acetate in a concentration of 20 mM, sucrose in a
concentration of 120 mM, polysorbate 20 in a concentration of 0.04%
(w/v), and pH 5.8; wherein said monoclonal antibody comprises a
light chain variable region comprising the amino acid sequence of
SEQ ID NO:7, and a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO:32.
41. (canceled)
42: The method of claim 40, wherein the medicament is which is for
intravenous (IV) administration.
43: The method of claim 40, wherein the cancer is locally advanced
or metastatic.
44: The method of claim 40, wherein the cancer is selected from the
group consisting of a solid tumor, a hematologic cancer, bladder
cancer, brain cancer, breast cancer, colon cancer, colorectal
cancer, gastric cancer, glioma, head cancer, leukemia, liver
cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian
cancer, melanoma, pancreatic cancer, renal cancer, salivary cancer,
stomach cancer, thymic epithelial cancer, thyroid cancer, and
squamous cell carcinoma of the head and neck.
45: The method of claim 44, wherein the cancer is bladder
cancer.
46: The method of claim 44, wherein the cancer is lung cancer.
47: The method of claim 46, wherein the lung is non-small cell lung
cancer.
48: The method of claim 44, wherein the cancer is breast
cancer.
49: The method of claim 40, wherein the subject has PD-L1 positive
cancer.
50: The method of claim 40, wherein the formulation is to be
administered in conjunction with another therapeutic agent to the
subject.
51: The method of claim 50, wherein the therapeutic agent is a
chemotherapeutic agent or an antibody treatment.
52: A method of treating cancer in a subject comprising
administering an effective amount of a stable aqueous
pharmaceutical formulation comprising an anti-programmed death
ligand 1 (anti-PD-L1) monoclonal antibody in a concentration of 60
mg/ml, histidine acetate in a concentration of 20 mM, sucrose in a
concentration of 120 mM, polysorbate 20 in a concentration of 0.04%
(w/v), and pH 5.8; wherein said monoclonal antibody comprises a
light chain comprising the amino acid sequence of SEQ ID NO:9, and
a heavy chain comprising the amino acid sequence of SEQ ID
NO:10.
53: The method of claim 52, wherein the medicament is which is for
intravenous (IV) administration.
54: The method of claim 52, wherein the cancer is locally advanced
or metastatic.
55: The method of claim 52, wherein the cancer is selected from the
group consisting of a solid tumor, a hematologic cancer, bladder
cancer, brain cancer, breast cancer, colon cancer, colorectal
cancer, gastric cancer, glioma, head cancer, leukemia, liver
cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian
cancer, melanoma, pancreatic cancer, renal cancer, salivary cancer,
stomach cancer, thymic epithelial cancer, thyroid cancer, and
squamous cell carcinoma of the head and neck.
56: The method of claim 55, wherein the cancer is bladder
cancer.
57: The method of claim 55, wherein the cancer is lung cancer.
58: The method of claim 57, wherein the lung is non-small cell lung
cancer.
59: The method of claim 55, wherein the cancer is breast
cancer.
60: The method of claim 52, wherein the subject has PD-L1 positive
cancer.
61: The method of claim 52, wherein the formulation is to be
administered in conjunction with another therapeutic agent to the
subject.
62: The method of claim 61, wherein the therapeutic agent is a
chemotherapeutic agent or an antibody treatment.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Application is a divisional of U.S. patent application
Ser. No. 15/081,785 filed Mar. 25, 2016 which is a continuation of
PCT/US2014/057821, filed Sep. 26, 2014, which claims the priority
benefit of U.S. provisional application Ser. No. 61/883,953, filed
Sep. 27, 2013, the disclosures of each of which is incorporated
herein by reference in their entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
146392022010SEQLIST.TXT, date recorded: Nov. 18, 2020, size: 19
KB).
FIELD OF THE INVENTION
[0003] This invention relates to stable aqueous pharmaceutical
formulations comprising anti-PDL1 antibodies.
BACKGROUND OF THE INVENTION
[0004] The provision of two distinct signals to T-cells is a widely
accepted model for lymphocyte activation of resting T lymphocytes
by antigen-presenting cells (APCs). Lafferty et al, Aust. J. Exp.
Biol. Med. ScL 53: 27-42 (1975). This model further provides for
the discrimination of self from non-self and immune tolerance.
Bretscher et al, Science 169: 1042-1049 (1970); Bretscher, P. A.,
P.N.A.S. USA 96: 185-190 (1999); Jenkins et al, J. Exp. Med. 165:
302-319 (1987). The primary signal, or antigen specific signal, is
transduced through the T-cell receptor (TCR) following recognition
of foreign antigen peptide presented in the context of the major
histocompatibility-complex (MHC). The second or co-stimulatory
signal is delivered to T-cells by co-stimulatory molecules
expressed on antigen-presenting cells (APCs), and induce T-cells to
promote clonal expansion, cytokine secretion and effector function.
Lenschow et al., Ann. Rev. Immunol. 14:233 (1996). In the absence
of co-stimulation, T-cells can become refractory to antigen
stimulation, do not mount an effective immune response, and further
may result in exhaustion or tolerance to foreign antigens.
[0005] In the two-signal model T-cells receive both positive and
negative secondary co-stimulatory signals. The regulation of such
positive and negative signals is critical to maximize the host's
protective immune responses, while maintaining immune tolerance and
preventing autoimmunity. Negative secondary signals seem necessary
for induction of T-cell tolerance, while positive signals promote
T-cell activation. While the simple two-signal model still provides
a valid explanation for naive lymphocytes, a host's immune response
is a dynamic process, and co-stimulatory signals can also be
provided to antigen-exposed T-cells. The mechanism of
co-stimulation is of therapeutic interest because the manipulation
of co-stimulatory signals has shown to provide a means to either
enhance or terminate cell-based immune response. Recently, it has
been discovered that T cell dysfunction or anergy occurs
concurrently with an induced and sustained expression of the
inhibitory receptor, programmed death 1 polypeptide (PD-1). As a
result, therapeutic targeting of PD-1 and other molecules which
signal through interactions with PD-1, such as programmed death
ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) are an area
of intense interest.
[0006] PD-L1 is overexpressed in many cancers and is often
associated with poor prognosis (Okazaki T et al., Intern. Immun
2007 19(7):813) (Thompson R H et al., Cancer Res 2006, 66(7):3381).
Interestingly, the majority of tumor infiltrating T lymphocytes
predominantly express PD-1, in contrast to T lymphocytes in normal
tissues and peripheral blood T lymphocytes indicating that
up-regulation of PD-1 on tumor-reactive T cells can contribute to
impaired antitumor immune responses (Blood 2009 114(8):1537). This
may be due to exploitation of PD-L1 signaling mediated by PD-L1
expressing tumor cells interacting with PD-1 expressing T cells to
result in attenuation of T cell activation and evasion of immune
surveillance (Sharpe et al., Nat Rev 2002) (Keir M E et al., 2008
Annu. Rev. Immunol. 26:677). Therefore, inhibition of the
PD-L1/PD-1 interaction may enhance CD8+ T cell-mediated killing of
tumors.
[0007] Therapeutic targeting PD-1 and other molecules which signal
through interactions with PD-1, such as programmed death ligand 1
(PD-L1) and programmed death ligand 2 (PD-L2) are an area of
intense interest. The inhibition of PD-L1 signaling has been
proposed as a means to enhance T cell immunity for the treatment of
cancer (e.g., tumor immunity) and infection, including both acute
and chronic (e.g., persistent) infection. However, as an optimal
therapeutic directed to a target in this pathway has yet to be
commercialized, a significant unmet medical need exists.
[0008] All references cited herein, including patent applications,
patent publications, and UniProtKB/Swiss-Prot Accession numbers are
herein incorporated by reference in their entirety, as if each
individual reference were specifically and individually indicated
to be incorporated by reference.
SUMMARY OF THE INVENTION
[0009] Provided herein are stable aqueous pharmaceutical
formulations comprising an antibody. The formulation comprises an
antibody (e.g., an anti-PDL1 antibody), a buffer, sucrose, and a
surfactant, wherein the formulation has a pH of about 5.0 to about
7.0.
[0010] In one aspect, provided herein is a stable aqueous
pharmaceutical formulation, the formulation comprising an anti-PDL1
monoclonal antibody in a concentration of about 40 mg/ml to about
125 mg/ml, histidine acetate or sodium acetate in a concentration
of about 15 mM to about 25 mM, sucrose in a concentration of about
60 mM to about 240 mM, polysorbate in a concentration of about
0.005% (w/v) to about 0.06% (w/v), and pH about 5.0 to about
6.3.
[0011] In some embodiments, the monoclonal antibody in the
formulation is about 40 mg/ml to about 80 mg/ml. In some
embodiments, the monoclonal antibody in the formulation is about 54
mg/ml to about 66 mg/ml. In some embodiments, the monoclonal
antibody in the formulation is about 60 mg/ml. In some embodiments,
the monoclonal antibody in the formulation is about 60 mg/ml to
about 125 mg/ml. In some embodiments, the monoclonal antibody in
the formulation is about 125 mg/ml.
[0012] In some embodiments, said histidine acetate or sodium
acetate in the formulation is in a concentration of about 17 mM to
about 22 mM. In some embodiments, said histidine acetate or sodium
acetate in the formulation is in a concentration of about 20
mM.
[0013] In some embodiments, said sucrose in the formulation is
about 60 mM to about 180 mM. In some embodiments, said sucrose in
the formulation is about 120 mM. In some embodiments, said sucrose
in the formulation is about 240 mM.
[0014] In some embodiments, the formulation has a pH of about 5.5
to about 6.1. In some embodiments, the formulation has a pH of
about 5.5 or about 5.8.
[0015] In some embodiments, said polysorbate in the formulation is
polysorbate 20. In some embodiments, said polysorbate (e.g.,
polysorbate 20) in the formulation is about 0.02% to about
0.04%.
[0016] In some embodiments, said monoclonal antibody in the
formulation is about 60 mg/ml, sucrose in the formulation is about
120 mM, and pH is about 5.8. In some embodiments, said monoclonal
antibody in the formulation is about 125 mg/ml, sucrose in the
formulation is about 240 mM, and pH is about 5.5.
[0017] In some embodiments, the formulation comprises a monoclonal
antibody (e.g., an anti-PDL1 antibody described herein) in an
amount of about 60 mg/mL, histidine acetate in a concentration of
about 20 mM, sucrose in a concentration of about 120 mM, and
polysorbate which is polysorbate 20 in a concentration of 0.04%
(w/v), and the formulation has a pH of about 5.8.
[0018] In some embodiments, the formulation comprises a monoclonal
antibody in an amount of about 125 mg/mL, histidine acetate in a
concentration of about 20 mM, sucrose in a concentration of about
240 mM, and polysorbate which is polysorbate 20 in a concentration
of 0.02%, and the formulation has a pH of about 5.5.
[0019] In some embodiments, said monoclonal antibody in the
formulation is not subject to prior lyophilization. In some
embodiments, said monoclonal antibody in the formulation is a full
length antibody. In some embodiments, said monoclonal antibody in
the formulation is an IgG1 antibody. In some embodiments, said
monoclonal antibody in the formulation is a humanized antibody. In
some embodiments, said monoclonal antibody in the formulation is an
antibody fragment comprising an antigen-binding region. In some
embodiments, the antibody fragment is a Fab or F(ab').sub.2
fragment.
[0020] In some embodiments, said monoclonal antibody in the
formulation comprises
TABLE-US-00001 (a) a light chain variable region comprising: (1)
HVR-L1 comprising the amino acid sequence (SEQ ID NO: 1)
RASQDVSTAVA; (2) HVR-L2 comprising the amino acid sequence (SEQ ID
NO: 2) SASFLYS; (3) HVR-L3 comprising the amino acid sequence (SEQ
ID NO: 3) QQYLYHPAT; and (b) a heavy chain variable region
comprising: (1) HVR-H1 comprising the amino acid sequence (SEQ ID
NO: 4) GFTFSDSWIH; (2) HVR-H2 comprising the amino acid sequence
(SEQ ID NO: 5) AWISPYGGSTYYADSVKG; (3) HVR-H3 comprising the amino
acid sequence (SEQ ID NO: 6) RHWPGGFDY.
[0021] In some embodiments, said monoclonal antibody in the
formulation comprises a light chain variable region comprising the
amino acid sequence of SEQ ID NO:7, and a heavy chain variable
region comprising the amino acid sequence of SEQ ID NO:8. In some
embodiments, said monoclonal antibody in the formulation comprises
a light chain variable region having at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence
identity to the light chain variable region having the amino acid
sequence of SEQ ID NO:7, and a heavy chain variable region having
at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, or 99% identity to the heavy chain variable region
having the amino acid sequence of SEQ ID NO:8. In some embodiments,
said monoclonal antibody in the formulation comprises a light chain
variable region comprising the amino acid sequence of SEQ ID NO:7,
and a heavy chain variable region comprising the amino acid
sequence of SEQ ID NO:32. In some embodiments, said monoclonal
antibody in the formulation comprises a light chain variable region
having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% sequence identity to the light chain
variable region having the amino acid sequence of SEQ ID NO:7, and
a heavy chain variable region having at least 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
to the heavy chain variable region having the amino acid sequence
of SEQ ID NO:32. In some embodiments, said monoclonal antibody in
the formulation comprises a light chain comprising the amino acid
sequence of SEQ ID NO:9, and a heavy comprising the amino acid
sequence of SEQ ID NO:10. In some embodiments, said monoclonal
antibody in the formulation comprises a light chain having at least
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% sequence identity to the light chain having the amino
acid sequence of SEQ ID NO:9, and a heavy chain having at least
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to the heavy chain having the amino acid
sequence of SEQ ID NO:10.
[0022] In some embodiments, the formulation comprising the antibody
is stored in a glass vial or a metal alloy container. In some
embodiments, the metal alloy is 316L stainless steel or hastelloy.
In some embodiments, the formulation is stable at 2-8.degree. C.
for at least 6 months, at least 12 months, at least 18 months or at
least 24 months. In some embodiments, the antibody in the
formulation retains, after storage, at least about 75%, at least
about 80%, at least about 85%, at least about 90% of the biological
activity before storage. In some embodiments, the biological
activity is measured by antibody binding to PD-L1.
[0023] In some embodiments, the formulation described herein is
sterile. In some embodiments, the formulation described herein is
suitable to be administered to a subject. In some embodiments, the
formulation described herein is for intravenous (IV)
administration.
[0024] In another aspect, provided herein is an article of
manufacture or kit comprising a container holding any of the stable
aqueous pharmaceutical formulation described above and herein. In
some embodiments, the container is a glass vial or a metal alloy
container. In some embodiments, the metal alloy is 316L stainless
steel or hastelloy.
[0025] In another aspect, provided herein is a method of treating a
disease or disorder in a subject comprising administering an
effective amount of the formulation described herein to the
subject, wherein the disease or disorder is selected from the group
consisting of infection, cancer, and inflammatory disease.
[0026] It is to be understood that one, some, or all of the
properties of the various embodiments described herein may be
combined to form other embodiments of the present invention. These
and other aspects of the invention will become apparent to one of
skill in the art. These and other embodiments of the invention are
further described by the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1A and FIG. 1B are a series of graphs showing
statistical analysis of stability data of .alpha.-PDL1 formulations
at 40.degree. C. by ICIEF using JMP software. FIG. 1A) Average main
peak rate loss from fractional factorial design of experiments
(DOE). FIG. 1B) Main peak analysis from fractional factorial DOE.
Main peak contains .alpha.-PDL1 charged species with the same pH as
the pI (isoelectric point) of the molecule.
[0028] FIG. 2A and FIG. 2B are a series of graphs showing
statistical analysis of stability data of .alpha.-PDL1 formulations
at 25.degree. C. by ICIEF using JMP software. FIG. 2A) Average main
peak rate loss from fractional factorial design of experiments
(DOE). FIG. 2B) Main peak analysis from fractional factorial DOE.
Main peak contains .alpha.-PDL1 charged species with the same pH as
the pI (isoelectric point) of the molecule.
[0029] FIG. 3A and FIG. 3B are is a series of graphs showing
statistical analysis of stability data of .alpha.-PDL1 formulations
at 40.degree. C. by SE-HPLC using JMP software. FIG. 3A) Average
main peak rate loss from fractional factorial design of experiments
(DOE). FIG. 3B) Main peak analysis from fractional factorial DOE.
Main peak contains .alpha.-PDL1 monomer.
[0030] FIG. 4A and FIG. 4B are a series of graphs showing
statistical analysis of stability data of .alpha.-PDL1 formulations
at 25.degree. C. by SE-HPLC using JMP software. FIG. 4A) Average
main peak rate loss from fractional factorial design of experiments
(DOE). FIG. 4B) Main peak analysis from fractional factorial DOE.
Main peak contains .alpha.-PDL1 monomer.
[0031] FIG. 5 is a graph showing lack of significant PS20
degradation of various .alpha.-PDL1 formulations stored at various
temperatures and time. Graph of percent (%) PS20 remaining in the
formulation as detected by evaporative light scattering detector
(ELSD) in F1 through F10 formulations. a is time zero (TO); b is
40.degree. C., 1M; c is 25.degree. C., 2M; d is 5.degree. C., 2M; e
is 5.degree. C., 6M; f is 5.degree. C., 6M, 20 cc glass vial (GV),
high fill; and g is 5.degree. C., 6M, 20 cc glass vial (GV), low
fill.
[0032] FIGS. 6A-6D are a series of graphs showing stability of
.alpha.-PDL1 formulations stored at -20.degree. C. or 5.degree. C.
for up to 6 months in a glass vial (GV). FIG. 6A) Graph of percent
(%) monomer in formulations after five freeze thaw cycles during
storage at -20.degree. C. for the indicated time. FIG. 6B) Graph of
percent (%) monomer in formulations stored at 5.degree. C. for the
indicated time. FIG. 6C) Graph of percent (%) main peak obtained
from formulation after five freeze thaw cycles during storage at
-20.degree. C. for the indicated time. FIG. 6D) Graph of percent
(%) main peak obtained from formulation stored at 5.degree. C. for
the indicated time.
[0033] FIG. 7A and FIG. 7B are a series of graphs showing stability
of an .alpha.-PDL1 formulation after three freeze thaw cycles and
storage in a stainless steel or hastelloy minican. FIG. 7A) Graph
of percent (%) monomer in the formulation after storage at the
indicated temperature for 3 months. FIG. 7B) Graph of percent (%)
main peak in the formulation after storage at the indicated
temperature for 3 months.
[0034] FIG. 8A and FIG. 8B are a series of graphs showing stability
of an .alpha.-PDL1 formulation storage in a 20 cc vial. FIG. 8A)
Graph of percent (%) monomer in the formulation after storage at
the indicated temperature for 3 months. FIG. 8B) Graph of percent
(%) main peak in the formulation after storage at the indicated
temperature for 3 months.
[0035] FIG. 9A and FIG. 9B are a series of graphs showing stability
of .alpha.-PDL1 formulations containing various concentration of
PS20 when agitated in glass vials. FIG. 9A) Graph of percent (%)
monomer in formulations after agitation for the indicated time at
room temperature. FIG. 9B) Graph of turbidity as measured by
absorbance at 350 nm after agitation for the indicated time at room
temperature.
[0036] FIG. 10 is a graph showing stability of .alpha.-PDL1
formulations stored in glass vials for a period of time at the
indicated temperatures and then subjected to agitation. Percent
monomer change in formulations was measured by SEC.
[0037] FIG. 11A and FIG. 11B are is a series of graphs showing
comparability of .alpha.-PDL1 loss rate per week with increasing
pH. FIG. 11A) Graph of percent (%) monomer loss per week in the
formulation after storage at 40.degree. C. FIG. 11B) Graph of
percent (%) main peak loss per week in the formulation after
storage at 40.degree. C.
DETAILED DESCRIPTION
I. Definitions
[0038] Before describing the invention in detail, it is to be
understood that this invention is not limited to particular
compositions or biological systems, which can, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting. As used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "a molecule" optionally includes a
combination of two or more such molecules, and the like.
[0039] The term "about" as used herein refers to the usual error
range for the respective value readily known to the skilled person
in this technical field. Reference to "about" a value or parameter
herein includes (and describes) embodiments that are directed to
that value or parameter per se.
[0040] It is understood that aspects and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially of" aspects and embodiments.
[0041] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of the active ingredient to be effective, and which
contains no additional components which are unacceptably toxic to a
subject to which the formulation would be administered. Such
formulations are sterile. "Pharmaceutically acceptable" excipients
(vehicles, additives) are those which can reasonably be
administered to a subject mammal to provide an effective dose of
the active ingredient employed.
[0042] A "sterile" formulation is asceptic or free or essentially
free from all living microorganisms and their spores.
[0043] A "frozen" formulation is one at a temperature below
0.degree. C. Generally, the frozen formulation is not freeze-dried,
nor is it subjected to prior, or subsequent, lyophilization. In
certain embodiments, the frozen formulation comprises frozen drug
substance for storage (in stainless steel tank) or frozen drug
product (in final vial configuration).
[0044] A "stable" formulation is one in which the protein therein
essentially retains its physical stability and/or chemical
stability and/or biological activity upon storage. Preferably, the
formulation essentially retains its physical and chemical
stability, as well as its biological activity upon storage. The
storage period is generally selected based on the intended
shelf-life of the formulation. Various analytical techniques for
measuring protein stability are available in the art and are
reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee
Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones,
A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. Stability
can be measured at a selected temperature for a selected time
period. Stability can be evaluated qualitatively and/or
quantitatively in a variety of different ways, including evaluation
of aggregate formation (for example using size exclusion
chromatography, by measuring turbidity, and/or by visual
inspection); by assessing charge heterogeneity using cation
exchange chromatography, image capillary isoelectric focusing
(icIEF) or capillary zone electrophoresis; amino-terminal or
carboxy-terminal sequence analysis; mass spectrometric analysis;
SDS-PAGE analysis to compare reduced and intact antibody; peptide
map (for example tryptic or LYS-C) analysis; evaluating biological
activity or antigen binding function of the antibody; etc.
Instability may involve any one or more of: aggregation,
deamidation (e.g., Asn deamidation), oxidation (e.g., Met
oxidation), isomerization (e.g., Asp isomeriation),
clipping/hydrolysis/fragmentation (e.g., hinge region
fragmentation), succinimide formation, unpaired cysteine(s),
N-terminal extension, C-terminal processing, glycosylation
differences, etc.
[0045] A protein "retains its physical stability" in a
pharmaceutical formulation if it shows no signs or very little of
aggregation, precipitation and/or denaturation upon visual
examination of color and/or clarity, or as measured by UV light
scattering or by size exclusion chromatography.
[0046] A protein "retains its chemical stability" in a
pharmaceutical formulation, if the chemical stability at a given
time is such that the protein is considered to still retain its
biological activity as defined below. Chemical stability can be
assessed by detecting and quantifying chemically altered forms of
the protein. Chemical alteration may involve size modification
(e.g. clipping) which can be evaluated using size exclusion
chromatography, SDS-PAGE and/or matrix-assisted laser desorption
ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for
example. Other types of chemical alteration include charge
alteration (e.g. occurring as a result of deamidation) which can be
evaluated by ion-exchange chromatography or icIEF, for example.
[0047] An antibody "retains its biological activity" in a
pharmaceutical formulation, if the biological activity of the
antibody at a given time is at least about 60% (within the errors
of the assay) of the biological activity exhibited at the time the
pharmaceutical formulation was prepared as determined in an assay
(e.g., an antigen binding assay). Other "biological activity"
assays for antibodies are elaborated herein below.
[0048] As used herein, "biological activity" of a monoclonal
antibody includes the ability of the antibody to bind to antigen
and resulting in a measurable biological response which can be
measured in vitro or in vivo.
[0049] A "deamidated" monoclonal antibody herein is one in which
one or more asparagine residue thereof has been derivitized, e.g.
to an aspartic acid or an iso-aspartic acid.
[0050] An "oxidized" monoclonal antibody herein is one in which one
or more tryptophan residue and/or one or more methionine thereof
has been oxidized.
[0051] A "glycated" monoclonal antibody herein is one in which one
or more lysine residue thereof has been glycated.
[0052] An antibody which is "susceptible to deamidation" is one
comprising one or more residue, which has been found to be prone to
deamidate.
[0053] An antibody which is "susceptible to oxidation" is one
comprising one or more residue, which has been found to be prone to
oxidize.
[0054] An antibody which is "susceptible to aggregation" is one
which has been found to aggregate with other antibody molecule(s),
especially upon freezing and/or agitation.
[0055] An antibody which is "susceptible to fragmentation" is one
which has been found to be cleaved into two or more fragments, for
example at a hinge region thereof.
[0056] By "reducing deamidation, oxidation, aggregation, or
fragmentation" is intended preventing or decreasing the amount of
deamidation, oxidation, aggregation, or fragmentation relative to
the monoclonal antibody formulated in a different formulation.
[0057] The antibody which is formulated is preferably essentially
pure and desirably essentially homogeneous (e.g., free from
contaminating proteins etc.). "Essentially pure" antibody means a
composition comprising at least about 90% by weight of the
antibody, based on total weight of proteins in the composition,
preferably at least about 95% by weight. "Essentially homogeneous"
antibody means a composition comprising at least about 99% by
weight of antibody, based on total weight of proteins in the
composition.
[0058] By "isotonic" is meant that the formulation of interest has
essentially the same osmotic pressure as human blood. Isotonic
formulations generally have an osmotic pressure from about 250 to
350 mOsm. Isotonicity can be measured using a vapor pressure or
ice-freezing type osmometer, for example.
[0059] As used herein, "buffer" refers to a buffered solution that
resists changes in pH by the action of its acid-base conjugate
components. The buffer of this invention preferably has a pH in the
range from about 4.5 to about 7.0, preferably from about 5.6 to
about 7.0, for example from 5.6 to 6.9, 5.7 to 6.8, 5.8 to 6.7, 5.9
to 6.6, 5.9 to 6.5, 6.0, 6.0 to 6.4, or 6.1 to 6.3. In one
embodiment the buffer has a pH 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2,
6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0. For example, sodium
phosphate is an example of buffers that will control the pH in this
range.
[0060] As used herein, a "surfactant" refers to a surface-active
agent, preferably a nonionic surfactant. Examples of surfactants
herein include polysorbate (for example, polysorbate 20 and,
polysorbate 80); poloxamer (e.g. poloxamer 188); Triton; sodium
dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl
glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine;
lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-,
myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,
linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-betaine (e.g. lauroamidopropyl);
myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.); polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc); etc. In one embodiment, the surfactant herein
is polysorbate 20.
[0061] In a pharmacological sense, in the context of the invention,
a "therapeutically effective amount" of an antibody refers to an
amount effective in the prevention or treatment of a disorder for
the treatment of which the antibody is effective. A "disorder" is
any condition that would benefit from treatment with the antibody.
This includes chronic and acute disorders or diseases including
those pathological conditions which predispose the mammal to the
disorder in question.
[0062] A "preservative" is a compound which can be optionally
included in the formulation to essentially reduce bacterial action
therein, thus facilitating the production of a multi-use
formulation, for example. Examples of potential preservatives
include octadecyldimethylbenzyl ammonium chloride, hexamethonium
chloride, benzalkonium chloride (a mixture of
alkylbenzyldimethylammonium chlorides in which the alkyl groups are
long-chain compounds), and benzethonium chloride. Other types of
preservatives include aromatic alcohols such as phenol, butyl and
benzyl alcohol, alkyl parabens such as methyl or propyl paraben,
catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. In
one embodiment, the preservative herein is benzyl alcohol.
[0063] As used herein, the term "treatment" refers to clinical
intervention designed to alter the natural course of the individual
or cell being treated during the course of clinical pathology.
Desirable effects of treatment include decreasing the rate of
disease progression, ameliorating or palliating the disease state,
and remission or improved prognosis. For example, an individual is
successfully "treated" if one or more symptoms associated with
cancer are mitigated or eliminated, including, but are not limited
to, reducing the proliferation of (or destroying) cancerous cells,
decreasing symptoms resulting from the disease, increasing the
quality of life of those suffering from the disease, decreasing the
dose of other medications required to treat the disease, delaying
the progression of the disease, and/or prolonging survival of
individuals.
[0064] As used herein, "delaying progression of a disease" means to
defer, hinder, slow, retard, stabilize, and/or postpone development
of the disease (such as cancer). This delay can be of varying
lengths of time, depending on the history of the disease and/or
individual being treated. As is evident to one skilled in the art,
a sufficient or significant delay can, in effect, encompass
prevention, in that the individual does not develop the disease.
For example, a late stage cancer, such as development of
metastasis, may be delayed.
[0065] An "effective amount" is at least the minimum amount
required to effect a measurable improvement or prevention of a
particular disorder. An effective amount herein may vary according
to factors such as the disease state, age, sex, and weight of the
patient, and the ability of the antibody to elicit a desired
response in the individual. An effective amount is also one in
which any toxic or detrimental effects of the treatment are
outweighed by the therapeutically beneficial effects. For
prophylactic use, beneficial or desired results include results
such as eliminating or reducing the risk, lessening the severity,
or delaying the onset of the disease, including biochemical,
histological and/or behavioral symptoms of the disease, its
complications and intermediate pathological phenotypes presenting
during development of the disease. For therapeutic use, beneficial
or desired results include clinical results such as decreasing one
or more symptoms resulting from the disease, increasing the quality
of life of those suffering from the disease, decreasing the dose of
other medications required to treat the disease, enhancing effect
of another medication such as via targeting, delaying the
progression of the disease, and/or prolonging survival. In the case
of cancer or tumor, an effective amount of the drug may have the
effect in reducing the number of cancer cells; reducing the tumor
size; inhibiting (i.e., slow to some extent or desirably stop)
cancer cell infiltration into peripheral organs; inhibit (i.e.,
slow to some extent and desirably stop) tumor metastasis;
inhibiting to some extent tumor growth; and/or relieving to some
extent one or more of the symptoms associated with the disorder. An
effective amount can be administered in one or more
administrations. For purposes of this invention, an effective
amount of drug, compound, or pharmaceutical composition is an
amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective amount of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective amount" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0066] As used herein, "in conjunction with" refers to
administration of one treatment modality in addition to another
treatment modality. As such, "in conjunction with" refers to
administration of one treatment modality before, during, or after
administration of the other treatment modality to the
individual.
[0067] A "disorder" is any condition that would benefit from
treatment including, but not limited to, chronic and acute
disorders or diseases including those pathological conditions which
predispose the mammal to the disorder in question.
[0068] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer. In one embodiment, the cell
proliferative disorder is a tumor.
[0069] "Tumor," as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer",
"cancerous", "cell proliferative disorder", "proliferative
disorder" and "tumor" are not mutually exclusive as referred to
herein.
[0070] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include, but not limited to, squamous cell cancer (e.g., epithelial
squamous cell cancer), lung cancer including small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung and
squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer and gastrointestinal stromal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, cancer of the urinary tract,
hepatoma, breast cancer, colon cancer, rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,
melanoma, superficial spreading melanoma, lentigo maligna melanoma,
acral lentiginous melanomas, nodular melanomas, multiple myeloma
and B-cell lymphoma (including low grade/follicular non-Hodgkin's
lymphoma (NHL); small lymphocytic (SL) NHL; intermediate
grade/follicular NHL; intermediate grade diffuse NHL; high grade
immunoblastic NHL; high grade lymphoblastic NHL; high grade small
non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;
AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia);
chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia
(ALL); hairy cell leukemia; chronic myeloblastic leukemia; and
post-transplant lymphoproliferative disorder (PTLD), as well as
abnormal vascular proliferation associated with phakomatoses, edema
(such as that associated with brain tumors), Meigs' syndrome,
brain, as well as head and neck cancer, and associated metastases.
In certain embodiments, cancers that are amenable to treatment by
the antibodies of the invention include breast cancer, colorectal
cancer, rectal cancer, non-small cell lung cancer, glioblastoma,
non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer,
liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi's
sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer,
mesothelioma, and multiple myeloma. In some embodiments, the cancer
is selected from: small cell lung cancer, gliblastoma,
neuroblastomas, melanoma, breast carcinoma, gastric cancer,
colorectal cancer (CRC), and hepatocellular carcinoma. Yet, in some
embodiments, the cancer is selected from: non-small cell lung
cancer, colorectal cancer, glioblastoma and breast carcinoma,
including metastatic forms of those cancers.
[0071] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and cyclosphosphamide
(CYTOXAN.RTM.); alkyl sulfonates such as busulfan, improsulfan and
piposulfan; aziridines such as benzodopa, carboquone, meturedopa,
and uredopa; ethylenimines and methylamelamines including
altretamine, triethylenemelamine, triethylenephosphoramide,
triethylenethiophosphoramide and trimethylomelamine; acetogenins
(especially bullatacin and bullatacinone);
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
chlorophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma1I and calicheamicin omegaIl (see, e.g., Nicolaou et al.,
Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral
alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including ADRIAMYCIN.RTM., morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin
HCl liposome injection (DOXIL.RTM.), liposomal doxorubicin TLC D-99
(MYOCET.RTM.), peglylated liposomal doxorubicin (CAELYX.RTM.), and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate, gemcitabine (GEMZAR.RTM.), tegafur (UFTORAL.RTM.),
capecitabine (XELODA.RTM.), an epothilone, and 5-fluorouracil
(5-FU); combretastatin; folic acid analogues such as denopterin,
methotrexate, pteropterin, trimetrexate; purine analogs such as
fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine
analogs such as ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM.
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine (ELDISINE.RTM., FILDESIN.RTM.); dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclitaxel
(TAXOL.RTM., Bristol-Myers Squibb Oncology, Princeton, N.J.),
albumin-engineered nanoparticle formulation of paclitaxel
(ABRAXANE.TM.), and docetaxel (TAXOTERE.RTM., Rhome-Poulene Rorer,
Antony, France); chloranbucil; 6-thioguanine; mercaptopurine;
methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,
ELOXATIN.RTM.), and carboplatin; vincas, which prevent tubulin
polymerization from forming microtubules, including vinblastine
(VELBAN.RTM.), vincristine (ONCOVIN.RTM.), vindesine
(ELDISINE.RTM., FILDESIN.RTM.), and vinorelbine (NAVELBINE.RTM.);
etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin;
novantrone; edatrexate; daunomycin; aminopterin; ibandronate;
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid, including bexarotene
(TARGRETIN.RTM.); bisphosphonates such as clodronate (for example,
BONEFOS.RTM. or OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095,
zoledronic acid/zoledronate (ZOMETA.RTM.), alendronate
(FOSAMAX.RTM.), pamidronate (AREDIA.RTM.), tiludronate
(SKELID.RTM.), or risedronate (ACTONEL.RTM.); troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); antisense
oligonucleotides, particularly those that inhibit expression of
genes in signaling pathways implicated in aberrant cell
proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and
epidermal growth factor receptor (EGF-R) (e.g., erlotinib
(Tarceva.TM.)); and VEGF-A that reduce cell proliferation; vaccines
such as THERATOPE.RTM. vaccine and gene therapy vaccines, for
example, ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and
VAXID.RTM. vaccine; topoisomerase 1 inhibitor (e.g.,
LURTOTECAN.RTM.); rmRH (e.g., ABARELIX.RTM.); BAY439006 (sorafenib;
Bayer); SU-11248 (sunitinib, SUTENT.RTM., Pfizer); perifosine,
COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome
inhibitor (e.g. PS341); bortezomib (VELCADE.RTM.); CCI-779;
tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as
oblimersen sodium (GENASENSE.RTM.); pixantrone; EGFR inhibitors;
tyrosine kinase inhibitors; serine-threonine kinase inhibitors such
as rapamycin (sirolimus, RAPAMUNE.RTM.); farnesyltransferase
inhibitors such as lonafarnib (SCH 6636, SARASAR.TM.); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above; as well as combinations of two or more of the above such
as CHOP, an abbreviation for a combined therapy of
cyclophosphamide, doxorubicin, vincristine, and prednisolone; and
FOLFOX, an abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovorin, and
pharmaceutically acceptable salts, acids or derivatives of any of
the above; as well as combinations of two or more of the above.
[0072] Chemotherapeutic agents as defined herein include
"anti-hormonal agents" or "endocrine therapeutics" which act to
regulate, reduce, block, or inhibit the effects of hormones that
can promote the growth of cancer. They may be hormones themselves,
including, but not limited to: anti-estrogens and selective
estrogen receptor modulators (SERMs), including, for example,
tamoxifen (including NOLVADEX.RTM. tamoxifen), raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.cndot.toremifene; aromatase inhibitors
that inhibit the enzyme aromatase, which regulates estrogen
production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, MEGASE.RTM. megestrol acetate,
AROMASIN.RTM. exemestane, formestanie, fadrozole, RIVISOR.RTM.
vorozole, FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole; and
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); antisense
oligonucleotides, particularly those which inhibit expression of
genes in signaling pathways implicated in abherant cell
proliferation, such as, for example, PKC-alpha, Raf and H-Ras;
ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME.RTM.
ribozyme) and a HER2 expression inhibitor; vaccines such as gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; PROLEUKIN.RTM.
rIL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; Vinorelbine and Esperamicins (see U.S. Pat. No. 4,675,187),
and pharmaceutically acceptable salts, acids or derivatives of any
of the above; as well as combinations of two or more of the
above.
[0073] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell either in
vitro or in vivo. In one embodiment, growth inhibitory agent is
growth inhibitory antibody that prevents or reduces proliferation
of a cell expressing an antigen to which the antibody binds. In
another embodiment, the growth inhibitory agent may be one which
significantly reduces the percentage of cells in S phase. Examples
of growth inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vincas (vincristine and vinblastine), taxanes, and
topoisomerase II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. Those agents that arrest G1
also spill over into S-phase arrest, for example, DNA alkylating
agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in Mendelsohn and Israel, eds., The
Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic drugs" by Murakami et al.
(W.B. Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes
(paclitaxel and docetaxel) are anticancer drugs both derived from
the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer),
derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0074] By "radiation therapy" is meant the use of directed gamma
rays or beta rays to induce sufficient damage to a cell so as to
limit its ability to function normally or to destroy the cell
altogether. It will be appreciated that there will be many ways
known in the art to determine the dosage and duration of treatment.
Typical treatments are given as a one-time administration and
typical dosages range from 10 to 200 units (Grays) per day.
[0075] A "subject" or an "individual" for purposes of treatment
refers to any animal classified as a mammal, including humans,
domestic and farm animals, and zoo, sports, or pet animals, such as
dogs, horses, cats, cows, etc. Preferably, the mammal is human.
[0076] The term "antibody" herein is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired biological activity.
[0077] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with research, diagnostic or
therapeutic uses for the antibody, and may include enzymes,
hormones, and other proteinaceous or nonproteinaceous solutes. In
some embodiments, an antibody is purified (1) to greater than 95%
by weight of antibody as determined by, for example, the Lowry
method, and in some embodiments, to greater than 99% by weight; (2)
to a degree sufficient to obtain at least 15 residues of N-terminal
or internal amino acid sequence by use of, for example, a spinning
cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using, for example, Coomassie blue or silver
stain. Isolated antibody includes the antibody in situ within
recombinant cells since at least one component of the antibody's
natural environment will not be present. Ordinarily, however,
isolated antibody will be prepared by at least one purification
step.
[0078] "Native antibodies" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies among the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (V.sub.H) followed by
a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end;
the constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed to form an interface
between the light chain and heavy chain variable domains.
[0079] The term "constant domain" refers to the portion of an
immunoglobulin molecule having a more conserved amino acid sequence
relative to the other portion of the immunoglobulin, the variable
domain, which contains the antigen binding site. The constant
domain contains the C.sub.H1, C.sub.H2 and C.sub.H3 domains
(collectively, CH) of the heavy chain and the CHL (or CL) domain of
the light chain.
[0080] The "variable region" or "variable domain" of an antibody
refers to the amino-terminal domains of the heavy or light chain of
the antibody. The variable domain of the heavy chain may be
referred to as "V.sub.H." The variable domain of the light chain
may be referred to as "V.sub.L." These domains are generally the
most variable parts of an antibody and contain the antigen-binding
sites.
[0081] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are used in the binding and specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed throughout the variable
domains of antibodies. It is concentrated in three segments called
hypervariable regions (HVRs) both in the light-chain and the
heavy-chain variable domains. The more highly conserved portions of
variable domains are called the framework regions (FR). The
variable domains of native heavy and light chains each comprise
four FR regions, largely adopting a beta-sheet configuration,
connected by three HVRs, which form loops connecting, and in some
cases forming part of, the beta-sheet structure. The HVRs in each
chain are held together in close proximity by the FR regions and,
with the HVRs from the other chain, contribute to the formation of
the antigen-binding site of antibodies (see Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, National
Institute of Health, Bethesda, Md. (1991)). The constant domains
are not involved directly in the binding of an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody-dependent cellular
toxicity.
[0082] The "light chains" of antibodies (immunoglobulins) from any
mammalian species can be assigned to one of two clearly distinct
types, called kappa (".kappa.") and lambda (".lamda."), based on
the amino acid sequences of their constant domains.
[0083] The term IgG "isotype" or "subclass" as used herein is meant
any of the subclasses of immunoglobulins defined by the chemical
and antigenic characteristics of their constant regions.
[0084] Depending on the amino acid sequences of the constant
domains of their heavy chains, antibodies (immunoglobulins) can be
assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these
may be further divided into subclasses (isotypes), e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and IgA.sub.2. The
heavy chain constant domains that correspond to the different
classes of immunoglobulins are called .alpha., .gamma., , .gamma.,
and .mu., respectively. The subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known and described generally in, for
example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B.
Saunders, Co., 2000). An antibody may be part of a larger fusion
molecule, formed by covalent or non-covalent association of the
antibody with one or more other proteins or peptides.
[0085] The terms "full length antibody," "intact antibody" and
"whole antibody" are used herein interchangeably to refer to an
antibody in its substantially intact form, not antibody fragments
as defined below. The terms particularly refer to an antibody with
heavy chains that contain an Fc region.
[0086] A "naked antibody" for the purposes herein is an antibody
that is not conjugated to a cytotoxic moiety or radiolabel.
[0087] "Antibody fragments" comprise a portion of an intact
antibody, preferably comprising the antigen binding region thereof.
In some embodiments, the antibody fragment described herein is an
antigen-binding fragment. Examples of antibody fragments include
Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies; linear
antibodies; single-chain antibody molecules; and multispecific
antibodies formed from antibody fragments.
[0088] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab').sub.2 fragment that has two antigen-combining
sites and is still capable of cross-linking antigen.
[0089] "Fv" is the minimum antibody fragment which contains a
complete antigen-binding site. In one embodiment, a two-chain Fv
species consists of a dimer of one heavy- and one light-chain
variable domain in tight, non-covalent association. In a
single-chain Fv (scFv) species, one heavy- and one light-chain
variable domain can be covalently linked by a flexible peptide
linker such that the light and heavy chains can associate in a
"dimeric" structure analogous to that in a two-chain Fv species. It
is in this configuration that the three HVRs of each variable
domain interact to define an antigen-binding site on the surface of
the VH-VL dimer. Collectively, the six HVRs confer antigen-binding
specificity to the antibody. However, even a single variable domain
(or half of an Fv comprising only three HVRs specific for an
antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding site.
[0090] The Fab fragment contains the heavy- and light-chain
variable domains and also contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0091] "Single-chain Fv" or "scFv" antibody fragments comprise the
VH and VL domains of antibody, wherein these domains are present in
a single polypeptide chain. Generally, the scFv polypeptide further
comprises a polypeptide linker between the VH and VL domains which
enables the scFv to form the desired structure for antigen binding.
For a review of scFv, see, e.g., Pluckthun, in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York, 1994), pp. 269-315.
[0092] The term "diabodies" refers to antibody fragments with two
antigen-binding sites, which fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain
(VL) in the same polypeptide chain (VH-VL). By using a linker that
is too short to allow pairing between the two domains on the same
chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites.
Diabodies may be bivalent or bispecific. Diabodies are described
more fully in, for example, EP 404,097; WO 1993/01161; Hudson et
al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl.
Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are
also described in Hudson et al., Nat. Med. 9:129-134 (2003).
[0093] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, e.g., the individual antibodies comprising the
population are identical except for possible mutations, e.g.,
naturally occurring mutations, that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies. In
certain embodiments, such a monoclonal antibody typically includes
an antibody comprising a polypeptide sequence that binds a target,
wherein the target-binding polypeptide sequence was obtained by a
process that includes the selection of a single target binding
polypeptide sequence from a plurality of polypeptide sequences. For
example, the selection process can be the selection of a unique
clone from a plurality of clones, such as a pool of hybridoma
clones, phage clones, or recombinant DNA clones. It should be
understood that a selected target binding sequence can be further
altered, for example, to improve affinity for the target, to
humanize the target binding sequence, to improve its production in
cell culture, to reduce its immunogenicity in vivo, to create a
multispecific antibody, etc., and that an antibody comprising the
altered target binding sequence is also a monoclonal antibody of
this invention. In contrast to polyclonal antibody preparations,
which typically include different antibodies directed against
different determinants (epitopes), each monoclonal antibody of a
monoclonal antibody preparation is directed against a single
determinant on an antigen. In addition to their specificity,
monoclonal antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins.
[0094] The modifier "monoclonal" indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the
invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler and Milstein, Nature,
256:495-97 (1975); Hongo et al., Hybridoma, 14 (3): 253-260 (1995),
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)),
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),
phage-display technologies (see, e.g., Clackson et al., Nature,
352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597
(1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et
al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl.
Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J.
Immunol. Methods 284(1-2): 119-132 (2004), and technologies for
producing human or human-like antibodies in animals that have parts
or all of the human immunoglobulin loci or genes encoding human
immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096;
WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad.
Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258
(1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
U.S. Pat. No. 5,661,016; Marks et al., Bio/Technology 10: 779-783
(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison,
Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14:
845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and
Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[0095] The monoclonal antibodies herein specifically include
"chimeric" antibodies in which a portion of the heavy and/or light
chain is identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc.
Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies
include PRIMATTZED.RTM. antibodies wherein the antigen-binding
region of the antibody is derived from an antibody produced by,
e.g., immunizing macaque monkeys with the antigen of interest.
[0096] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. In one embodiment, a humanized antibody
is a human immunoglobulin (recipient antibody) in which residues
from a HVR of the recipient are replaced by residues from a HVR of
a non-human species (donor antibody) such as mouse, rat, rabbit, or
nonhuman primate having the desired specificity, affinity, and/or
capacity. In some instances, FR residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies may comprise residues that are
not found in the recipient antibody or in the donor antibody. These
modifications may be made to further refine antibody performance.
In general, a humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin, and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally will also comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see, e.g., Jones et al.,
Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329
(1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See
also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma &
Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions
23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433
(1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.
[0097] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Human antibodies can be
produced using various techniques known in the art, including
phage-display libraries. Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also
available for the preparation of human monoclonal antibodies are
methods described in Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr.
Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be
prepared by administering the antigen to a transgenic animal that
has been modified to produce such antibodies in response to
antigenic challenge, but whose endogenous loci have been disabled,
e.g., immunized xenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and
6,150,584 regarding XENOMOUSE.TM. technology). See also, for
example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562
(2006) regarding human antibodies generated via a human B-cell
hybridoma technology.
[0098] A "species-dependent antibody" is one which has a stronger
binding affinity for an antigen from a first mammalian species than
it has for a homologue of that antigen from a second mammalian
species. Normally, the species-dependent antibody "binds
specifically" to a human antigen (e.g., has a binding affinity (Kd)
value of no more than about 1.times.10.sup.-7 M, preferably no more
than about 1.times.10.sup.-8 M and preferably no more than about
1.times.10.sup.-9 M) but has a binding affinity for a homologue of
the antigen from a second nonhuman mammalian species which is at
least about 50 fold, or at least about 500 fold, or at least about
1000 fold, weaker than its binding affinity for the human antigen.
The species-dependent antibody can be any of the various types of
antibodies as defined above, but preferably is a humanized or human
antibody.
[0099] The term "hypervariable region," "HVR," or "HV," when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six HVRs; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). In native
antibodies, H3 and L3 display the most diversity of the six HVRs,
and H3 in particular is believed to play a unique role in
conferring fine specificity to antibodies. See, e.g., Xu et al.,
Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular
Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003).
Indeed, naturally occurring camelid antibodies consisting of a
heavy chain only are functional and stable in the absence of light
chain. See, e.g., Hamers-Casterman et al., Nature 363:446-448
(1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).
[0100] A number of HVR delineations are in use and are encompassed
herein. The Kabat Complementarity Determining Regions (CDRs) are
based on sequence variability and are the most commonly used (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Chothia refers instead to the location of the structural
loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM
HVRs represent a compromise between the Kabat HVRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software. The "contact" HVRs are based on an analysis of
the available complex crystal structures. The residues from each of
these HVRs are noted below.
TABLE-US-00002 Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34
L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97
L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B
(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia
Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102
H96-H101 H93-H101
[0101] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34
(L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and
26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3)
in the VH. The variable domain residues are numbered according to
Kabat et al., supra, for each of these definitions.
[0102] "Framework" or "FR" residues are those variable domain
residues other than the HVR residues as herein defined.
[0103] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat," and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., supra. Using this numbering system,
the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or HVR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc. according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0104] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g.,
Kabat et al., Sequences of Immunological Interest. 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The "EU numbering system" or "EU index" is generally used
when referring to a residue in an immunoglobulin heavy chain
constant region (e.g., the EU index reported in Kabat et al.,
supra). The "EU index as in Kabat" refers to the residue numbering
of the human IgG1 EU antibody.
[0105] The expression "linear antibodies" refers to the antibodies
described in Zapata et al. (1995 Protein Eng, 8(10):1057-1062).
Briefly, these antibodies comprise a pair of tandem Fd segments
(VH-CH1-VH-CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions. Linear
antibodies can be bispecific or monospecific.
[0106] As use herein, the term "specifically binds to" or is
"specific for" refers to measurable and reproducible interactions
such as binding between a target and an antibody, which is
determinative of the presence of the target in the presence of a
heterogeneous population of molecules including biological
molecules. For example, an antibody that specifically binds to a
target (which can be an epitope) is an antibody that binds this
target with greater affinity, avidity, more readily, and/or with
greater duration than it binds to other targets. In one embodiment,
the extent of binding of an antibody to an unrelated target is less
than about 10% of the binding of the antibody to the target as
measured, e.g., by a radioimmunoassay (RIA). In certain
embodiments, an antibody that specifically binds to a target has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM. In certain
embodiments, an antibody specifically binds to an epitope on a
protein that is conserved among the protein from different species.
In another embodiment, specific binding can include, but does not
require exclusive binding.
II. Antibody Formulations and Preparation
[0107] The invention herein relates to stable aqueous formulations
comprising an antibody, such as an anti-PDL1 antibody. In some
embodiments, the formulation comprises an antibody (e.g., a
monoclonal antibody), sucrose, a buffer, and a surfactant, wherein
the formulation has a pH of about 5.0 to about 7.0. In some
embodiments, the antibody (e.g., an anti-PDL1 antibody described
herein) in the formulation is in an amount of about 40 mg/ml to
about 125 mg/ml. In some embodiments, the buffer is histidine
(e.g., histidine acetate) or sodium acetate. In some embodiments,
the buffer in the formulation is in a concentration of about 15 mM
to about 25 mM. In some embodiments, sucrose in the formulation is
about 60 mM to about 240 mM. In some embodiments, the surfactant in
the formulation is polysorbate (e.g, polysorbate 20). In some
embodiments, polysorbate in the formulation is in a concentration
of about 0.005% (w/v) to about 0.06% (w/v). In some embodiments,
the formulation has a pH of about 5.0 to about 6.3. In some
embodiments, provided herein is stable aqueous pharmaceutical
formulation, the formulation comprising an anti-PDL1 monoclonal
antibody in a concentration of about 40 mg/ml to about 125 mg/ml,
histidine acetate or sodium acetate in a concentration of about 15
mM to about 25 mM, sucrose in a concentration of about 60 mM to
about 240 mM, polysorbate in a concentration of about 0.005% (w/v)
to about 0.06% (w/v), and pH about 5.0 to about 6.3. In some
embodiments, the formulation comprises an anti-PDL1 monoclonal
antibody in amount of about 125 mg/ml, sucrose in a concentration
of about 240 mM, and pH of about 5.5. In some embodiments, the
formulation comprises an anti-PDL1 monoclonal antibody in amount of
about 60 mg/ml, sucrose in a concentration of about 120 mM, and pH
of about 5.8.
[0108] In some embodiments, the antibody in the formulation is
stable at -20.degree. C. for at least about 6 months, at least
about 12 months, at least about 18 months, at least two years, at
least three years, or at least four years. In some embodiments, the
antibody in the formulation is stable at 2-8.degree. C. for at
least about 6 months, at least about 12 months, at least about 18
months, at least two years, or at least three years. In some
embodiments, after storage, the antibody retains at least about
60%, at least about 65%, at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, or at
least about 95% of its biological activity (e.g., binding to the
target, or therapeutic potency) exhibited before storage, i.e., at
the time the pharmaceutical formulation was prepared.
[0109] In certain embodiments, the formulation is stable at about
40.degree. C. for at least about 1, 2, 3, 4, 5, 6, 7, 14, 21, 28,
or more days. In certain embodiments, the formulation is stable at
about 40.degree. C. for at least about 1, 2, 3, 4, 5, 6, 7, 8, or
more weeks. In certain embodiments, the formulation is stable at
about 25.degree. C. for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months.
In certain embodiments, the formulation is stable at about
5.degree. C. for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months. In
certain embodiments, the formulation is stable at about -20.degree.
C. for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or
more months. In certain embodiments, the formulation is stable at
5.degree. C. or -20.degree. C. for at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, or more months. Furthermore, the
formulation is preferably stable following freezing (to, e.g.,
-20.degree. C., -40.degree. C. or -70.degree. C.) and thawing of
the formulation, for example following 1, 2 3, 4, or 5 cycles of
freezing and thawing.
[0110] A. Antibodies (Such as Anti-PDL1 Antibodies)
[0111] In some embodiments, the antibody in the formulation
comprises at least one tryptophan (e.g., at least two, at least
three, or at least four) in the heavy and/or light chain sequence.
In some embodiments, amino acid tryptophan is in the CDR regions,
framework regions and/or constant regions of the antibody. In some
embodiments, the antibody comprises two or three tryptophan
residues in the CDR regions. In some embodiments, the antibody in
the formulation is an anti-PDL1 antibody. PD-L1 (programmed cell
death 1 ligand 1), also known as PDL1, B7-H1, B7-4, CD274, and
B7-H, is a transmembrane protein, and its interaction with PD-1
inhibits T-cell activation and cytokine production. In some
embodiments, the anti-PDL1 antibody described herein binds to human
PD-L1. Examples of anti-PDL1 antibodies that can be formulated
using the formulations described herein are described in PCT patent
application WO 2010/077634 A1 and U.S. Pat. No. 8,217,149, which
are incorporated herein by reference.
[0112] In some embodiments, the anti-PDL1 antibody is capable of
inhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and
B7-1. In some embodiments, the anti-PDL1 antibody is a monoclonal
antibody. In some embodiments, the anti-PDL1 antibody is an
antibody fragment selected from the group consisting of Fab,
Fab'-SH, Fv, scFv, and (Fab').sub.2 fragments. In some embodiments,
the anti-PDL1 antibody is a humanized antibody. In some
embodiments, the anti-PDL1 antibody is a human antibody.
[0113] Anti-PDL1 antibodies described in WO 2010/077634 A1 and U.S.
Pat. No. 8,217,149 may be formulated in the formulations described
herein. In some embodiments, the anti-PDL1 antibody comprises a
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO:30 and a light chain variable region comprising the amino
acid sequence of SEQ ID NO:31.
[0114] In one embodiment, the anti-PDL1 antibody comprises a heavy
chain variable region polypeptide comprising an HVR-H1, HVR-H2 and
HVR-H3 sequence, wherein:
TABLE-US-00003 (a) the HVR-H1 sequence is (SEQ ID NO: 11)
GFTFSX.sub.1SWIH; (b) the HVR-H2 sequence is (SEQ ID NO: 12)
AWIX.sub.2PYGGSX.sub.3YYADSVKG; (c) the HVR-H3 sequence is (SEQ ID
NO: 13) RHWPGGPDY;
[0115] further wherein: X.sub.1 is D or G; X.sub.2 is S or L;
X.sub.3 is T or S.
[0116] In one specific aspect, X.sub.1 is D; X.sub.2 is S and
X.sub.3 is T. In another aspect, the polypeptide further comprises
variable region heavy chain framework sequences juxtaposed between
the HVRs according to the formula:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a further aspect, the framework
sequences are VH subgroup III consensus framework. In a still
further aspect, at least one of the framework sequences is the
following:
TABLE-US-00004 HC-FR1 is (SEQ ID NO: 14) EVQLVESGGGLVQPGGSLRLSCAAS
HC-FR2 is (SEQ ID NO: 15) WVRQAPGKGLEWV HC-FR3 is (SEQ ID NO: 16)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 is (SEQ ID NO: 17)
WGQGTLVTVSA.
[0117] In a still further aspect, the heavy chain polypeptide is
further combined with a variable region light chain comprising an
HVR-L1, HVR-L2 and HVR-L3, wherein:
TABLE-US-00005 (a) the HVR-L1 sequence is (SEQ ID NO: 18)
RASQX.sub.4X.sub.5X.sub.6TX.sub.7X.sub.8A; (b) the HVR-L2 sequence
is (SEQ ID NO: 19) SASX.sub.9LX.sub.10S; (c) the HVR-L3 sequence is
(SEQ ID NO: 20) QQX.sub.11X.sub.12X.sub.13X.sub.14PX.sub.15T;
[0118] further wherein: X.sub.4 is D or V; X.sub.5 is V or I;
X.sub.6 is S or N; X.sub.7 is A or F; X.sub.8 is V or L; X.sub.9 is
F or T; X.sub.10 is Y or A; X.sub.11 is Y, G, F, or S; X.sub.12 is
L, Y, F or W; X.sub.13 is Y, N, A, T, G, F or I; X.sub.14 is H, V,
P, T or I; X.sub.15 is A, W, R, P or T.
[0119] In a still further aspect, X.sub.4 is D; X.sub.5 is V;
X.sub.6 is 5; X.sub.7 is A; X.sub.8 is V; X.sub.9 is F; X.sub.10 is
Y; X.sub.11 is Y; X.sub.12 is L; X.sub.13 is Y; X.sub.14 is H;
X.sub.15 is A. In a still further aspect, the light chain further
comprises variable region light chain framework sequences
juxtaposed between the HVRs according to the formula:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
a still further aspect, the framework sequences are derived from
human consensus framework sequences. In a still further aspect, the
framework sequences are VL kappa I consensus framework. In a still
further aspect, at least one of the framework sequence is the
following:
TABLE-US-00006 LC-FR1 is (SEQ ID NO: 21) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 is (SEQ ID NO: 22) WYQQKPGKAPKLLIY LC-FR3 is (SEQ ID NO: 23)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 is (SEQ ID NO: 24)
FGQGTKVEIKR.
[0120] In another embodiment, provided is an isolated anti-PDL1
antibody or antigen binding fragment comprising a heavy chain and a
light chain variable region sequence, wherein:
TABLE-US-00007 (a) the heavy chain comprises and HVR-H1, HVR-H2 and
HVR-H3, wherein further: (i) the HVR-H1 sequence is (SEQ ID NO: 11)
GFTFSX.sub.1SWIH; (ii) the HVR-H2 sequence is (SEQ ID NO: 12)
AWIX2PYGGSX.sub.3YYADSVKG (iii) the HVR-H3 sequence is (SEQ ID NO:
13) RHWPGGFDY, and (b) the light chain comprises and HVR-L1, HVR-L2
and HVR-L3, wherein further: (i) the HVR-L1 sequence is (SEQ ID NO:
18) RASQX.sub.4X.sub.5X.sub.6TX.sub.7X.sub.8A (ii) the HVR-L2
sequence is (SEQ ID NO: 19) SASX.sub.9LX.sub.10S; and (iii) the
HVR-L sequence is (SEQ ID NO: 20)
QQX.sub.11X.sub.12X.sub.13X.sub.14PX.sub.15T;
[0121] Further wherein: X.sub.1 is D or G; X.sub.2 is S or L;
X.sub.3 is T or S; X.sub.4 is D or V; X.sub.5 is V or I; X.sub.6 is
S or N; X.sub.7 is A or F; X.sub.8 is V or L; X.sub.9 is F or T;
X.sub.10 is Y or A; X.sub.11 is Y, G, F, or S; X.sub.12 is L, Y, F
or W; X.sub.13 is Y, N, A, T, G, F or I; X.sub.14 is H, V, P, T or
I; X.sub.15 is A, W, R, P or T.
[0122] In a specific aspect, X.sub.1 is D; X.sub.2 is S and X.sub.3
is T. In another aspect, X.sub.4 is D; X.sub.5 is V; X.sub.6 is S;
X.sub.7 is A; X.sub.8 is V; X.sub.9 is F; X.sub.10 is Y; X.sub.11
is Y; X.sub.12 is L; X.sub.13 is Y; X.sub.14 is H; X.sub.15 is A.
In yet another aspect, X.sub.1 is D; X.sub.2 is S and X.sub.3 is T,
X.sub.4 is D; X.sub.5 is V; X.sub.6 is S; X.sub.7 is A; X.sub.8 is
V; X.sub.9 is F; X.sub.10 is Y; X.sub.11 is Y; X.sub.12 is L;
X.sub.13 is Y; X.sub.14 is H and X.sub.15 is A.
[0123] In a further aspect, the heavy chain variable region
comprises one or more framework sequences juxtaposed between the
HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-1-R1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
a still further aspect, the framework sequences are derived from
human consensus framework sequences. In a still further aspect, the
heavy chain framework sequences are derived from a Kabat subgroup
I, II, or III sequence. In a still further aspect, the heavy chain
framework sequence is a VH subgroup III consensus framework. In a
still further aspect, one or more of the heavy chain framework
sequences is the following:
TABLE-US-00008 HC-FR1 is (SEQ ID NO: 14) EVQLVESGGGLVQPGGSLRLSCAAS
HC-FR2 is (SEQ ID NO: 15) WVRQAPGKGLEWV HC-FR3 is (SEQ ID NO: 16)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 is (SEQ ID NO: 17)
WGQGTLVTVSA.
[0124] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00009 LC-FR1 is (SEQ ID NO: 21) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 is (SEQ ID NO: 22) WYQQKPGKAPKLLIY LC-FR3 is (SEQ ID NO: 23)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 is (SEQ ID NO: 24)
FGQGTKVEIKR.
[0125] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect the minimal effector
function results from an "effector-less Fc mutation" or
aglycosylation. In still a further embodiment, the effector-less Fc
mutation is an N297A or D265A/N297A substitution in the constant
region.
[0126] In yet another embodiment, provided is an anti-PDL1 antibody
comprising a heavy chain and a light chain variable region
sequence, wherein:
TABLE-US-00010 (a) the heavy chain further comprises and HVR-H1,
HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity
to (SEQ ID NO: 25) GFTFSDSWIH, (SEQ ID NO: 26) AWISPYGGSTYYADSVKG
and (SEQ ID NO: 13) RHWPGGFDY, respectively or (b) the light chain
further comprises an HVR-L1, HVR-L2 and an HVR-L3 sequence having
at least 85% sequence identity to (SEQ ID NO: 27) RASQDVSTAVA. (SEQ
ID NO: 28) SASFLYS and (SEQ ID NO: 29) QQYLYHPAT, respectively.
[0127] In a specific aspect, the sequence identity is 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
In another aspect, the heavy chain variable region comprises one or
more framework sequences juxtaposed between the HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-1-R3)-(HVR-L3)-(LC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a still further aspect, the heavy
chain framework sequences are derived from a Kabat subgroup I, II,
or III sequence. In a still further aspect, the heavy chain
framework sequence is a VH subgroup III consensus framework. In a
still further aspect, one or more of the heavy chain framework
sequences is the following:
TABLE-US-00011 HC-FR1 is (SEQ ID NO: 14) EVQLVESGGGLVQPGGSLRLSCAAS
HC-FR2 is (SEQ ID NO: 15) WVRQAPGKGLEWV HC-FR3 is (SEQ ID NO: 16)
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 is (SEQ ID NO: 17)
WGQGTLVTVSA.
[0128] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00012 LC-FR1 is (SEQ ID NO: 21) DIQMTQSPSSLSASVGDRVTITC
LC-FR2 is (SEQ ID NO: 22) WYQQKPGKAPKLLIY LC-FR3 is (SEQ ID NO: 23)
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC LC-FR4 is (SEQ ID NO: 24)
FGQGTKVEIKR.
[0129] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect the minimal effector
function results from an "effector-less Fc mutation" or
aglycosylation. In still a further embodiment, the effector-less Fc
mutation is an N297A or D265A/N297A substitution in the constant
region.
[0130] In a still further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and a light chain
variable region sequence, wherein:
TABLE-US-00013 (a) the heavy chain sequence has at least 85%
sequence identity to the heavy chain sequence: (SEQ ID NO: 30)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWV
AWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC
ARRHWPGGFDYWGQGTLVTVSA, or (b) the light chain sequences has at
least 85% sequence identity to the light chain sequence: (SEQ ID
NO: 31) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLI
YSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPA TFGQGTKVEIKR.
[0131] In a specific aspect, the sequence identity is 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
In another aspect, the heavy chain variable region comprises one or
more framework sequences juxtaposed between the HVRs as:
(HC-1-R1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4),
and the light chain variable regions comprises one or more
framework sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a further aspect, the heavy chain
framework sequences are derived from a Kabat subgroup I, II, or III
sequence. In a still further aspect, the heavy chain framework
sequence is a VH subgroup III consensus framework. In a still
further aspect, one or more of the heavy chain framework sequences
is the following:
TABLE-US-00014 (SEQ ID NO: 14) HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS
(SEQ ID NO: 15) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 16) HC-FR3
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 17) HC-FR4
WGQGTLVTVSA.
[0132] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00015 (SEQ ID NO: 21) LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ
ID NO: 22) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 23) LC-FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24) LC-FR4
FGQGTKVEIKR.
[0133] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect, the minimal effector
function results from production in prokaryotic cells. In a still
further specific aspect the minimal effector function results from
an "effector-less Fc mutation" or aglycosylation. In still a
further embodiment, the effector-less Fc mutation is an N297A or
D265A/N297A substitution in the constant region.
[0134] In another further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and a light chain
variable region sequence, wherein:
TABLE-US-00016 (a) the heavy chain sequence has at least 85%
sequence identity to the heavy chain sequence: (SEQ ID NO: 32)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVA
WISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARR
HWPGGFDYWGQGTLVTVSS, or (b) the light chain sequences has at least
85% sequence identity to the light chain sequence: (SEQ ID NO: 31)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIY SASF
LYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTK VEIKR.
[0135] In a specific aspect, the sequence identity is 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
In another aspect, the heavy chain variable region comprises one or
more framework sequences juxtaposed between the HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).
[0136] In yet another aspect, the framework sequences are derived
from human consensus framework sequences. In a further aspect, the
heavy chain framework sequences are derived from a Kabat subgroup
I, II, or III sequence. In a still further aspect, the heavy chain
framework sequence is a VH subgroup III consensus framework. In a
still further aspect, one or more of the heavy chain framework
sequences is the following:
TABLE-US-00017 (SEQ ID NO: 14) HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS
(SEQ ID NO: 15) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 16) HC-FR3
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 33) HC-FR4
WGQGTLVTVSS.
[0137] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00018 (SEQ ID NO: 21) LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ
ID NO: 22) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 23) LC-FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24) LC-FR4
FGQGTKVEIKR.
[0138] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect, the minimal effector
function results from production in prokaryotic cells. In a still
further specific aspect the minimal effector function results from
an "effector-less Fc mutation" or aglycosylation. In still a
further embodiment, the effector-less Fc mutation is an N297A or
D265A/N297A substitution in the constant region.
[0139] In a further aspect, the heavy chain variable region
comprises one or more framework sequences juxtaposed between the
HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In
a still further aspect, the framework sequences are derived from
human consensus framework sequences. In a still further aspect, the
heavy chain framework sequences are derived from a Kabat subgroup
I, II, or III sequence. In a still further aspect, the heavy chain
framework sequence is a VH subgroup III consensus framework. In a
still further aspect, one or more of the heavy chain framework
sequences is the following:
TABLE-US-00019 (SEQ ID NO: 34) HC-FR1
EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO: 35) HC-FR2
WVRQAPGKGLEWVA (SEQ ID NO: 16) HC-FR3
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 33) HC-FR4
WGQGTLVTVSS.
[0140] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00020 (SEQ ID NO: 21) LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ
ID NO: 22) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 23) LC-FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 36) LC-FR4
FGQGTKVEIK.
[0141] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect the minimal effector
function results from an "effector-less Fc mutation" or
aglycosylation. In still a further embodiment, the effector-less Fc
mutation is an N297A or D265A/N297A substitution in the constant
region.
[0142] In yet another embodiment, provided is an anti-PDL1 antibody
comprising a heavy chain and a light chain variable region
sequence, wherein:
TABLE-US-00021 (c) the heavy chain further comprises and HVR-H1,
HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity
to (SEQ ID NO: 4) GFTFSDSWIH, (SEQ ID NO: 5) AWISPYGGSTYYADSVKG and
(SEQ ID NO: 6) RHWPGGFDY, respectively, or (d) the light chain
further comprises an HVR-L1, HVR-L2 and an HVR-L3 sequence having
at least 85% sequence identity to (SEQ ID NO: 1) RASQDVSTAVA, (SEQ
ID NO: 2) SASFLYS and (SEQ ID NO: 3) QQYLYHPAT, respectively.
[0143] In a specific aspect, the sequence identity is 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
In another aspect, the heavy chain variable region comprises one or
more framework sequences juxtaposed between the HVRs as:
(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and
the light chain variable regions comprises one or more framework
sequences juxtaposed between the HVRs as:
(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-1-R3)-(HVR-L3)-(LC-FR4). In
yet another aspect, the framework sequences are derived from human
consensus framework sequences. In a still further aspect, the heavy
chain framework sequences are derived from a Kabat subgroup I, II,
or III sequence. In a still further aspect, the heavy chain
framework sequence is a VH subgroup III consensus framework. In a
still further aspect, one or more of the heavy chain framework
sequences is the following:
TABLE-US-00022 (SEQ ID NO: 34) HC-FR1 EVQLVESGGGLVQPGGSLRLSCAAS
(SEQ ID NO: 35) HC-FR2 WVRQAPGKGLEWV (SEQ ID NO: 16) HC-FR3
RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 33) HC-FR4
WGQGTLVTVSSASTK.
[0144] In a still further aspect, the light chain framework
sequences are derived from a Kabat kappa I, II, II or IV subgroup
sequence. In a still further aspect, the light chain framework
sequences are VL kappa I consensus framework. In a still further
aspect, one or more of the light chain framework sequences is the
following:
TABLE-US-00023 (SEQ ID NO: 21) LC-FR1 DIQMTQSPSSLSASVGDRVTITC (SEQ
ID NO: 22) LC-FR2 WYQQKPGKAPKLLIY (SEQ ID NO: 23) LC-FR3
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 24) LC-FR4
FGQGTKVEIKR
[0145] In a still further specific aspect, the antibody further
comprises a human or murine constant region. In a still further
aspect, the human constant region is selected from the group
consisting of IgG1, IgG2, IgG2, IgG3, IgG4. In a still further
specific aspect, the human constant region is IgG1. In a still
further aspect, the murine constant region is selected from the
group consisting of IgG1, IgG2A, IgG2B, IgG3. In a still further
aspect, the murine constant region if IgG2A. In a still further
specific aspect, the antibody has reduced or minimal effector
function. In a still further specific aspect the minimal effector
function results from an "effector-less Fc mutation" or
aglycosylation. In still a further embodiment, the effector-less Fc
mutation is an N297A or D265A/N297A substitution in the constant
region.
[0146] In a still further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and a light chain
variable region sequence, wherein:
TABLE-US-00024 (a) the heavy chain sequence has at least 85%
sequence identity to the heavy chain sequence: (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSSASTK, or (b) the light chain sequences has at
least 85% sequence identity to the light chain sequence: (SEQ ID
NO: 7) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.
[0147] In some embodiments, provided is an isolated anti-PDL1
antibody comprising a heavy chain and a light chain variable region
sequence, wherein the light chain variable region sequence has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% sequence identity to the amino acid sequence of SEQ ID
NO:7. In some embodiments, provided is an isolated anti-PDL1
antibody comprising a heavy chain and a light chain variable region
sequence, wherein the heavy chain variable region sequence has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% sequence identity to the amino acid sequence of SEQ ID
NO:8. In some embodiments, provided is an isolated anti-PDL1
antibody comprising a heavy chain and a light chain variable region
sequence, wherein the light chain variable region sequence has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% sequence identity to the amino acid sequence of SEQ ID
NO:7 and the heavy chain variable region sequence has at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least
99% sequence identity to the amino acid sequence of SEQ ID
NO:8.
[0148] In a still further embodiment, provided is an isolated
anti-PDL1 antibody comprising a heavy chain and a light chain
sequence, wherein:
TABLE-US-00025 (a) the heavy chain sequence has at least 85%
sequence identity to the heavy chain sequence: (SEQ ID NO: 10)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG, or (b) the light
chain sequences has at least 85% sequence identity to the light
chain sequence: (SEQ ID NO: 9)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
[0149] In some embodiments, provided is an isolated anti-PDL1
antibody comprising a heavy chain and a light chain sequence,
wherein the light chain sequence has at least 85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99% sequence identity
to the amino acid sequence of SEQ ID NO:9. In some embodiments,
provided is an isolated anti-PDL1 antibody comprising a heavy chain
and a light chain sequence, wherein the heavy chain sequence has at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% sequence identity to the amino acid sequence of SEQ ID
NO:10. In some embodiments, provided is an isolated anti-PDL1
antibody comprising a heavy chain and a light chain sequence,
wherein the light chain sequence has at least 85%, at least 86%, at
least 87%, at least 88%, at least 89%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99% sequence identity
to the amino acid sequence of SEQ ID NO:9 and the heavy chain
sequence has at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, or at least 99% sequence identity to the amino acid
sequence of SEQ ID NO:10.
[0150] In some embodiments, the isolated anti-PDL1 antibody is an
oxidized monoclonal antibody. In some embodiments, the oxidized
monoclonal antibody in the formulation comprises a light chain
comprising the amino acid sequence of SEQ ID NO:9, and a heavy
comprising the amino acid sequence of SEQ ID NO:10. In some
embodiments, the oxidized monoclonal antibody in the formulation
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:10, wherein one or more of W33, W50, or W101 is oxidized. In
some embodiments, the oxidized monoclonal antibody in the
formulation comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO:10, wherein one or more of M253 and M429 is
oxidized. In some embodiments, the oxidized monoclonal antibody
retains at least about 60%, at least about 65%, at least about 70%,
at least about 75%, at least about 80%, at least about 85%, at
least about 90%, or at least about 95% of its biological activity
(e.g., binding to the target, or therapeutic potency) exhibited
before storage, i.e., at the time the pharmaceutical formulation
was prepared.
[0151] In some embodiments, the isolated anti-PDL1 antibody is a
glycated monoclonal antibody. In some embodiments, the glycated
monoclonal antibody in the formulation comprises a light chain
comprising the amino acid sequence of SEQ ID NO:9, and a heavy
comprising the amino acid sequence of SEQ ID NO:10. In some
embodiments, the glycated monoclonal antibody in the formulation
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:10, wherein one or more of lysine is glycated. In some
embodiments, the glycated monoclonal antibody in the formulation
comprises a heavy chain comprising the amino acid sequence of SEQ
ID NO:10, wherein K65 is glycated.
[0152] In some embodiments, the isolated anti-PDL1 antibody is
aglycosylated.
[0153] In any of the embodiments herein, the isolated anti-PDL1
antibody can bind to a human PD-L1, for example a human PD-L1 as
shown in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1, or a variant
thereof.
[0154] In a still further embodiment, provided is an isolated
nucleic acid encoding any of the antibodies described herein. In
some embodiments, the nucleic acid further comprises a vector
suitable for expression of the nucleic acid encoding any of the
previously described anti-PDL1 antibodies. In a still further
specific aspect, the vector is in a host cell suitable for
expression of the nucleic acid. In a still further specific aspect,
the host cell is a eukaryotic cell or a prokaryotic cell. In a
still further specific aspect, the eukaryotic cell is a mammalian
cell, such as Chinese Hamster Ovary (CHO).
[0155] The antibody or antigen binding fragment thereof, may be
made using methods known in the art, for example, by a process
comprising culturing a host cell containing nucleic acid encoding
any of the previously described anti-PDL1 antibodies or
antigen-binding fragment in a form suitable for expression, under
conditions suitable to produce such antibody or fragment, and
recovering the antibody or fragment.
[0156] B. Antibody Preparation
[0157] The antibody in the formulation is prepared using techniques
available in the art for generating antibodies, exemplary methods
of which are described in more detail in the following
sections.
[0158] The antibody is directed against an antigen of interest
(i.e., PD-L1, such as human PD-L1). Preferably, the antigen is a
biologically important polypeptide and administration of the
antibody to a mammal suffering from a disorder can result in a
therapeutic benefit in that mammal.
[0159] (i) Antigen Preparation
[0160] Soluble antigens or fragments thereof, optionally conjugated
to other molecules, can be used as immunogens for generating
antibodies. For transmembrane molecules, such as receptors,
fragments of these (e.g. the extracellular domain of a receptor)
can be used as the immunogen. Alternatively, cells expressing the
transmembrane molecule can be used as the immunogen. Such cells can
be derived from a natural source (e.g. cancer cell lines) or may be
cells which have been transformed by recombinant techniques to
express the transmembrane molecule. Other antigens and forms
thereof useful for preparing antibodies will be apparent to those
in the art.
[0161] (ii) Certain Antibody-Based Methods
[0162] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen to a protein that is immunogenic in the
species to be immunized, e.g., keyhole limpet hemocyanin, serum
albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a
bifunctional or derivatizing agent, for example, maleimidobenzoyl
sulfosuccinimide ester (conjugation through cysteine residues),
N-hydroxysuccinimide (through lysine residues), glutaraldehyde,
succinic anhydride, SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R
and R.sup.1 are different alkyl groups.
[0163] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same antigen, but conjugated to a
different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
[0164] Monoclonal antibodies of the invention can be made using the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), and further described, e.g., in Hongo et al., Hybridoma, 14
(3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas
563-681 (Elsevier, N.Y., 1981), and Ni, Xiandai Mianyixue,
26(4):265-268 (2006) regarding human-human hybridomas. Additional
methods include those described, for example, in U.S. Pat. No.
7,189,826 regarding production of monoclonal human natural IgM
antibodies from hybridoma cell lines. Human hybridoma technology
(Trioma technology) is described in Vollmers and Brandlein,
Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and
Brandlein, Methods and Findings in Experimental and Clinical
Pharmacology, 27(3):185-91 (2005).
[0165] For various other hybridoma techniques, see, e.g., US
2006/258841; US 2006/183887 (fully human antibodies), US
2006/059575; US 2005/287149; US 2005/100546; US 2005/026229; and
U.S. Pat. Nos. 7,078,492 and 7,153,507. An exemplary protocol for
producing monoclonal antibodies using the hybridoma method is
described as follows. In one embodiment, a mouse or other
appropriate host animal, such as a hamster, is immunized to elicit
lymphocytes that produce or are capable of producing antibodies
that will specifically bind to the protein used for immunization.
Antibodies are raised in animals by multiple subcutaneous (sc) or
intraperitoneal (ip) injections of a polypeptide of the invention
or a fragment thereof, and an adjuvant, such as monophosphoryl
lipid A (MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem.
Research, Inc., Hamilton, Mont.). A polypeptide of the invention
(e.g., antigen) or a fragment thereof may be prepared using methods
well known in the art, such as recombinant methods, some of which
are further described herein. Serum from immunized animals is
assayed for anti-antigen antibodies, and booster immunizations are
optionally administered. Lymphocytes from animals producing
anti-antigen antibodies are isolated. Alternatively, lymphocytes
may be immunized in vitro.
[0166] Lymphocytes are then fused with myeloma cells using a
suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell. See, e.g., Goding, Monoclonal Antibodies:
Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma
cells may be used that fuse efficiently, support stable high-level
production of antibody by the selected antibody-producing cells,
and are sensitive to a medium such as HAT medium. Exemplary myeloma
cells include, but are not limited to, murine myeloma lines, such
as those derived from MOPC-21 and MPC-11 mouse tumors available
from the Salk Institute Cell Distribution Center, San Diego, Calif.
USA, and SP-2 or X63-Ag8-653 cells available from the American Type
Culture Collection, Rockville, Md. USA. Human myeloma and
mouse-human heteromyeloma cell lines also have been described for
the production of human monoclonal antibodies (Kozbor, J. Immunol.,
133:3001 (1984); Brodeur et al., Monoclonal Antibody Production
Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New
York, 1987)).
[0167] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium, e.g., a medium that contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
Preferably, serum-free hybridoma cell culture methods are used to
reduce use of animal-derived serum such as fetal bovine serum, as
described, for example, in Even et al., Trends in Biotechnology,
24(3), 105-108 (2006).
[0168] Oligopeptides as tools for improving productivity of
hybridoma cell cultures are described in Franek, Trends in
Monoclonal Antibody Research, 111-122 (2005). Specifically,
standard culture media are enriched with certain amino acids
(alanine, serine, asparagine, proline), or with protein hydrolyzate
fractions, and apoptosis may be significantly suppressed by
synthetic oligopeptides, constituted of three to six amino acid
residues. The peptides are present at millimolar or higher
concentrations.
[0169] Culture medium in which hybridoma cells are growing may be
assayed for production of monoclonal antibodies that bind to an
antibody of the invention. The binding specificity of monoclonal
antibodies produced by hybridoma cells may be determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoadsorbent assay
(ELISA). The binding affinity of the monoclonal antibody can be
determined, for example, by Scatchard analysis. See, e.g., Munson
et al., Anal. Biochem., 107:220 (1980).
[0170] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods. See, e.g., Goding, supra. Suitable culture media
for this purpose include, for example, D-MEM or RPMI-1640 medium.
In addition, hybridoma cells may be grown in vivo as ascites tumors
in an animal. Monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography. One
procedure for isolation of proteins from hybridoma cells is
described in US 2005/176122 and U.S. Pat. No. 6,919,436. The method
includes using minimal salts, such as lyotropic salts, in the
binding process and preferably also using small amounts of organic
solvents in the elution process.
[0171] (iii) Certain Library Screening Methods
[0172] Antibodies of the invention can be made by using
combinatorial libraries to screen for antibodies with the desired
activity or activities. For example, a variety of methods are known
in the art for generating phage display libraries and screening
such libraries for antibodies possessing the desired binding
characteristics. Such methods are described generally in Hoogenboom
et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al.,
ed., Human Press, Totowa, N.J., 2001). For example, one method of
generating antibodies of interest is through the use of a phage
antibody library as described in Lee et al., J. Mol. Biol. (2004),
340(5):1073-93.
[0173] In principle, synthetic antibody clones are selected by
screening phage libraries containing phage that display various
fragments of antibody variable region (Fv) fused to phage coat
protein. Such phage libraries are panned by affinity chromatography
against the desired antigen. Clones expressing Fv fragments capable
of binding to the desired antigen are adsorbed to the antigen and
thus separated from the non-binding clones in the library. The
binding clones are then eluted from the antigen, and can be further
enriched by additional cycles of antigen adsorption/elution. Any of
the antibodies of the invention can be obtained by designing a
suitable antigen screening procedure to select for the phage clone
of interest followed by construction of a full length antibody
clone using the Fv sequences from the phage clone of interest and
suitable constant region (Fc) sequences described in Kabat et al.,
Sequences of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
[0174] In certain embodiments, the antigen-binding domain of an
antibody is formed from two variable (V) regions of about 110 amino
acids, one each from the light (VL) and heavy (VH) chains, that
both present three hypervariable loops (HVRs) or
complementarity-determining regions (CDRs). Variable domains can be
displayed functionally on phage, either as single-chain Fv (scFv)
fragments, in which VH and VL are covalently linked through a
short, flexible peptide, or as Fab fragments, in which they are
each fused to a constant domain and interact non-covalently, as
described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
As used herein, scFv encoding phage clones and Fab encoding phage
clones are collectively referred to as "Fv phage clones" or "Fv
clones."
[0175] Repertoires of VH and VL genes can be separately cloned by
polymerase chain reaction (PCR) and recombined randomly in phage
libraries, which can then be searched for antigen-binding clones as
described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
Libraries from immunized sources provide high-affinity antibodies
to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned to
provide a single source of human antibodies to a wide range of
non-self and also self antigens without any immunization as
described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally,
naive libraries can also be made synthetically by cloning the
unrearranged V-gene segments from stem cells, and using PCR primers
containing random sequence to encode the highly variable CDR3
regions and to accomplish rearrangement in vitro as described by
Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
[0176] In certain embodiments, filamentous phage is used to display
antibody fragments by fusion to the minor coat protein pIII. The
antibody fragments can be displayed as single chain Fv fragments,
in which VH and VL domains are connected on the same polypeptide
chain by a flexible polypeptide spacer, e.g. as described by Marks
et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab fragments, in
which one chain is fused to pIII and the other is secreted into the
bacterial host cell periplasm where assembly of a Fab-coat protein
structure which becomes displayed on the phage surface by
displacing some of the wild type coat proteins, e.g. as described
in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).
[0177] In general, nucleic acids encoding antibody gene fragments
are obtained from immune cells harvested from humans or animals. If
a library biased in favor of anti-antigen clones is desired, the
subject is immunized with antigen to generate an antibody response,
and spleen cells and/or circulating B cells other peripheral blood
lymphocytes (PBLs) are recovered for library construction. In one
embodiment, a human antibody gene fragment library biased in favor
of anti-antigen clones is obtained by generating an anti-antigen
antibody response in transgenic mice carrying a functional human
immunoglobulin gene array (and lacking a functional endogenous
antibody production system) such that antigen immunization gives
rise to B cells producing human antibodies against antigen. The
generation of human antibody-producing transgenic mice is described
below.
[0178] Additional enrichment for anti-antigen reactive cell
populations can be obtained by using a suitable screening procedure
to isolate B cells expressing antigen-specific membrane bound
antibody, e.g., by cell separation using antigen affinity
chromatography or adsorption of cells to fluorochrome-labeled
antigen followed by flow-activated cell sorting (FACS).
[0179] Alternatively, the use of spleen cells and/or B cells or
other PBLs from an unimmunized donor provides a better
representation of the possible antibody repertoire, and also
permits the construction of an antibody library using any animal
(human or non-human) species in which antigen is not antigenic. For
libraries incorporating in vitro antibody gene construction, stem
cells are harvested from the subject to provide nucleic acids
encoding unrearranged antibody gene segments. The immune cells of
interest can be obtained from a variety of animal species, such as
human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,
bovine, equine, and avian species, etc.
[0180] Nucleic acid encoding antibody variable gene segments
(including VH and VL segments) are recovered from the cells of
interest and amplified. In the case of rearranged VH and VL gene
libraries, the desired DNA can be obtained by isolating genomic DNA
or mRNA from lymphocytes followed by polymerase chain reaction
(PCR) with primers matching the 5' and 3' ends of rearranged VH and
VL genes as described in Orlandi et al., Proc. Natl. Acad. Sci.
(USA), 86: 3833-3837 (1989), thereby making diverse V gene
repertoires for expression. The V genes can be amplified from cDNA
and genomic DNA, with back primers at the 5' end of the exon
encoding the mature V-domain and forward primers based within the
J-segment as described in Orlandi et al. (1989) and in Ward et al.,
Nature, 341: 544-546 (1989). However, for amplifying from cDNA,
back primers can also be based in the leader exon as described in
Jones et al., Biotechnol., 9: 88-89 (1991), and forward primers
within the constant region as described in Sastry et al., Proc.
Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To maximize
complementarity, degeneracy can be incorporated in the primers as
described in Orlandi et al. (1989) or Sastry et al. (1989). In
certain embodiments, library diversity is maximized by using PCR
primers targeted to each V-gene family in order to amplify all
available VH and VL arrangements present in the immune cell nucleic
acid sample, e.g. as described in the method of Marks et al., J.
Mol. Biol., 222: 581-597 (1991) or as described in the method of
Orum et al., Nucleic Acids Res., 21: 4491-4498 (1993). For cloning
of the amplified DNA into expression vectors, rare restriction
sites can be introduced within the PCR primer as a tag at one end
as described in Orlandi et al. (1989), or by further PCR
amplification with a tagged primer as described in Clackson et al.,
Nature, 352: 624-628 (1991).
[0181] Repertoires of synthetically rearranged V genes can be
derived in vitro from V gene segments. Most of the human VH-gene
segments have been cloned and sequenced (reported in Tomlinson et
al., J. Mol. Biol., 227: 776-798 (1992)), and mapped (reported in
Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned
segments (including all the major conformations of the H1 and H2
loop) can be used to generate diverse VH gene repertoires with PCR
primers encoding H3 loops of diverse sequence and length as
described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388
(1992). VH repertoires can also be made with all the sequence
diversity focused in a long H3 loop of a single length as described
in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992).
Human V.kappa. and V.lamda.. segments have been cloned and
sequenced (reported in Williams and Winter, Eur. J. Immunol., 23:
1456-1461 (1993)) and can be used to make synthetic light chain
repertoires. Synthetic V gene repertoires, based on a range of VH
and VL folds, and L3 and H3 lengths, will encode antibodies of
considerable structural diversity. Following amplification of
V-gene encoding DNAs, germline V-gene segments can be rearranged in
vitro according to the methods of Hoogenboom and Winter, J. Mol.
Biol., 227: 381-388 (1992).
[0182] Repertoires of antibody fragments can be constructed by
combining VH and VL gene repertoires together in several ways. Each
repertoire can be created in different vectors, and the vectors
recombined in vitro, e.g., as described in Hogrefe et al., Gene,
128: 119-126 (1993), or in vivo by combinatorial infection, e.g.,
the loxP system described in Waterhouse et al., Nucl. Acids Res.,
21: 2265-2266 (1993). The in vivo recombination approach exploits
the two-chain nature of Fab fragments to overcome the limit on
library size imposed by E. coli transformation efficiency. Naive VH
and VL repertoires are cloned separately, one into a phagemid and
the other into a phage vector. The two libraries are then combined
by phage infection of phagemid-containing bacteria so that each
cell contains a different combination and the library size is
limited only by the number of cells present (about 10.sup.12
clones). Both vectors contain in vivo recombination signals so that
the VH and VL genes are recombined onto a single replicon and are
co-packaged into phage virions. These huge libraries provide large
numbers of diverse antibodies of good affinity (K.sub.d.sup.-1 of
about 10.sup.-8 M).
[0183] Alternatively, the repertoires may be cloned sequentially
into the same vector, e.g. as described in Barbas et al., Proc.
Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together
by PCR and then cloned, e.g. as described in Clackson et al.,
Nature, 352: 624-628 (1991). PCR assembly can also be used to join
VH and VL DNAs with DNA encoding a flexible peptide spacer to form
single chain Fv (scFv) repertoires. In yet another technique, "in
cell PCR assembly" is used to combine VH and VL genes within
lymphocytes by PCR and then clone repertoires of linked genes as
described in Embleton et al., Nucl. Acids Res., 20: 3831-3837
(1992).
[0184] The antibodies produced by naive libraries (either natural
or synthetic) can be of moderate affinity (K.sub.d.sup.-1 of about
10.sup.6 to 10.sup.7 M.sup.-1), but affinity maturation can also be
mimicked in vitro by constructing and reselecting from secondary
libraries as described in Winter et al. (1994), supra. For example,
mutation can be introduced at random in vitro by using error-prone
polymerase (reported in Leung et al., Technique 1: 11-15 (1989)) in
the method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or
in the method of Gram et al., Proc. Natl. Acad. Sci USA, 89:
3576-3580 (1992). Additionally, affinity maturation can be
performed by randomly mutating one or more CDRs, e.g. using PCR
with primers carrying random sequence spanning the CDR of interest,
in selected individual Fv clones and screening for higher affinity
clones. WO 9607754 (published 14 Mar. 1996) described a method for
inducing mutagenesis in a complementarity determining region of an
immunoglobulin light chain to create a library of light chain
genes. Another effective approach is to recombine the VH or VL
domains selected by phage display with repertoires of naturally
occurring V domain variants obtained from unimmunized donors and
screen for higher affinity in several rounds of chain reshuffling
as described in Marks et al., Biotechnol., 10: 779-783 (1992). This
technique allows the production of antibodies and antibody
fragments with affinities of about 10.sup.-9 M or less.
[0185] Screening of the libraries can be accomplished by various
techniques known in the art. For example, antigen can be used to
coat the wells of adsorption plates, expressed on host cells
affixed to adsorption plates or used in cell sorting, or conjugated
to biotin for capture with streptavidin-coated beads, or used in
any other method for panning phage display libraries.
[0186] The phage library samples are contacted with immobilized
antigen under conditions suitable for binding at least a portion of
the phage particles with the adsorbent. Normally, the conditions,
including pH, ionic strength, temperature and the like are selected
to mimic physiological conditions. The phages bound to the solid
phase are washed and then eluted by acid, e.g. as described in
Barbas et al., Proc. Natl. Acad. Sci USA, 88: 7978-7982 (1991), or
by alkali, e.g. as described in Marks et al., J. Mol. Biol., 222:
581-597 (1991), or by antigen competition, e.g. in a procedure
similar to the antigen competition method of Clackson et al.,
Nature, 352: 624-628 (1991). Phages can be enriched 20-1,000-fold
in a single round of selection. Moreover, the enriched phages can
be grown in bacterial culture and subjected to further rounds of
selection.
[0187] The efficiency of selection depends on many factors,
including the kinetics of dissociation during washing, and whether
multiple antibody fragments on a single phage can simultaneously
engage with antigen. Antibodies with fast dissociation kinetics
(and weak binding affinities) can be retained by use of short
washes, multivalent phage display and high coating density of
antigen in solid phase. The high density not only stabilizes the
phage through multivalent interactions, but favors rebinding of
phage that has dissociated. The selection of antibodies with slow
dissociation kinetics (and good binding affinities) can be promoted
by use of long washes and monovalent phage display as described in
Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a
low coating density of antigen as described in Marks et al.,
Biotechnol., 10: 779-783 (1992).
[0188] It is possible to select between phage antibodies of
different affinities, even with affinities that differ slightly,
for antigen. However, random mutation of a selected antibody (e.g.
as performed in some affinity maturation techniques) is likely to
give rise to many mutants, most binding to antigen, and a few with
higher affinity. With limiting antigen, rare high affinity phage
could be competed out. To retain all higher affinity mutants,
phages can be incubated with excess biotinylated antigen, but with
the biotinylated antigen at a concentration of lower molarity than
the target molar affinity constant for antigen. The high
affinity-binding phages can then be captured by streptavidin-coated
paramagnetic beads. Such "equilibrium capture" allows the
antibodies to be selected according to their affinities of binding,
with sensitivity that permits isolation of mutant clones with as
little as two-fold higher affinity from a great excess of phages
with lower affinity. Conditions used in washing phages bound to a
solid phase can also be manipulated to discriminate on the basis of
dissociation kinetics.
[0189] Anti-antigen clones may be selected based on activity. In
certain embodiments, the invention provides anti-antigen antibodies
that bind to living cells that naturally express antigen or bind to
free floating antigen or antigen attached to other cellular
structures. Fv clones corresponding to such anti-antigen antibodies
can be selected by (1) isolating anti-antigen clones from a phage
library as described above, and optionally amplifying the isolated
population of phage clones by growing up the population in a
suitable bacterial host; (2) selecting antigen and a second protein
against which blocking and non-blocking activity, respectively, is
desired; (3) adsorbing the anti-antigen phage clones to immobilized
antigen; (4) using an excess of the second protein to elute any
undesired clones that recognize antigen-binding determinants which
overlap or are shared with the binding determinants of the second
protein; and (5) eluting the clones which remain adsorbed following
step (4). Optionally, clones with the desired blocking/non-blocking
properties can be further enriched by repeating the selection
procedures described herein one or more times.
[0190] DNA encoding hybridoma-derived monoclonal antibodies or
phage display Fv clones of the invention is readily isolated and
sequenced using conventional procedures (e.g. by using
oligonucleotide primers designed to specifically amplify the heavy
and light chain coding regions of interest from hybridoma or phage
DNA template). Once isolated, the DNA can be placed into expression
vectors, which are then transfected into host cells such as E. coli
cells, simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of the desired monoclonal antibodies in the
recombinant host cells. Review articles on recombinant expression
in bacteria of antibody-encoding DNA include Skerra et al., Curr.
Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs,
130: 151 (1992).
[0191] DNA encoding the Fv clones of the invention can be combined
with known DNA sequences encoding heavy chain and/or light chain
constant regions (e.g. the appropriate DNA sequences can be
obtained from Kabat et al., supra) to form clones encoding full or
partial length heavy and/or light chains. It will be appreciated
that constant regions of any isotype can be used for this purpose,
including IgG, IgM, IgA, IgD, and IgE constant regions, and that
such constant regions can be obtained from any human or animal
species. An Fv clone derived from the variable domain DNA of one
animal (such as human) species and then fused to constant region
DNA of another animal species to form coding sequence(s) for
"hybrid," full length heavy chain and/or light chain is included in
the definition of "chimeric" and "hybrid" antibody as used herein.
In certain embodiments, an Fv clone derived from human variable DNA
is fused to human constant region DNA to form coding sequence(s)
for full- or partial-length human heavy and/or light chains.
[0192] DNA encoding anti-antigen antibody derived from a hybridoma
of the invention can also be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of homologous murine sequences derived from the
hybridoma clone (e.g. as in the method of Morrison et al., Proc.
Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNA encoding a
hybridoma- or Fv clone-derived antibody or fragment can be further
modified by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide. In this manner, "chimeric" or
"hybrid" antibodies are prepared that have the binding specificity
of the Fv clone or hybridoma clone-derived antibodies of the
invention.
[0193] (iv) Humanized and Human Antibodies
[0194] Various methods for humanizing non-human antibodies are
known in the art. For example, a humanized antibody has one or more
amino acid residues introduced into it from a source which is
non-human. These non-human amino acid residues are often referred
to as "import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0195] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., Proc. Natl. Acad Sci. USA, 89:4285
(1992); Presta et al., J. Immunol., 151:2623 (1993)).
[0196] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties. To achieve this goal, according to one
embodiment of the method, humanized antibodies are prepared by a
process of analysis of the parental sequences and various
conceptual humanized products using three-dimensional models of the
parental and humanized sequences. Three-dimensional immunoglobulin
models are commonly available and are familiar to those skilled in
the art. Computer programs are available which illustrate and
display probable three-dimensional conformational structures of
selected candidate immunoglobulin sequences. Inspection of these
displays permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0197] Human antibodies of the invention can be constructed by
combining Fv clone variable domain sequence(s) selected from
human-derived phage display libraries with known human constant
domain sequence(s) as described above. Alternatively, human
monoclonal antibodies of the invention can be made by the hybridoma
method. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described,
for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et
al., Monoclonal Antibody Production Techniques and Applications,
pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et
al., J. Immunol., 147: 86 (1991).
[0198] It is possible to produce transgenic animals (e.g., mice)
that are capable, upon immunization, of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (J.sub.H) gene
in chimeric and germ-line mutant mice results in complete
inhibition of endogenous antibody production. Transfer of the human
germ-line immunoglobulin gene array in such germ-line mutant mice
will result in the production of human antibodies upon antigen
challenge. See, e.g., Jakobovits et al, Proc. Natl. Acad. Sci. USA,
90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggermann et al., Year in Immuno., 7:33 (1993); and Duchosal et
al. Nature 355:258 (1992).
[0199] Gene shuffling can also be used to derive human antibodies
from non-human, e.g. rodent, antibodies, where the human antibody
has similar affinities and specificities to the starting non-human
antibody. According to this method, which is also called "epitope
imprinting", either the heavy or light chain variable region of a
non-human antibody fragment obtained by phage display techniques as
described herein is replaced with a repertoire of human V domain
genes, creating a population of non-human chain/human chain scFv or
Fab chimeras. Selection with antigen results in isolation of a
non-human chain/human chain chimeric scFv or Fab wherein the human
chain restores the antigen binding site destroyed upon removal of
the corresponding non-human chain in the primary phage display
clone, i.e. the epitope governs (imprints) the choice of the human
chain partner. When the process is repeated in order to replace the
remaining non-human chain, a human antibody is obtained (see PCT WO
93/06213 published Apr. 1, 1993). Unlike traditional humanization
of non-human antibodies by CDR grafting, this technique provides
completely human antibodies, which have no FR or CDR residues of
non-human origin.
[0200] (v) Antibody Fragments
[0201] Antibody fragments may be generated by traditional means,
such as enzymatic digestion, or by recombinant techniques. In
certain circumstances there are advantages of using antibody
fragments, rather than whole antibodies. The smaller size of the
fragments allows for rapid clearance, and may lead to improved
access to solid tumors. For a review of certain antibody fragments,
see Hudson et al. (2003) Nat. Med. 9:129-134.
[0202] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising salvage receptor binding epitope residues
are described in U.S. Pat. No. 5,869,046. Other techniques for the
production of antibody fragments will be apparent to the skilled
practitioner. In certain embodiments, an antibody is a single chain
Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and
5,587,458. Fv and scFv are the only species with intact combining
sites that are devoid of constant regions; thus, they may be
suitable for reduced nonspecific binding during in vivo use. scFv
fusion proteins may be constructed to yield fusion of an effector
protein at either the amino or the carboxy terminus of an scFv. See
Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment
may also be a "linear antibody", e.g., as described in U.S. Pat.
No. 5,641,870, for example. Such linear antibodies may be
monospecific or bispecific.
[0203] (vi) Multispecific Antibodies
[0204] Multispecific antibodies have binding specificities for at
least two different epitopes, where the epitopes are usually from
different antigens. While such molecules normally will only bind
two different epitopes (i.e. bispecific antibodies, BsAbs),
antibodies with additional specificities such as trispecific
antibodies are encompassed by this expression when used herein.
Bispecific antibodies can be prepared as full length antibodies or
antibody fragments (e.g. F(ab').sub.2 bispecific antibodies).
[0205] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0206] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3
regions. It is typical to have the first heavy-chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0207] In one embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690. For further details of
generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymology, 121:210 (1986).
[0208] According to another approach described in WO96/27011, the
interface between a pair of antibody molecules can be engineered to
maximize the percentage of heterodimers which are recovered from
recombinant cell culture. One interface comprises at least a part
of the C.sub.H 3 domain of an antibody constant domain. In this
method, one or more small amino acid side chains from the interface
of the first antibody molecule are replaced with larger side chains
(e.g. tyrosine or tryptophan). Compensatory "cavities" of identical
or similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g. alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0209] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0210] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science, 229: 81 (1985) describe a
procedure wherein intact antibodies are proteolytically cleaved to
generate F(ab').sub.2 fragments. These fragments are reduced in the
presence of the dithiol complexing agent sodium arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0211] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med., 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody.
[0212] Various techniques for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
V.sub.H and V.sub.L domains of one fragment are forced to pair with
the complementary V.sub.L and V.sub.H domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for
making bispecific antibody fragments by the use of single-chain Fv
(sFv) dimers has also been reported. See Gruber et al, J. Immunol,
152:5368 (1994).
[0213] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tuft et al. J.
Immunol. 147: 60 (1991).
[0214] (vii) Single-Domain Antibodies
[0215] In some embodiments, an antibody of the invention is a
single-domain antibody. A single-domain antibody is a single
polypeptide chain comprising all or a portion of the heavy chain
variable domain or all or a portion of the light chain variable
domain of an antibody. In certain embodiments, a single-domain
antibody is a human single-domain antibody (Domantis, Inc.,
Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1). In one
embodiment, a single-domain antibody consists of all or a portion
of the heavy chain variable domain of an antibody.
[0216] (viii) Antibody Variants
[0217] In some embodiments, amino acid sequence modification(s) of
the antibodies described herein are contemplated. For example, it
may be desirable to improve the binding affinity and/or other
biological properties of the antibody Amino acid sequence variants
of the antibody may be prepared by introducing appropriate changes
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. The amino acid alterations may be introduced in
the subject antibody amino acid sequence at the time that sequence
is made.
[0218] (ix) Antibody Derivatives
[0219] The antibodies of the invention can be further modified to
contain additional nonproteinaceous moieties that are known in the
art and readily available. In certain embodiments, the moieties
suitable for derivatization of the antibody are water soluble
polymers. Non-limiting examples of water soluble polymers include,
but are not limited to, polyethylene glycol (PEG), copolymers of
ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,
polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,
poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,
polyaminoacids (either homopolymers or random copolymers), and
dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol homopolymers, prolypropylene oxide/ethylene
oxide copolymers, polyoxyethylated polyols (e.g., glycerol),
polyvinyl alcohol, and mixtures thereof. Polyethylene glycol
propionaldehyde may have advantages in manufacturing due to its
stability in water. The polymer may be of any molecular weight, and
may be branched or unbranched. The number of polymers attached to
the antibody may vary, and if more than one polymer are attached,
they can be the same or different molecules. In general, the number
and/or type of polymers used for derivatization can be determined
based on considerations including, but not limited to, the
particular properties or functions of the antibody to be improved,
whether the antibody derivative will be used in a therapy under
defined conditions, etc.
[0220] (x) Vectors, Host Cells, and Recombinant Methods
[0221] Antibodies may also be produced using recombinant methods.
For recombinant production of an anti-antigen antibody, nucleic
acid encoding the antibody is isolated and inserted into a
replicable vector for further cloning (amplification of the DNA) or
for expression. DNA encoding the antibody may be readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of the antibody). Many
vectors are available. The vector components generally include, but
are not limited to, one or more of the following: a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence.
[0222] (a) Signal Sequence Component
[0223] An antibody of the invention may be produced recombinantly
not only directly, but also as a fusion polypeptide with a
heterologous polypeptide, which is preferably a signal sequence or
other polypeptide having a specific cleavage site at the N-terminus
of the mature protein or polypeptide. The heterologous signal
sequence selected preferably is one that is recognized and
processed (e.g., cleaved by a signal peptidase) by the host cell.
For prokaryotic host cells that do not recognize and process a
native antibody signal sequence, the signal sequence is substituted
by a prokaryotic signal sequence selected, for example, from the
group of the alkaline phosphatase, penicillinase, 1pp, or
heat-stable enterotoxin II leaders. For yeast secretion the native
signal sequence may be substituted by, e.g., the yeast invertase
leader, a factor leader (including Saccharomyces and Kluyveromyces
.alpha.-factor leaders), or acid phosphatase leader, the C.
albicans glucoamylase leader, or the signal described in WO
90/13646. In mammalian cell expression, mammalian signal sequences
as well as viral secretory leaders, for example, the herpes simplex
gD signal, are available.
[0224] (b) Origin of Replication
[0225] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria, yeast, and viruses. The origin of
replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2p, plasmid origin is suitable for
yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or
BPV) are useful for cloning vectors in mammalian cells. Generally,
the origin of replication component is not needed for mammalian
expression vectors (the SV40 origin may typically be used only
because it contains the early promoter.
[0226] (c) Selection Gene Component
[0227] Expression and cloning vectors may contain a selection gene,
also termed a selectable marker. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli.
[0228] One example of a selection scheme utilizes a drug to arrest
growth of a host cell. Those cells that are successfully
transformed with a heterologous gene produce a protein conferring
drug resistance and thus survive the selection regimen. Examples of
such dominant selection use the drugs neomycin, mycophenolic acid
and hygromycin.
[0229] Another example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up antibody-encoding nucleic acid, such as DHFR, glutamine
synthetase (GS), thymidine kinase, metallothionein-I and -II,
preferably primate metallothionein genes, adenosine deaminase,
ornithine decarboxylase, etc.
[0230] For example, cells transformed with the DHFR gene are
identified by culturing the transformants in a culture medium
containing methotrexate (Mtx), a competitive antagonist of DHFR.
Under these conditions, the DHFR gene is amplified along with any
other co-transformed nucleic acid. A Chinese hamster ovary (CHO)
cell line deficient in endogenous DHFR activity (e.g., ATCC
CRL-9096) may be used.
[0231] Alternatively, cells transformed with the GS gene are
identified by culturing the transformants in a culture medium
containing L-methionine sulfoximine (Msx), an inhibitor of GS.
Under these conditions, the GS gene is amplified along with any
other co-transformed nucleic acid. The GS selection/amplification
system may be used in combination with the DHFR
selection/amplification system described above.
[0232] Alternatively, host cells (particularly wild-type hosts that
contain endogenous DHFR) transformed or co-transformed with DNA
sequences encoding an antibody of interest, wild-type DHFR gene,
and another selectable marker such as aminoglycoside
3'-phosphotransferase (APH) can be selected by cell growth in
medium containing a selection agent for the selectable marker such
as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or
G418. See U.S. Pat. No. 4,965,199.
[0233] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., Nature,
282:39 (1979)). The trp1 gene provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12
(1977). The presence of the trp1 lesion in the yeast host cell
genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
[0234] In addition, vectors derived from the 1.6 .mu.m circular
plasmid pKD1 can be used for transformation of Kluyveromyces
yeasts. Alternatively, an expression system for large-scale
production of recombinant calf chymosin was reported for K. lactis.
Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy
expression vectors for secretion of mature recombinant human serum
albumin by industrial strains of Kluyveromyces have also been
disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).
[0235] (d) Promoter Component
[0236] Expression and cloning vectors generally contain a promoter
that is recognized by the host organism and is operably linked to
nucleic acid encoding an antibody. Promoters suitable for use with
prokaryotic hosts include the phoA promoter, .beta.-lactamase and
lactose promoter systems, alkaline phosphatase promoter, a
tryptophan (trp) promoter system, and hybrid promoters such as the
tac promoter. However, other known bacterial promoters are
suitable. Promoters for use in bacterial systems also will contain
a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding an antibody.
[0237] Promoter sequences are known for eukaryotes. Virtually all
eukaryotic genes have an AT-rich region located approximately 25 to
30 bases upstream from the site where transcription is initiated.
Another sequence found 70 to 80 bases upstream from the start of
transcription of many genes is a CNCAAT region where N may be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA
sequence that may be the signal for addition of the poly A tail to
the 3' end of the coding sequence. All of these sequences are
suitably inserted into eukaryotic expression vectors.
[0238] Examples of suitable promoter sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase or other
glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0239] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657. Yeast enhancers also are advantageously used with yeast
promoters.
[0240] Antibody transcription from vectors in mammalian host cells
can be controlled, for example, by promoters obtained from the
genomes of viruses such as polyoma virus, fowlpox virus, adenovirus
(such as Adenovirus 2), bovine papilloma virus, avian sarcoma
virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian
Virus 40 (SV40), or from heterologous mammalian promoters, e.g.,
the actin promoter or an immunoglobulin promoter, from heat-shock
promoters, provided such promoters are compatible with the host
cell systems.
[0241] The early and late promoters of the SV40 virus are
conveniently obtained as an SV40 restriction fragment that also
contains the SV40 viral origin of replication. The immediate early
promoter of the human cytomegalovirus is conveniently obtained as a
HindIII E restriction fragment. A system for expressing DNA in
mammalian hosts using the bovine papilloma virus as a vector is
disclosed in U.S. Pat. No. 4,419,446. A modification of this system
is described in U.S. Pat. No. 4,601,978. See also Reyes et al.,
Nature 297:598-601 (1982) on expression of human .beta.-interferon
cDNA in mouse cells under the control of a thymidine kinase
promoter from herpes simplex virus. Alternatively, the Rous Sarcoma
Virus long terminal repeat can be used as the promoter.
[0242] (e) Enhancer Element Component
[0243] Transcription of a DNA encoding an antibody of this
invention by higher eukaryotes is often increased by inserting an
enhancer sequence into the vector. Many enhancer sequences are now
known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing
elements for activation of eukaryotic promoters. The enhancer may
be spliced into the vector at a position 5' or 3' to the
antibody-encoding sequence, but is preferably located at a site 5'
from the promoter.
[0244] (f) Transcription Termination Component
[0245] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
antibody. One useful transcription termination component is the
bovine growth hormone polyadenylation region. See WO94/11026 and
the expression vector disclosed therein.
[0246] (g) Selection and Transformation of Host Cells
[0247] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0248] Full length antibody, antibody fusion proteins, and antibody
fragments can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) that by itself shows effectiveness in tumor cell
destruction. Full length antibodies have greater half-life in
circulation. Production in E. coli is faster and more cost
efficient. For expression of antibody fragments and polypeptides in
bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S.
Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523 (Simmons
et al.), which describes translation initiation region (TIR) and
signal sequences for optimizing expression and secretion. See also
Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,
Humana Press, Totowa, N.J., 2003), pp. 245-254, describing
expression of antibody fragments in E. coli. After expression, the
antibody may be isolated from the E. coli cell paste in a soluble
fraction and can be purified through, e.g., a protein A or G column
depending on the isotype. Final purification can be carried out
similar to the process for purifying antibody expressed e.g., in
CHO cells.
[0249] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors. Saccharomyces cerevisiae, or common
baker's yeast, is the most commonly used among lower eukaryotic
host microorganisms. However, a number of other genera, species,
and strains are commonly available and useful herein, such as
Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
laths, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger. For a review discussing the use of
yeasts and filamentous fungi for the production of therapeutic
proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414
(2004).
[0250] Certain fungi and yeast strains may be selected in which
glycosylation pathways have been "humanized," resulting in the
production of an antibody with a partially or fully human
glycosylation pattern. See, e.g., Li et al., Nat. Biotech.
24:210-215 (2006) (describing humanization of the glycosylation
pathway in Pichia pastoris); and Gerngross et al., supra.
[0251] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains and
variants and corresponding permissive insect host cells from hosts
such as Spodoptera frugiperda (caterpillar), Aedes aegypti
(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster
(fruitfly), and Bombyx mori have been identified. A variety of
viral strains for transfection are publicly available, e.g., the
L-1 variant of Autographa californica NPV and the Bm-5 strain of
Bombyx mori NPV, and such viruses may be used as the virus herein
according to the invention, particularly for transfection of
Spodoptera frugiperda cells.
[0252] Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula),
and tobacco can also be utilized as hosts. See, e.g., U.S. Pat.
Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429
(describing PLANTIBODIES.TM. technology for producing antibodies in
transgenic plants).
[0253] Vertebrate cells may be used as hosts, and propagation of
vertebrate cells in culture (tissue culture) has become a routine
procedure. Examples of useful mammalian host cell lines are monkey
kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture, Graham et al., J. Gen Virol. 36:59 (1977));
baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells
(TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells
(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC
CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2);
canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells
(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75);
human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT
060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.
Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human
hepatoma line (Hep G2). Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR.sup.- CHO
cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980));
and myeloma cell lines such as NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp.
255-268.
[0254] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0255] (h) Culturing the Host Cells
[0256] The host cells used to produce an antibody of this invention
may be cultured in a variety of media. Commercially available media
such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),
(Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium
((DMEM), Sigma) are suitable for culturing the host cells. In
addition, any of the media described in Ham et al., Meth. Enz.
58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S.
Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469;
WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be used as
culture media for the host cells. Any of these media may be
supplemented as necessary with hormones and/or other growth factors
(such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate),
buffers (such as HEPES), nucleotides (such as adenosine and
thymidine), antibiotics (such as GENTAMYCIN.TM. drug), trace
elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an
equivalent energy source. Any other necessary supplements may also
be included at appropriate concentrations that would be known to
those skilled in the art. The culture conditions, such as
temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0257] (xi) Purification of Antibody
[0258] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, are removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10:163-167 (1992) describe a procedure for isolating antibodies
which are secreted to the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0259] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography,
hydrophobic interaction chromatography, gel electrophoresis,
dialysis, and affinity chromatography, with affinity chromatography
being among one of the typically preferred purification steps. The
suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2, or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
[0260] In general, various methodologies for preparing antibodies
for use in research, testing, and clinical are well-established in
the art, consistent with the above-described methodologies and/or
as deemed appropriate by one skilled in the art for a particular
antibody of interest.
[0261] C. Selecting Biologically Active Antibodies
[0262] Antibodies produced as described above may be subjected to
one or more "biological activity" assays to select an antibody with
beneficial properties from a therapeutic perspective or selecting
formulations and conditions that retain biological activity of the
antibody. The antibody may be tested for its ability to bind the
antigen against which it was raised. For example, for an anti-PDL1
antibody, the antigen binding properties of the antibody can be
evaluated in an assay that detects the ability to bind to PDL1. In
some embodiments, the binding of the antibody may be determined by
saturation binding; ELISA; and/or competition assays (e.g. RIA's),
for example. Also, the antibody may be subjected to other
biological activity assays, e.g., in order to evaluate its
effectiveness as a therapeutic. Such assays are known in the art
and depend on the target antigen and intended use for the antibody.
For example, the biological effects of PD-L1 blockade by the
antibody can be assessed in CD8+ T cells, a lymphocytic
choriomeningitis virus (LCMV) mouse model and/or a syngeneic tumor
model e.g., as described in U.S. Pat. No. 8,217,149.
[0263] To screen for antibodies which bind to a particular epitope
on the antigen of interest (e.g., those which block binding of the
anti-PDL1 antibody of the example to PD-L1), a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed. Alternatively, epitope
mapping, e.g. as described in Champe et al., J. Biol. Chem.
270:1388-1394 (1995), can be performed to determine whether the
antibody binds an epitope of interest.
[0264] D. Preparation of the Formulations
[0265] After preparation of the antibody of interest (e.g.,
techniques for producing antibodies which can be formulated as
disclosed herein will be elaborated below and are known in the
art), the pharmaceutical formulation comprising it is prepared. In
certain embodiments, the antibody to be formulated has not been
subjected to prior lyophilization and the formulation of interest
herein is an aqueous formulation. In certain embodiments, the
antibody is a full length antibody. In one embodiment, the antibody
in the formulation is an antibody fragment, such as an
F(ab').sub.2, in which case problems that may not occur for the
full length antibody (such as clipping of the antibody to Fab) may
need to be addressed. The therapeutically effective amount of
antibody present in the formulation is determined by taking into
account the desired dose volumes and mode(s) of administration, for
example. From about 25 mg/mL to about 150 mg/mL, or from about 30
mg/mL to about 140 mg/mL, or from about 35 mg/mL to about 130
mg/mL, or from about 40 mg/mL to about 120 mg/mL, or from about 50
mg/mL to about 130 mg/mL, or from about 50 mg/mL to about 125
mg/mL, or from about 50 mg/mL to about 120 mg/mL, or from about 50
mg/mL to about 110 mg/mL, or from about 50 mg/mL to about 100
mg/mL, or from about 50 mg/mL to about 90 mg/mL, or from about 50
mg/mL to about 80 mg/mL, or from about 54 mg/mL to about 66 mg/mL
is an exemplary antibody concentration in the formulation.
[0266] An aqueous formulation is prepared comprising the antibody
in a pH-buffered solution. The buffer of this invention has a pH in
the range from about 5.0 to about 7.0. In certain embodiments the
pH is in the range from about 5.0 to about 6.5, the pH is in the
range from about 5.0 to about 6.4, in the range from about 5.0 to
about 6.3, the pH is in the range from about 5.0 to about 6.2, the
pH is in the range from about 5.0 to about 6.1, the pH is in the
range from about 5.5 to about 6.1, the pH is in the range from
about 5.0 to about 6.0, the pH is in the range from about 5.0 to
about 5.9, the pH is in the range from about 5.0 to about 5.8, the
pH is in the range from about 5.1 to about 6.0, the pH is in the
range from about 5.2 to about 6.0, the pH is in the range from
about 5.3 to about 6.0, the pH is in the range from about 5.4 to
about 6.0, the pH is in the range from about 5.5 to about 6.0, the
pH is in the range from about 5.6 to about 6.0, the pH is in the
range from about 5.7 to about 6.0, or the pH is in the range from
about 5.8 to about 6.0. In certain embodiments of the invention,
the formulation has a pH of 6.0 or about 6.0. In certain
embodiments of the invention, the formulation has a pH of 5.9 or
about 5.9. In certain embodiments of the invention, the formulation
has a pH of 5.8 or about 5.8. In certain embodiments of the
invention, the formulation has a pH of 5.7 or about 5.7. In certain
embodiments of the invention, the formulation has a pH of 5.6 or
about 5.6. In certain embodiments of the invention, the formulation
has a pH of 5.5 or about 5.5. In certain embodiments of the
invention, the formulation has a pH of 5.4 or about 5.4. In certain
embodiments of the invention, the formulation has a pH of 5.3 or
about 5.3. In certain embodiments of the invention, the formulation
has a pH of 5.2 or about 5.2. Examples of buffers that will control
the pH within this range include histidine (such as L-histidine) or
sodium acetate. In certain embodiments, the buffer contains
histidine acetate or sodium acetate in the concentration of about
15 mM to about 25 mM. In certain embodiments of the invention, the
buffer contains histidine acetate or sodium acetate in the
concentration of about 15 mM to about 25 mM, about 16 mM to about
25 mM, about 17 mM to about 25 mM, about 18 mM to about 25 mM,
about 19 mM to about 25 mM, about 20 mM to about 25 mM, about 21 mM
to about 25 mM, about 22 mM to about 25 mM, about 15 mM, about 16
mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21
mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM. In one
embodiment, the buffer is histidine acetate or sodium acetate in an
amount of about 20 mM, pH 5.0. In one embodiment, the buffer is
histidine acetate or sodium acetate in an amount of about 20 mM, pH
5.1. In one embodiment, the buffer is histidine acetate or sodium
acetate in an amount of about 20 mM, pH 5.2. In one embodiment, the
buffer is histidine acetate or sodium acetate in an amount of about
20 mM, pH 5.3. In one embodiment, the buffer is histidine acetate
or sodium acetate in an amount of about 20 mM, pH 5.4. In one
embodiment, the buffer is histidine acetate or sodium acetate in an
amount of about 20 mM, pH 5.5. In one embodiment, the buffer is
histidine acetate or sodium acetate in an amount of about 20 mM, pH
5.6. In one embodiment, the buffer is histidine acetate or sodium
acetate in an amount of about 20 mM, pH 5.7. In one embodiment, the
buffer is histidine acetate or sodium acetate in an amount of about
20 mM, pH 5.8. In one embodiment, the buffer is histidine acetate
or sodium acetate in an amount of about 20 mM, pH 5.9. In one
embodiment, the buffer is histidine acetate or sodium acetate in an
amount of about 20 mM, pH 6.0. In one embodiment, the buffer is
histidine acetate or sodium acetate in an amount of about 20 mM, pH
6.1. In one embodiment, the buffer is histidine acetate or sodium
acetate in an amount of about 20 mM, pH 6.2. In one embodiment, the
buffer is histidine acetate or sodium acetate in an amount of about
20 mM, pH 6.3. In one embodiment, the buffer is histidine acetate
or sodium acetate in an amount of about 25 mM, pH 5.2. In one
embodiment, the buffer is histidine acetate or sodium acetate in an
amount of about 25 mM, pH 5.3. In one embodiment, the buffer is
histidine acetate or sodium acetate in an amount of about 25 mM, pH
5.4. In one embodiment, the buffer is histidine acetate or sodium
acetate in an amount of about 25 mM, pH 5.5. In one embodiment, the
buffer is histidine acetate or sodium acetate in an amount of about
25 mM, pH 5.6. In one embodiment, the buffer is histidine acetate
or sodium acetate in an amount of about 25 mM, pH 5.7. In one
embodiment, the buffer is histidine acetate or sodium acetate in an
amount of about 25 mM, pH 5.8. In one embodiment, the buffer is
histidine acetate or sodium acetate in an amount of about 25 mM, pH
5.9. In one embodiment, the buffer is histidine acetate or sodium
acetate in an amount of about 25 mM, pH 6.0. In one embodiment, the
buffer is histidine acetate or sodium acetate in an amount of about
25 mM, pH 6.1. In one embodiment, the buffer is histidine acetate
or sodium acetate in an amount of about 25 mM, pH 6.2. In one
embodiment, the buffer is histidine acetate or sodium acetate in an
amount of about 25 mM, pH 6.3.
[0267] The formulation further comprises sucrose in an amount of
about 60 mM to about 240 mM. In some embodiments, sucrose in the
formulation is about 60 mM to about 230 mM, about 60 mM to about
220 mM, about 60 mM to about 210 mM, about 60 mM to about 200 mM,
about 60 mM to about 190 mM, about 60 mM to about 180 mM, about 60
mM to about 170 mM, about 60 mM to about 160 mM, about 60 mM to
about 150 mM, about 60 mM to about 140 mM, about 80 mM to about 240
mM, about 90 mM to about 240 mM, about 100 mM to about 240 mM,
about 110 mM to about 240 mM, about 120 mM to about 240 mM, about
130 mM to about 240 mM, about 140 mM to about 240 mM, about 150 mM
to about 240 mM, about 160 mM to about 240 mM, about 170 mM to
about 240 mM, about 180 mM to about 240 mM, about 190 mM to about
240 mM, about 200 mM to about 240 mM, about 80 mM to about 160 mM,
about 100 mM to about 140 mM, or about 110 mM to about 130 mM. In
some embodiments, sucrose in the formulation is about 60 mM, about
70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about
120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM,
about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210
mM, about 220 mM, about 230 mM, or about 240 mM.
[0268] In some embodiments, the antibody concentration in the
formulation is about 40 mg/ml to about 125 mg/ml. In some
embodiments, the antibody concentration in the formulation is about
40 mg/ml to about 120 mg/ml, about 40 mg/ml to about 110 mg/ml,
about 40 mg/ml to about 100 mg/ml, about 40 mg/ml to about 90
mg/ml, about 40 mg/ml to about 80 mg/ml, about 40 mg/ml to about 70
mg/ml, about 50 mg/ml to about 120 mg/ml, about 60 mg/ml to about
120 mg/ml, about 70 mg/ml to about 120 mg/ml, about 80 mg/ml to
about 120 mg/ml, about 90 mg/ml to about 120 mg/ml, or about 100
mg/ml to about 120 mg/ml. In some embodiments, the antibody
concentration in the formulation is about 60 mg/ml. In some
embodiments, the antibody concentration in the formulation is about
65 mg/ml. In some embodiments, the antibody concentration in the
formulation is about 70 mg/ml. In some embodiments, the antibody
concentration in the formulation is about 75 mg/ml. In some
embodiments, the antibody concentration in the formulation is about
80 mg/ml. In some embodiments, the antibody concentration in the
formulation is about 85 mg/ml. In some embodiments, the antibody
concentration in the formulation is about 90 mg/ml. In some
embodiments, the antibody concentration in the formulation is about
95 mg/ml. In some embodiments, the antibody concentration in the
formulation is about 100 mg/ml. In some embodiments, the antibody
concentration in the formulation is about 110 mg/ml. In some
embodiments, the antibody concentration in the formulation is about
125 mg/ml.
[0269] In some embodiments, a surfactant is added to the antibody
formulation. Exemplary surfactants include nonionic surfactants
such as polysorbates (e.g. polysorbates 20, 80 etc) or poloxamers
(e.g. poloxamer 188, etc.). The amount of surfactant added is such
that it reduces aggregation of the formulated antibody and/or
minimizes the formation of particulates in the formulation and/or
reduces adsorption. For example, the surfactant may be present in
the formulation in an amount from about 0.001% to about 0.5% (w/v).
In some embodiments, the surfactant (e.g., polysorbate 20) is from
about 0.005% to about 0.2%, from about 0.005% to about 0.1%, from
about 0.005% to about 0.09%, from about 0.005% to about 0.08%, from
about 0.005% to about 0.07%, from about 0.005% to about 0.06%, from
about 0.005% to about 0.05%, from about 0.005% to about 0.04%, from
about 0.008% to about 0.06%, from about 0.01% to about 0.06%, from
about 0.02% to about 0.06%, from about 0.01% to about 0.05%, or
from about 0.02% to about 0.04%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.005% or about 0.005%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.006% or about 0.006%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.007% or about 0.007%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.008% or about 0.008%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.009% or about 0.009%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.01% or about 0.01%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.02% or about 0.02%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.03% or about 0.03%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.04% or about 0.04%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.05% or about 0.05%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.06% or about 0.06%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.07% or about 0.07%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.08% or about 0.08%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.1% or about 0.1%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.2% or about 0.2%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.3% or about 0.3%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.4% or about 0.4%. In certain embodiments, the
surfactant (e.g., polysorbate 20) is present in the formulation in
an amount of 0.5% or about 0.5%.
[0270] In one embodiment, the formulation contains the
above-identified agents (e.g., antibody, buffer, sucrose, and/or
surfactant) and is essentially free of one or more preservatives,
such as benzyl alcohol, phenol, m-cresol, chlorobutanol and
benzethonium Cl. In another embodiment, a preservative may be
included in the formulation, particularly where the formulation is
a multidose formulation. The concentration of preservative may be
in the range from about 0.1% to about 2%, preferably from about
0.5% to about 1%. One or more other pharmaceutically acceptable
carriers, excipients or stabilizers such as those described in
Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980) may be included in the formulation provided that they do not
adversely affect the desired characteristics of the formulation.
Acceptable carriers, excipients or stabilizers are nontoxic to
recipients at the dosages and concentrations employed and include;
additional buffering agents; co-solvents; anti-oxidants including
ascorbic acid and methionine; chelating agents such as EDTA; metal
complexes (e.g. Zn-protein complexes); biodegradable polymers such
as polyesters; and/or salt-forming counterions. Exemplary
pharmaceutically acceptable carriers herein further include
insterstitial drug dispersion agents such as soluble neutral-active
hyaluronidase glycoproteins (sHASEGP), for example, human soluble
PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX.RTM.,
Baxter International, Inc.). Certain exemplary sHASEGPs and methods
of use, including rHuPH20, are described in US Patent Publication
Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is
combined with one or more additional glycosaminoglycanases such as
chondroitinases.
[0271] The formulation herein may also contain more than one
protein as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect the other protein. For example, where the antibody
is anti-PDL1, it may be combined with another agent (e.g., a
chemotherapeutic agent, and anti-neoplastic agent).
[0272] In some embodiments, the physical stability, chemical
stability, or biological activity of the antibody in the
formulation is evaluated or measured. Any methods known in the art
and described in the Examples herein may be used to evaluate the
stability and biological activity of the antibody in the
formulation. For example, stability of the antibody in the
formulation can be measured by, but not limited to, size exclusion
chromatography (SEC or SE-HPLC), imaged capillary isoelectric
focusing (ICIEF), peptide mapping, small-volume light obscuration
(HIAC) assay, and capillary electrophoresis (CE) techniques such as
CE-sodium dodecyl sulfate (CE-SDS) and CE-glycan analysis. In some
embodiments, the antibody in the formulation is stable at
-20.degree. C. for at least about 6 months, at least about 8
months, at least about 10 months, at least about 12 months, at
least about 14 months, at least about 16 months, at least about 18
months, at least about 20 months, at least about 21 months, at
least about 22 months, at least about 23 months, at least about 24
months, at least about 3 years, or at least about 4 years. In some
embodiments, the antibody in the formulation is stable at 2.degree.
C. to 8.degree. C. (e.g., 5.degree. C.) for at least about 6
months, at least about 8 months, at least about 10 months, at least
about 12 months, at least about 14 months, at least about 16
months, at least about 18 months, at least about 20 months, at
least about 21 months, at least about 22 months, at least about 23
months, or at least about 24 months. In some embodiments, the
stability of the antibody (i.e., an antibody monomer) is measured
by size exclusion chromatography in the formulation after storage.
In some embodiments, the stability of the antibody is (i.e., an
antibody monomer) measured by imaged capillary isoelectric focusing
in the formulation after storage. In some embodiments, the percent
of antibody monomer in the formulation as compared to total protein
(e.g., including antibody and aggregates) is greater than about
60%, about 65%, about 70%, about 75%, about 80%, about 85%, about
86%, about 87%, about 88%, about 89%, about 90%, about 91%, about
92%, about 93%, about 94% or about 95% after storage at -20.degree.
C. for at least about 6 months, at least about 12 months, at least
about 18 months, or at least about 24 months. In some embodiments,
the percent of antibody monomer in the formulation as compared to
(e.g., including antibody and aggregates) is greater than about
60%, about 65%, about 70%, about 75%, about 80%, about 85%, about
86%, about 87%, about 88%, about 89%, about 90%, about 91%, about
92%, about 93%, about 94% or about 95% after storage at 2.degree.
C. to 8.degree. C. (e.g., 5.degree. C.) for at least about 6
months, at least about 12 months, at least about 18 months, or at
least about 24 months. In some embodiments, the percent of antibody
monomer in the formulation as compared to (e.g., including antibody
and aggregates) is greater than about 60%, about 65%, about 70%,
about 75%, about 80%, about 85%, about 86%, about 87%, about 88%,
about 89%, about 90%, about 91%, about 92%, about 93%, about 94% or
about 95% after agitation at room temperature (e.g., about
15.degree. C. to 25.degree. C.) for at least about 2 hours, at
least about 4 hours, at least about 6 hours, at least about 8
hours, at least about 10 hours, at least about 12 hours, at least
about 14 hours, at least about 16 hours, at least about 18 hours,
at least about 20 hours, or at least about 24 hours. In some
embodiments, the percent of total aggregates (e.g., high molecular
weight species and low molecular weight species) in the formulation
is less than any of about 0.1%, about 0.2%, about 0.3%, about 0.4%,
about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about
1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,
about 8%, about 9%, or about 10% after storage at -20.degree. C.
for at least about 6 months, at least about 12 months, at least
about 18 months, or at least about 24 months. In some embodiments,
the percent of total aggregates (e.g., high molecular weight
species and low molecular weight species) in the formulation is
less than any of about 0.1%, about 0.2%, about 0.3%, about 0.4%,
about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about
1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,
about 8%, about 9%, or about 10% after storage at 2.degree. C. to
8.degree. C. (e.g., 5.degree. C.) for at least about 6 months, at
least about 12 months, at least about 18 months, or at least about
24 months. In some embodiments, the percent of total aggregates
(e.g., high molecular weight species and low molecular weight
species) in the formulation is less than any of about 0.1%, about
0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%,
about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%,
about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%
after agitation at room temperature (e.g., about 15.degree. C. to
25.degree. C.) for at least about 2 hours, at least about 4 hours,
at least about 6 hours, at least about 8 hours, at least about 10
hours, at least about 12 hours, at least about 14 hours, at least
about 16 hours, at least about 18 hours, at least about 20 hours,
or at least about 24 hours. In any of the embodiments herein, the
stable formulation can be stored in a glass vial, a metal alloy
container, or an intravenous (IV) bag. In some embodiments, the
metal alloy is 316L stainless steel or hastelloy.
[0273] The formulations to be used for in vivo administration
should be sterile. This is readily accomplished by filtration
through sterile filtration membranes, prior to, or following,
preparation of the formulation.
III. Methods of Treatment and Administration of Antibody
Formulations
[0274] The formulation is administered to a mammal in need of
treatment with the antibody, preferably a human, in accord with
known methods, such as intravenous administration (e.g., as a bolus
or by continuous infusion over a period of time), by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes. In
one embodiment, the formulation is administered to the mammal by
intravenous administration. For such purposes, the formulation may
be injected using a syringe or via an IV line, for example. In one
embodiment, the formulation is administered to the mammal by
subcutaneous administration.
[0275] The appropriate dosage ("therapeutically effective amount")
of the antibody will depend, for example, on the condition to be
treated, the severity and course of the condition, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, the type of antibody used, and the discretion of the
attending physician. The antibody is suitably administered to the
patient at one time or over a series of treatments and may be
administered to the patient at any time from diagnosis onwards. The
antibody may be administered as the sole treatment or in
conjunction with other drugs or therapies useful in treating the
condition in question.
[0276] As a general proposition, the therapeutically effective
amount of the antibody administered to human will be in the range
of about 0.01 to about 50 mg/kg of patient body weight whether by
one or more administrations. In some embodiments, the antibody used
is about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg,
about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about
0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to
about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5
mg/kg, or about 0.01 to about 1 mg/kg administered daily, for
example. In some embodiments, the antibody is administered at 15
mg/kg. However, other dosage regimens may be useful. In one
embodiment, an anti-PDL1 antibody described herein is administered
to a human at a dose of about 100 mg, about 200 mg, about 300 mg,
about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800
mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg,
about 1300 mg or about 1400 mg on day 1 of 21-day cycles. The dose
may be administered as a single dose or as multiple doses (e.g., 2
or 3 doses), such as infusions. The dose of the antibody
administered in a combination treatment may be reduced as compared
to a single treatment. The progress of this therapy is easily
monitored by conventional techniques.
[0277] The formulations containing anti-PDL1 antibody described
herein can be used in a variety of in vitro and in vivo diagnostic
and therapeutic applications. For example, the formulation
containing the antibody may be administered to a subject or an
individual for treating a disease or disorder (e.g., disease or
disorder mediated by the PD-1 and PD-L1 interaction).
[0278] In some embodiments, the disease or disorder is cancer. In
some embodiments, the cancer is locally advanced or metastatic. In
some embodiments, the cancer is selected from the group consisting
of a solid tumor, a hematologic cancer, bladder cancer, brain
cancer, breast cancer, colon cancer, colorectal cancer, gastric
cancer, glioma, head cancer, leukemia, liver cancer, lung cancer
(e.g., non-small cell lung cancer), lymphoma, myeloma, neck cancer,
ovarian cancer, melanoma, pancreatic cancer, renal cancer, salivary
cancer, stomach cancer, thymic epithelial cancer, thyroid cancer,
and squamous cell carcinoma of the head and neck. In some
embodiments, the subject or individual treated has PD-L1 positive
cancer cells (e.g., detected by IHC).
[0279] In some embodiments, the disease or disorder is infection.
In some embodiments, the infection is a persistent infection. In
some embodiments, the infection is a viral infection, a bacterial
infection, a fungal infection, a helminth infection, or a protozoan
infection. In some embodiments, the viral infection is selected
from the group consisting of cytomegalovirus Epstein-Barr virus,
hepatitis B, hepatitis C virus, herpes virus, measles virus,
influenza, human immunodeficiency virus, human T lymphotropic
virus, lymphocytic choriomeningitis virus, respiratory syncytial
virus, and/or rhinovirus. In some embodiments, the bacterial
infection is selected from the group consisting of Helicobacter
spp., Mycobacterium spp., Porphyromonas spp., Chlamydia spp.,
Salmonella spp., Listeria spp., Streptococcus spp., Haemophilus
spp., Neisseria spp., Klebsiella spp., Borrelia spp., Bacterioides
spp., and Treponema spp. In some embodiments, the protozoan
infection is selected from the group consisting of Leishmania spp.,
Plasmodium falciparum, Schistosoma spp., Toxoplasma spp.,
Trypanosoma spp., and Taenia spp. In some embodiments, the fungal
infection is selected from the group consisting of blastomycosis,
coccidioiodmycosis, histoplamsosis, candidiasis, cryptococcosis,
aspergillossi, mucomycosis and pneumocystosis.
[0280] In some embodiments, the disease or disorder is an
inflammatory disease. In some embodiments, the inflammatory disease
is selected from the group consisting of acute disseminated
encephalomyelitis, Addison's disease, Alzheimer's disease,
ankylosing spondylitis, antiphospholipid antibody syndrome,
atherosclerosis, autoimmune hemolytic anemia, autoimmune hepatitis,
arthritis, Behcet's disease, Berger's disease, Bullous pemphigoid,
Celiac disease, Chagas' disease, cholangitis, Crohn's disease,
Dermatomyositis, Diabetes mellitus type 1, glomerulonephritis,
Goodpasture's syndrome, graft-versus-host disease, Graves' disease,
Guillain-Barre syndrome, Hashimoto's disease, hives, hyper IgE
syndrome, idiopathic thrombocytopenic purpura, lupus erythematosus,
lupus nephritis, multiple sclerosis, myasthenia gravis, organ
transplant rejection, Parkinson's disease, pemphigus, pernicious
anaemia, polymyositis, primary biliary cirrhosis, psoriasis,
Raynaud's syndrome, rheumatoid arthritis, scleroderma, Sjogren's
syndrome, temporal arteritis, thyroiditis, ulcerative colitis,
uveitis, vasculitis, and Wegener's granulomatosis.
[0281] In some embodiments, the formulation containing the antibody
may be administered in conjunction with another therapeutic agent
to a subject or an individual for treating a disease or disorder.
For example, for treating cancer, the anti-PDL1 antibody
formulation described herein may administered in conjunction with
another anti-cancer treatment (e.g., a chemotherapy or a different
antibody treatment).
IV. Articles of Manufacture or Kits
[0282] In another embodiment of the invention, an article of
manufacture or a kit is provided comprising a container which holds
the aqueous pharmaceutical formulation of the invention and
optionally provides instructions for its use. Suitable containers
include, for example, bottles, vials, bags and syringes. The
container may be formed from a variety of materials such as glass,
plastic (such as polyvinyl chloride or polyolefin), or metal alloy
(such as stainless steel or hastelloy). An exemplary container is a
300 cc metal alloy container (e.g., for storing at -20.degree. C.).
Another exemplary container may be 10-50 cc glass vial (e.g., for
storing at 2-8.degree. C.). For example, the container may be 10
cc, 15 cc, 20 cc, or 50 cc glass vials. The container holds the
formulation and the label on, or associated with, the container may
indicate directions for use. The article of manufacture may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use. In some
embodiments, the article of manufacture further includes one or
more of another agent (e.g., a chemotherapeutic agent, and
anti-neoplastic agent). Suitable containers for the one or more
agent include, for example, bottles, vials, bags and syringes.
[0283] The specification is considered to be sufficient to enable
one skilled in the art to practice the invention. Various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description and fall within the scope of the
appended claims. All publications, patents, and patent applications
cited herein are hereby incorporated by reference in their entirety
for all purposes.
EXAMPLES
[0284] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that various modifications or changes in light
thereof will be suggested to persons skilled in the art and are to
be included within the spirit and purview of this application and
scope of the appended claims.
Example 1: Formulation Development of an Anti-PDL1 Antibody
[0285] Anti-PDL1 antibody (.alpha.-PDL1) is a CHO-derived
aglycosylated IgG1 antibody intended to restore T cell function
through inhibition of PDL1/PD1 interactions. Challenges at the
outset of development included potential Trp oxidation and
glycation in or near CDR regions and some methionine oxidation.
Pre-robustness studies indicated a higher pH than previously
targeted (pH 5.5) was optimal. The target dosing was a fixed dose
but a weight based dose was also contemplated. Analytical studies
were conducted to analyze stability of various formulations and a
formulation (60 mg/mL .alpha.-PDL1, 20 mM His AcO pH 5.8, 120 mM
sucrose, 0.04% PS20) was selected. Initial formulation studies
support up to three years of stability in Drug Substance (DS) and
Drug Product (DS).
[0286] Methods and Materials
Production of .alpha.-PDL1 Formulations
[0287] .alpha.-PDL1 material that had undergone
ultrafiltration/diafiltration was subjected to formulation
development studies. The material was dialyzed into various
formulation buffers using 10000 Dalton dialysis cassettes. After
dialysis, protein concentrations were adjusted to reach target
concentrations and 10% PS20 stock solution was spiked in to achieve
targeted PS20 concentrations. The formulated material was filled
aseptically into 2-cc Forma Vitrum glass vials with 1 mL fill
volume and sealed with a 13 mm Daikyo 777-1 stopper. Samples were
stored upright at either 5.degree. C., 25.degree. C. or 40.degree.
C.
Color, Appearance, and Clarity (CAC)
[0288] Sample color, appearance, and clarity were determined by
visual inspection under a white fluorescence light with black and
white background at room temperature as described in the European
Pharmacopoeia (EP) methods (Council of Europe. European
Pharmacopoeia, 2008, 7.sup.th Ed., EP 2.2.2 and EP 2.2.1). A 3 cc
glass vial was filled with 1 mL of each sample tested. A negative
control (purified water) with the corresponding sample volume was
used for comparison.
Protein Concentration Measurements
[0289] The protein concentration was determined by measurement of
the UV-absorbance on an Agilent 8453 spectrophotometer (Santa
Clara, Calif.) via volumetric sample dilution to approximately 0.5
mg/mL with 0.9% saline. The samples were blanked against 0.9%
saline and the absorbance was measured at the A.sub.max of
approximately 280 nm and also at 320 nm. The difference between
A.sub.max and A.sub.320 was calculated to obtain the corrected
A.sub.max used to determine the final protein concentration with an
absorptivity of 1.5 mL cm.sup.-1 mg.sup.-1.
Turbidity Measurements
[0290] The average optical density at 350 nm of the samples was
measured in a quartz cuvette with a 1-cm path length on an Agilent
8453 spectrophotometer. Purified water was used as a blank.
Light Obscuration Method for Subvisible Particles (HIAC Assay)
[0291] Particulate counts of samples were performed using light
obscuration measured by the HIAC-Royco model 9703 (HACH, Loveland,
Colo.). Average cumulative numbers of particles per milliliter
.gtoreq.2 .mu.m, .gtoreq.5 .mu.m, .gtoreq.10 .mu.m and .gtoreq.25
.mu.m were tabulated for each sample using PharmSpec v2.0. Four
readings, consuming a total of 1.6 mL of each sample, were
performed per test, with the first reading discarded, and the
remaining 3 readings averaged.
Size Exclusion Chromatography (SEC or SE-HPLC)
[0292] Size variant distribution was determined by size exclusion
chromatography (SEC) using a TosoHaas Bioscience column G3000 SWXL
(South San Francisco, Calif.) at 30.degree. C. on an Agilent 1200
HPLC (Santa Clara, Calif., USA). All samples were injected
undiluted at 50 .mu.g onto the column and eluted over 60 minutes
with UV absorption at 280 nm. Two different SEC methods were used
for sample testing. Method 1 used 0.20 M potassium phosphate, 0.25
M potassium chloride, pH 6.2, while method 2 used 0.20 M potassium
phosphate, 0.25 M potassium chloride, pH 6.2 with 10% (v/v)
isopropanol as the mobile phase. Results are reported as relative
percent peak area of the total area under the curve.
Imaged Capillary Isoelectric Focusing (ICIEF)
[0293] The distribution of charge variants was assessed by iCIEF
using an iCE280 analyzer (ProteinSimple) with a fluorocarbon coated
capillary cartridge (100 .mu.m.times.5 cm). The ampholyte solution
consisted of a mixture of 0.35% methyl cellulose (MC), 0.75%
Pharmalyte 3-10 carrier ampholytes, 4.2% Pharmalyte 8-10.5 carrier
ampholytes, and 0.2% pI marker 7.40 and 0.15% pI marker 9.77 in
purified water. The anolyte was 80 mM phosphoric acid, and the
catholyte was 100 mM sodium hydroxide, both in 0.10%
methylcellulose. Samples were diluted in purified water and CpB was
added to each diluted sample at an enzyme to substrate ratio of
1:100 followed by incubation at 37.degree. C. for 20 minutes. The
CpB treated samples were mixed with the ampholyte solution and then
focused by introducing a potential of 1500 V for one minute,
followed by a potential of 3000 V for 10 minutes. An image of the
focused .alpha.-PDL1 charge variants was obtained by passing 280 nm
ultraviolet light through the capillary and into the lens of a
charge coupled device digital camera. This image was then analyzed
to determine the distribution of the various charge variants.
Peptide Mapping
[0294] A peptide mapping technique was used to monitor tryptophan
(W) and methionine (M) oxidation. To generate .alpha.-PDL1 peptide
maps, the protein was digested with trypsin after exposing the
protein to dithiothreitol (DTT) and iodoacetic acid (IAA), in a
process that reduces the disulfide bonds and alters the resultant
free thiols to produce carboxymethyl derivatives. The resulting
peptides were separated by reversed-phase high-performance liquid
chromatography (RP-HPLC) and monitored at 214 nm. Masses of the
tryptic peptides were determined by LC-MS analysis of the separated
digest mixture using a ThermoFisher Scientific LTQ-Orbitrap mass
spectrometer.
[0295] Results
Selection of Buffer System
[0296] During formulation development, two buffer systems were
evaluated. One was 20 mM histidine acetate with 240 mM sucrose at
pH 5.5, the other one was 200 mM arginine succinate at pH 5.5. The
accelerated stability study revealed that .alpha.-PDL1 has better
stability in histidine acetate buffer compared to arginine
succinate buffer (Table 1). Therefore histidine acetate was chosen
for further development of formulations.
TABLE-US-00026 TABLE 1 Zero-Order Degradation Rates of .alpha.-PDL1
for ICIEF and SE-HPLC Main Peak in Histidine Acetate and Arginine
Succinate buffers at 30.degree. C. Rate of % Main Peak Decrease per
Month at 30 C. Buffers ICIEF SE-HPLC Histidine Acetate* 5.7 1.0
Arginine Succinate** 17.6 1.5 Note: All formulations were stored
for up to 1 month at 30.degree. C. Analysis was performed using
ICIEF and SE-HPLC; *150 mg/mL .alpha.-PDL1 in 20 mM L-histidine
acetate, 240 mM sucrose, and 0.02% (w/v) polysorbate 20 at pH 5.5;
**150 mg/mL .alpha.-PDL1 in 200 mM arginine succinate, 0.02% (w/v)
polysorbate 20 at pH 5.5.
Selection of Stabilizer
[0297] Sucrose (120 mM) was selected as the stabilizer for the
.alpha.-PDL1 liquid formulation based on its ability to protect the
protein from freeze/thaw induced aggregation as well as function as
a cryoprotectant during long-term frozen storage of the Drug
Substance (DS) and subsequent Drug Product (DP) storage at
2.degree. C.-8.degree. C.
[0298] During formulation development, .alpha.-PDL1 at 50 mg/mL in
20 mM L-histidine acetate, pH 5.5, 0.02% (w/v) polysorbate 20, and
various concentrations of sucrose ranging from 0 mM to 120 mM was
subjected to five freeze/thaw cycles. Product quality measured by
SE-HPLC indicated that 60 mM sucrose was sufficient to prevent a
freeze/thaw induced increase in .alpha.-PDL1 HMWS (Table 2). Also,
120 mM sucrose was shown to maintain stability of the Drug
Substance when stored frozen at -20.degree. C. for at least 6
months (Table 3). Therefore, based upon results from the
freeze/thaw studies as well as the long-term stability of Drug
Substance stored at -20.degree. C., sucrose at a concentration of
120 mM was chosen as the cryoprotectant for the .alpha.-PDL1 liquid
formulation.
TABLE-US-00027 TABLE 2 Effect of Sucrose Concentration on Stability
of .alpha.-PDL1 SE-HPLC Percent High-Molecular-Weight Species
during Freezing and Thawing Sucrose Conc. SE-HPLC (mM) F/T cycles %
HMWS % Monomer CAC pH T0 NA 1.2 98.8 SY, CL, PFVP 5.6 0 mM 5 1.4
98.6 SY, CL, PFVP 5.7 60 mM 5 1.2 98.8 SY, CL, PFVP 5.7 120 mM 5
1.2 98.8 SY, CL, PFVP 5.6 Note: All formulations contain 50 mg/mL
.alpha.-PDL1, 20 mM L-histidine acetate, 0.02% (w/v) polysorbate
20, pH 5.5. Analysis was performed using SE-HPLC; F/T =
freeze/thaw; HMWS = high-molecular-weight species; SY = slightly
yellow; CL = clear; PFVP = practically free of visible
particles.
TABLE-US-00028 TABLE 3 Long Term Stability Data for .alpha.-PDL1
Drug Substance Development Batch Q12589 Q12631 SEC Q12695 ICIEF Sum
of Mon- Sum of CE-SDS-NGS (non-reduced) Q12708 Q12398 Acidic Main
Basic HMW omer LMW Sum of Main Sum of Potency Time Strength Region
Peak Region Forms Peak Forms Pre- Peak Post- (% Temp (days/ Q12005
Q12003 (mg/ (area (area (area (area (area (area Peaks (% (% Peaks
(% relative (.degree. C.) months) CAC pH mL) %) %) %) %) %) %) CPA)
CPA) CPA) potency) NA T = 0/0 SY, CL, 5.9 60.1 17.3 79.7 3.0 0.7
99.2 0.1 2.7 97.0 0.3 107 PFVP -20.degree. C. 30/1 SY, CL, 5.9 62.9
16.9 80.2 2.9 0.6 99.3 0.1 2.8 97.0 0.2 109 PFVP -20.degree. C.
61/2 SY, CL, 5.9 61.4 16.5 80.8 2.7 0.6 99.4 0.1 2.5 97.3 0.3 NT
PFVP -20.degree. C. 91/3 SY, CL, 5.9 62.5 18.1 79.0 3.0 0.6 99.3
0.1 2.8 97.1 0.2 96 PFVP -20.degree. C. 183/6 SY, CL, 5.9 61.1 17.9
79.0 3.1 0.6 99.4 0.1 3.1 96.6 0.3 100 PFVP 5.degree. C. 30/1 SY,
CL, 5.9 61.1 18.1 79.0 2.9 0.7 99.2 0.1 2.6 97.0 0.4 101 PFVP
5.degree. C. 61/2 SY, CL, 5.9 62.3 17.4 79.8 2.8 0.8 99.2 0.1 2.9
96.7 0.4 NT PFVP 5.degree. C. 91/3 SY, CL, 5.9 63.9 17.4 80.1 2.5
0.9 99.0 0.1 3.0 96.5 0.5 107 PFVP 5.degree. C. 183/6 SY, CL, 5.9
59.5 19.7 77.4 3.0 1.1 98.8 0.1 3.3 95.9 0.8 102 PFVP Note: All
formulations contain 60 mg/mL .alpha.-PDL1 in 20 mM L-histidine
acetate, 120 mM sucrose, 0.04% PS20, pH 5.8. 25cc 316L stainless
steel mini-cans were used for this study; NA = not applicable; CAC
= color, appearance, and clarity; SY = slightly yellow, CL = clear,
PFVP = practically free of visible particulates; HMW = high
molecular weight; LMW = low molecular weight; ICIEF = imaged
capillary isoelectric focusing; CE-SDS = capillary electrophoresis
sodium dodecyl sulfate; NT = not tested; TBD = to be
determined.
Pre-Formulation Robustness Studies: Selection of Protein
Concentration, pH and Polysorbate 20 Concentration
[0299] A fractional factorial design of experiments (DOE) design
was used to further examine the effects of .alpha.-PDL1 formulation
parameters on protein stability. A total of twelve different
.alpha.-PDL1 formulations were tested (ten experiments and two
center points). The three factors varied in the study were pH range
of 5.0-6.0 with 0.5 unit intervals, protein concentration range of
40-120 mg/mL, and polysorbate 20 concentration range of
0.005%-0.06% (w/v) (Table 4). All formulations were buffered by 20
mM histidine acetate with 120 mM sucrose except the last two
formulations as indicated in Table 4. The 25 mM histidine acetate
formulation was evaluated since it was considered to be a worst
case scenario in terms of oxidation risk. The 20 mM sodium acetate
buffer was evaluated as a back-up buffer system and compared to
histidine acetate buffer. The formulations were stored at
25.degree. C. for 2 months and 40.degree. C. for 1 month. The
stability data from the above studies were statistically analyzed
for interactions between the formulation parameters using JMP
software (JMP, Version 9, SAS Institute Inc., Cary, N.C.).
TABLE-US-00029 TABLE 4 .alpha.-PDL1 Drug Substance and Drug Product
Formulations Evaluated in the DOE study anti-PDL1 Solution PS20
His-Acetate Sucrose Formulation (mg/mL) pH (% w/v) (mM) (mM)
F1.sup.a 50 5.5 0.04 20 120 F2.sup.a 100 5.5 0.04 20 120 F3 40 6.0
0.06 20 120 F4 120 5.0 0.06 20 120 F5 120 6.0 0.005 20 120 F6 40
5.0 0.06 20 120 F7 120 5.0 0.005 20 120 F8 40 6.0 0.005 20 120 F9
40 5.0 0.005 20 120 F10 120 6.0 0.06 20 120 F11.sup.b 50 5.5 0.06
25 120 F12.sup.c 50 5.5 0.04 20 (Na-Ace) 120 Note: .sup.aCenter
points; .sup.bWorst case scenario: low protein concentration, high
PS20 concentration, high histidine concentration; .sup.c20 mM
sodium acetate (Na-Ace) buffer was tested.
[0300] In comparison to pH 5.0 and 5.5, the formulation at pH 6.0
has slightly slower main peak loss rate, as determined by ICIEF at
40.degree. C. and 25.degree. C. (FIG. 1A-B and FIG. 2A-B,
respectively). No significant impact of concentration on main peak
loss was observed by ICIEF. Analysis of formulation F1 showed that
an acidic variant increase contributed primarily to main peak loss
in ICIEF while the contribution to peak loss by a basic charge
variant was not significant. Under the same storage conditions, the
formulation at pH 6.0 also had a slower monomer peak loss rate, as
measured by SE-HPLC at 40.degree. C. and 25.degree. C. (FIG. 3A-B
and FIG. 4A-B, respectively). Analysis of formulation F1 showed
that both HMWS and LMWS formation contributed to monomer loss in
SEC at elevated temperatures (i.e., 40.degree. C. and 25.degree.
C.). Both the SEC and ICIEF pH rate profiles revealed that pH
5.5-6.0 is the optimal pH range for .alpha.-PDL1. To be within
optimal protein stability above pH 5.5 and to allow for a .+-.0.3
pH unit range in the formulated Drug Substance and Drug Product, a
target of pH 5.8 was chosen.
[0301] The above formulation studies also revealed that 120 mg/mL
of .alpha.-PDL1 formulations at pH range of 5.0-6.0 had a slightly
higher but non-significant monomer peak loss rate due to higher
HMWS formation rate compared to 40 mg/mL formulations at the same
pH, as determined by SE-HPLC (FIG. 3A-B and FIG. 4A-B). Based on
these data and to support a formulation with improved product
stability and to facilitate patient dosing, .alpha.-PDL1 at a
concentration of 60 mg/mL was selected.
[0302] No impact on protein stability was observed with polysorbate
20 (PS20) concentrations ranging from 0.005%-0.06% (w/v) as
indicated in the above statistical analysis (FIGS. 1-4).
[0303] It has been known that hydrogen peroxide impurity contained
in polysorbate 20 raw material can cause tryptophan (W) and
methionine (M) oxidation. L-histidine can also increase the above
oxidation risk. The samples of selected worst case scenario
formulations containing higher concentrations of polysorbate 20 and
L-histidine were analyzed by peptide mapping. Results of the
analysis showed that even the combination of higher histidine
concentration (25 mM histidine acetate buffer) and higher amount of
PS20 (0.06% PS20) didn't demonstrate significant oxidation risk
(Table 5) and histidine buffer is suitable for use to formulate
.alpha.-PDL1.
TABLE-US-00030 TABLE 5 Percentage of Trp and M.sup.253 oxidation in
Selected Formulations by Peptide Map Selected Formulations %
Oxidation Conc. Buffer PS20 W CDR W CDR W CDR LC27 (mg/mL) (mM) (%)
Time points HC2 HC4 HC10 M253 F1 50 20 mM His-Ace 0.04 T0 0.1 0.1
0.1 5.5 F3 40 20 mM His-Ace 0.06 25 C, 2M 0.2 0.2 0.2 6.4 F10 120
20 mM His-Ace 0.06 25 C, 2M 0.2 0.1 0.2 6.7 F11 50 25 mM His-Ace
0.06 25 C, 2M 0.2 0.2 0.2 6.6 Note: All formulations were stored
for up to 1 month at 40.degree. C. Analysis was performed using
Peptide map. W = Tryptophan; M = Methionine
[0304] To assess the possible degradation of PS20 in the
formulation upon storage, Formulations F1 to F10 (Table 4) were
stored at 40.degree. C. for 1 month, 25.degree. C. for 2 months,
5.degree. C. for 2 months or 5.degree. C. for 6 months. No PS20
degradation was observed in the evaluated formulations at the any
of the elevated (i.e., 40.degree. C. and 25.degree. C.) and
5.degree. C. storage temperature. Altering the fill volume of
selected formulations (i.e., F1, F2, F3, and F6) to 7 ml (high
fill) or 4 ml (low fill) and then storing at 5.degree. C. for 6
months also did not have a significant impact on the PS20
degradation rate (FIG. 5).
[0305] The formation of sub-visible particles (SbVP) in the
different formulations when stored at 5.degree. C. for 6 months was
assessed by the HIAC assay as a measure of stability (Table 6). No
measureable change in SbVP was observed in the formulation
tested.
TABLE-US-00031 TABLE 6 HIAC data for SbVP formation after 6 months
storage at 5.degree. C. Time Point Particle Size (Cumulative
Counts/mL) Sample (month) 2 .mu.M 5 .mu.M 10 .mu.M 25 .mu.M F1 0
802 193 61 5 6 1190 278 80 6 F2 0 799 146 43 12 6 370 112 29 2 F3 0
485 133 34 4 6 163 52 14 2 F4 0 211 65 31 8 6 181 48 8 1 F5 0 872
359 195 79 6 340 89 23 1 F6 0 233 61 16 3 6 116 34 16 3 F7 0 134 29
13 4 6 144 42 9 0 F8 0 433 118 34 1 6 564 98 23 2 F9 0 498 114 17 1
6 144 21 6 0 F10 0 610 124 23 0 6 248 75 28 3 Note: Two 1 mL fill
vials were combined together to perform a small volume HIAC
assay.
[0306] Stability of the formulations was further investigated with
a freeze thaw experiment. Formulations F1 through F10 (Table 4)
were subjected to either five freeze thaw cycles during storage at
-20.degree. C. or were stored at an elevated storage temperature of
5.degree. C. from 0 to 6 months and subsequently analyzed by SEC
and ICIEF for percentage of .alpha.-PDL1 monomer (FIGS. 6A and B)
and percentage of main peak in formulation (FIGS. 6C and D). No
significant change in percent monomer and percent main peak was
observed after the freeze thaw cycles and storage at the indicated
time points.
[0307] The Drug Substance stability in the F2 formulation (Table 4)
was assessed by conducting five freeze thaw cycles during storage
in a stainless steel minican at -20.degree. C. for up to 6 months
followed by stability measurement by CAC, SEC, and ICIEF (Table 7).
No change was observed after 6 months storage at -20.degree. C.
TABLE-US-00032 TABLE 7 Drug Substance stability in a stainless
steel minican stored at -20.degree. C. Q12005 Q12589 Q12631 CAC SEC
ICIEF Time Points F/T Cycles Clarity (% monomer) (% main peak) T0 0
CL/SY 98.6 80.1 1M 1 CL/SY 98.6 79.1 2M 2 CL/SY 98.7 80.2 3M 3
CL/SY 98.8 80.9 6M 5 CL/SY 98.6 80.2 Note: F/T = freeze/thaw; SY =
slightly yellow; CL = clear.
[0308] The Drug Substance stability in a formulation containing 100
mg/mL .alpha.-PDL1, 20 mM histidine acetate, 120 mM sucrose, 0.04%
PS20, pH 5.6 was assessed by conducting three freeze thaw cycles
followed by storage in a stainless steel minican or hastelloy
minican at -20.degree. C., 5.degree. C., or 25.degree. C. for up to
3 months followed by stability measurement by SEC (FIGS. 7A and B).
No difference was observed between storage in stainless steel and
hastelloy minicans at pH 5.6. The Drug Substance was stable for up
to 3 months at -20.degree. C. after three freeze thaw cycles.
Despite slight differences in stainless steel and hastelloy
minicans, both were appropriate for use for drug substance
storage.
[0309] The Drug Product stability in a formulation containing 50
mg/mL .alpha.-PDL1, 20 mM histidine acetate, 120 mM sucrose, 0.04%
PS20, pH 5.6 was assessed when stored as 16 mL fill in a 20 cc vial
at -5.degree. C., 25.degree. C., or 40.degree. C. for up to 3
months followed by stability measurement with SEC and ICIEF (FIGS.
8A and B). No change was observed at 5.degree. C. after three
months of storage. The pH 5.6 degradation rate per month at
40.degree. C. was 0.66% and 22% by SEC and ICIEF analysis,
respectively.
[0310] Assessment of the buffer in the F12 formulation indicated
that the sodium acetate buffer provided similar protein stability
as histidine acetate buffer, based on main peak degradation rates
measured by SE-HPLC and ICIEF (Table 8). The two formulations
tested were 50 mg/mL .alpha.-PDL1 in 20 mM L-histidine acetate, 120
mM sucrose, and 0.04% (w/v) polysorbate 20 at pH 5.5 and 0 mg/mL
.alpha.-PDL1 in 20 mM sodium acetate, 120 mM sucrose, and 0.04%
(w/v) polysorbate 20 at pH 5.5.
TABLE-US-00033 TABLE 8 Zero-Order Degradation Rates of .alpha.-PDL1
for ICIEF and SE-HPLC Main Peak in Histidine Acetate and Sodium
Acetate buffers at 40.degree. C. .alpha.-PDL1 Rate of % Main Peak
Concentration Decrease per Month (mg/mL) ICIEF SE-HPLC Histidine
Acetate 23 0.67 Sodium Acetate 21 0.74 Note: All formulations were
stored for up to 1 month at 40.degree. C.
[0311] Overall, the DoE designed stability studies revealed that at
40.degree. C. no significant impact of concentration on main peak
loss was observed by ICIEF, while lower pH has a slightly faster
main peak rate loss (FIG. 1A-B). At 40.degree. C. no significant
interactions were observed by SE-HPLC either, however, the higher
concentration formulations show a faster monomer loss (FIG. 3A-B).
It was also found that lower pH has a faster monomer rate loss.
Similar results were observed at 25.degree. C. (FIG. 2A-B and FIG.
4A-B). The statistical analysis revealed no practically meaningful
interactions (linkage) between any of the tested formulation
parameters.
Agitation and Thermal Stress Studies
[0312] Stability of the drug product in the presence of increasing
concentrations of PS20 when undergoing agitation stress in glass
vials was investigated. A formulation containing 57 mg/mL in 20 mM
histidine acetate, 120 mM sucrose, pH 5.5 was assessed in a 1 mL
fill in 2 cc glass vials with various concentrations of PS20
ranging from 0.005% to 0.06%. Glass vials were agitated at 70 rpm
for 3 days at room temperature prior to measurement of stability by
SEC (FIG. 9A) and turbidity (FIG. 9B) measurements. Formulation
with PS20 levels between 0.005-0.06% had no change in stability
during agitation. However, formulations lacking PS20 showed an
increase in monomer loss due to an HMWS increase. In this
experiment, 0.005% PS20 was sufficient to protect protein from
agitation stress in glass vials.
[0313] Stability of the drug product formulations (Table 4) when
stored at various temperature and time and then undergoing
agitation stress in glass vials was investigated. Formulations
F1-F10 were each assessed in a 1 mL fill in 2 cc glass vial. Glass
vials were agitated at 70 rpm for 1 day at room temperature prior
to measurement of stability by SEC (FIG. 10). In this experiment,
agitation has no impact on the stability of drug product when
stored for a length of time at 40.degree. C., 25.degree. C. or
5.degree. C.
[0314] In order to support IV bag transportation which often occurs
in hospital settings, an IV bag agitation study was performed with
.alpha.-PDL1 formulated in 20 mM histidine acetate, 240 mM sucrose,
pH 5.5 with 0.005%-0.02% (w/v) polysorbate 20. The most commonly
available 250 mL polyvinyl chloride (PVC) or polyolefin (PO) IV
bags containing isotonic sodium chloride solution (0.9% NaCl) were
evaluated by injecting 400-600 mg of .alpha.-PDL1 solutions and
agitated using orbital shaker at 100 rpm at 5.degree. C. for up to
6 hours. The results of the study supported weight-based dosing and
demonstrated that a minimum of 0.015% (w/v) of polysorbate 20 in
protein solution is needed in order to prevent visible particles
formation (related to protein precipitation) during transportation
(Table 9). In addition, to mitigate the risk of polysorbate 20
degradation over shelf life, the polysorbate 20 concentration was
increased from 0.02% (w/v) to 0.04% (w/v).
TABLE-US-00034 TABLE 9 IV Bag Agitation Study with Different Amount
of PS20 in .alpha.-PDLI Drug Product Subvisible particles % PS20 in
SE-HPLC (ppmL) DP Samples CAC % HMWS % Monomer .gtoreq.10 um
.gtoreq.25 um 0.005% 250 mL PO bag, T0 CO, CL, PFVP NT NT NT NT 250
mL PO bag, agitation at Visible particles NT NT NT NT 5.degree. C.
for 2 hours observed Experiment stopped 250 mL PVC bag, T0 CO, CL,
PFVP NT NT NT NT 250 mL PVC bag, agitation at Visible particles NT
NT NT NT 5.degree. C. for 2 hours observed Experiment stopped 0.01%
250 mL PO bag, T0 CO, CL, PFVP NT NT NT NT 250 mL PO bag, agitation
at Visible particles NT NT NT NT 5.degree. C. for 2 hours observed
Experiment stopped 250 mL PVC bag, T0 CO, CL, PFVP NT NT NT NT 250
mL PVC bag, agitation at CO, CL, PFVP NT NT NT NT 5.degree. C. for
4 hours 0.015% 250 mL PO bag, T0 CO, CL, PFVP 1.2 98.8 21 2 250 mL
PO bag, agitation at CO, CL, PFVP 1.3 98.7 195 19 5.degree. C. for
4 hours 250 mL PVC bag, T0 CO, CL, PFVP 1.2 98.8 16 0 250 mL PVC
bag, agitation at CO, CL, PFVP 1.2 98.8 24 2 5.degree. C. for 4
hours Note: All formulations 50 mg/mL .alpha.-PDL1 in 20 mM
L-histidine acetate, 240 mM sucrose at pH 5.5. Analysis was
performed using SE-HPLC. NT = not tested; CAC = color, appearance,
and clarity; CO = Colorless; CL = Clear; PFVP = Practically Free of
Visible Particulates.
Stability Assessment of .alpha.-PDL1 Formulations
[0315] An additional pH screen was conducted on the materials
produced from a Master Cell Bank and a Working Cell Bank across a
pH range of 5.2 to 6.3 in a formulation containing 20 mM histidine
acetate, 120 mM sucrose, and 0.04% PS20 (Table 10). Analysis by
SE-HPLC and ICIEF showed that pH 5.7-6.3 was chemically and
physically fairly stable and an allowed range of pH 5.5-6.3 in the
formulation was appropriate (FIGS. 11A and B). Higher pH reduced
monomer and main peak degradation rates, with rates flattening out
between about pH 5.7 and 6.3.
TABLE-US-00035 TABLE 10 pH screen of Formulations Concentration
Temperature (mg/mL) pH Container (.degree. C.) Time Points 120 5.2,
5.7, 6.0, 6.3 1 mL fill in 40 T0, 1 week, 2 2cc vial week, 1 month
40 5.2, 5.7, 6.0, 6.3 1 mL fill in 40 T0, 1 week, 2 2cc vial week,
1 month
[0316] The effect of formulation excipients on tryptophan (W) and
methionine (M) oxidation in .alpha.-PDL1 formulations was
investigated. Peptide mapping showed there was no significant
oxidation increase. Formulations containing 20 mM histidine
acetate, 120 mM sucrose, 0.04% PS20 with a solution pH of 5.8
showed no apparent tryptophan and methionine oxidation increase
when the formulation was stored for one month at elevated
temperatures for either the Drug Product or Drug Substance (Table
11).
TABLE-US-00036 TABLE 11 Percentage of Trp, M.sup.253 and M.sup.429
oxidation in Selected Formulations by Peptide Map % Oxidation W CDR
W CDR W CDR Sample H2 H4 H10 m.sup.253 m.sup.429 DP, 50 mg/mL, T0
0.35 0.26 0.12 4.86 0.92 DP, 50 mg/mL, 40.degree. C., 0.63 0.26
0.31 5.85 1.10 T = 1M DS, 100 mg/mL, SS, 25.degree. C., 0.52 0.27
0.28 5.61 1.17 T = 1M Note: All formulations of .alpha.-PDL1
contained 20 mM L-histidine acetate, 120 mM sucrose, 0.04% PS20, pH
5.8.
[0317] Based on the results from these formulation studies and
statistical analysis, a liquid formulation consisting of 60 mg/mL
.alpha.-PDL1 in 20 mM histidine acetate, 120 mM sucrose, 0.04%
polysorbate 20 with a target pH 5.8 was selected for clinical
studies.
[0318] The dosage for clinical trials will be conducted as a flat
dose of 1200 mg .alpha.-PDL1 per patient. A vial configuration of
nominal 20 mL fill (1200 mg .alpha.-PDL1) in a 20 cc glass vial was
selected to meet the target product profile.
[0319] Freeze/thaw studies were conducted with the intended
formulation containing 60 mg/mL .alpha.-PDL1 in 20 mM L-histidine
acetate, 120 mM sucrose, and 0.02% (w/v) polysorbate 20 at pH 5.8.
Assay results after five freeze/thaw cycles confirmed 120 mM of
sucrose protected .alpha.-PDL1 from freeze/thaw-induced aggregation
(Table 12). Similarly long-term stability of the intended liquid
formulation indicated that it is stable for over 6 months at
2-8.degree. C. (Table 13). Continuous monitoring over 36 months is
underway for this formulation. Target formulation and tested study
ranges for .alpha.-PDL1 Drug Substance and Drug Product are shown
in Table 14.
TABLE-US-00037 TABLE 12 Representative Freeze/Thaw Stability Data
for .alpha.-PDL1 Drug Substance Development Batch SE-HPLC CE SDS
NGS No. ICIEF Sum of Sum of (non-reduced) Sum of Freeze- Acidic
Main Basic HMW LMW Sum of Main Post- Potency Thaw Strength Region
Peak Region Forms Monomer Forms Pre-Peaks Peak Peaks (% specific
Cycles CAC (mg/mL) pH (area %) (area %) (area %) (area %) (area %)
(area %) (% CPA) (% CPA) (% CPA) activity) NA CL/SY/PFVP 60.1 5.9
19 78 3 0.5 99.4 0.1 2.9 97.0 0.1 107 5 CL/SY/PFVP 62.0 5.9 20 77 3
0.5 99.4 0.1 2.7 97.1 0.2 111 Note: Batch PP400L-02142013 contains
60 mg/mL .alpha.-PDL1 in 20 mM L-histidine acetate, 120 mM sucrose,
and 0.04% (w/v) polysorbate 20 at pH 5.8. CL = Clear; SY = Slightly
Yellow; PFVP = Practically Free of Visible Particulates; NA = not
applicable, ICIEF = imaged capillary isoelectric focusing; CE-SDS =
capillary electrophoresis sodium dodecyl sulfate; HMW = high
molecular weight; LMW = low molecular weight.
TABLE-US-00038 TABLE 13 Stability Data for .alpha.-PDL1 Drug
Development Batch CE SDS NGS SE-HPLC (non-reduced) Imaged cIEF Sum
of Mon- Sum of Sum of Sum of Acidic Main Basic HMW omer LMW Pre-
Main Post- Potency Sub-Visible Time Region Peak Region Forms Peak
Forms Peaks Peak Peaks (% Particles.sup.a Temp (days/ Strength
(area (area (area (area (area (area (% (% (% specific (ppmL)
(.degree. C.) months) CAC pH (mg/mL) %) %) %) %) %) %) CPA) CPA)
CPA) activity) .gtoreq.10 um .gtoreq.25 um NA T = 0/0 SY/CL/ 5.9
59.9 18.1 78.9 2.9 0.6 99.3 0.1 2.7 97.0 0.3 99 37 30 PFVP 5 30/1
SY/CL/ 5.9 59.9 18.3 78.6 3.1 0.6 99.3 0.1 2.7 96.9 0.4 NT 26 2
PFVP 5 61/2 SY/CL/ 5.9 61.7 18.4 78.9 2.7 0.7 99.3 0.1 2.8 96.9 0.4
NT 3 0 PFVP 5 91/3 SY/CL/ 5.9 61.7 17.1 80.1 2.8 0.7 99.2 0.1 2.7
97.0 0.4 102 18 3 PFVP 5 183/6 SY/CL/ 5.9 60.8 18.4 78.6 3.0 0.7
99.2 0.1 3.1 96.5 0.4 101 3 0 PFVP Batch PP400L-02142013-DP
contains 60 mg/mL .alpha.-PDL1 in 20 mM L-histidine acetate, 120 mM
sucrose, and 0.04% (w/v) polysorbate 20 at pH 5.8. NA = not
applicable; CAC = color, appearance, and clarity; SY = slightly
yellow, CL = clear, PFVP = practically free of visible
particulates; HMW = high molecular weight; LMW = low molecular
weight; ICIEF = imaged capillary isoelectric focusing; CE-SDS =
capillary electrophoresis sodium dodecyl sulfate, NT = not
tested.
TABLE-US-00039 TABLE 14 Target formulation and tested study ranges
for .alpha.-PDL1 drug substance and drug product Tested Formulation
Parameter Target Range .alpha.-PDL1 Concentration 60 mg/mL 40-120
mg/mL L-Histidine Acetate 20 mM 20 mM Concentration Solution pH 5.8
5.0-6.0 Sucrose Concentration 120 mM 0-240 mM Polysorbate 20 0.04%
0.005%-0.06%.sup.a Concentration (w/v)
[0320] Since .alpha.-PDL1 drug product (60 mg/mL) will be
administered by infusion after dilution in isotonic sodium chloride
solution (0.9% NaCl), compatibility and stability of the active
ingredient was tested under the following simulated preparation and
administration conditions: 1) Dilution of .alpha.-PDL1 drug product
in infusion bags containing 0.9% NaCl in the range of 2.4-9.6 mg/ml
(nominal concentration after dilution) to cover the dose range in
the clinical study; 2) Short-term exposure to infusion bags
containing isotonic sodium chloride solution (bag product-contact
surface material consisting of PVC or Polyolefin); 3) Use of IV
infusion lines with (product-contacting surfaces of PVC or
Polyolefin); and 4) Use of 0.2 .mu.m in-line filters (filter
membrane of PES).
[0321] Samples were tested after 24 hours of storage at 2.degree.
C.-8.degree. C. or after 24 hours at 30.degree. C. with exposure to
diffused light. The samples were tested using appropriate stability
indicating methods including: purity by SE-HPLC and ICIEF, protein
concentration (by UV), subvisible particles by light obscuration,
color, clarity/opalescence, and pH (Table 15).
TABLE-US-00040 TABLE 15 Stability of .alpha.-PDL1 diluted and
stored at 5.degree. C. or 30.degree. C. for 24 hours in 0.9% NaCl
infusion bags with and without 0.2 .mu.m in-line filters ICIEF
SE-HPLC Particulates Strength Turbidity % % % % % % (counts/mL)
Sample CAC (mg/mL) A.sub.350 Acidic Main Peak Basic HMWS Monomer
LMWS pH .gtoreq.10 um .gtoreq.25 um 2.4 mg/mL in PVC bag, T0 CL,
CO, 2.1 0.01 19.5 75.7 4.8 0.4 99.5 0.1 5.9 25 1 PFVP 2.4 mg/mL in
PVC bag, CL, CO, 2.2 0.02 19.6 75.5 4.9 0.4 99.5 0.1 5.8 32 0 t =
5.degree. C., 24 hrs before PFVP infusion 2.4 mg/mL in PVC bag, CL,
CO, 2.2 0.01 19.3 76.6 4.1 0.3 99.5 0.1 5.8 32 0 t = 30.degree. C.,
24 hrs before PFVP infusion 2.4 mg/mL in PVC bag, CL, CO, 2.1 0.04
19.5 76.4 4.1 0.4 99.5 0.1 5.8 44 1 t = 5.degree. C., 24 hrs,
passing PFVP through infusion set without in-line filter 2.4 mg/mL
in PVC bag, CL, CO, 2.1 0.01 19.3 76.7 4.1 0.3 99.5 0.1 5.9 4 0 t =
5.degree. C., 24 hrs, passing PFVP through infusion set with in-
line filter 2.4 mg/mL in PVC bag, CL, CO, 2.1 0.02 20.0 75.7 4.3
0.3 99.6 0.1 5.9 29 0 t = 30.degree. C., 24 hrs passing PFVP
through infusion set without in-line filter 2.4 mg/mL in PVC bag,
CL, CO, 2.0 0.04 19.5 76.4 4.1 0.3 99.6 0.1 6.0 5 0 t = 30.degree.
C., 24 hrs passing PFVP through infusion set with in- line filter
2.4 mg/mL in PO bag, T0 CL, CO, 2.1 0.01 18.6 77.3 4.1 0.4 99.5 0.1
6.1 5 0 PFVP 2.4 mg/mL in PO bag, CL, CO, 2.1 0.03 17.8 77.8 4.4
0.4 99.5 0.1 5.9 3 0 t = 5.degree. C., 24 hrs before PFVP infusion
2.4 mg/mL in PO bag, CL, CO, 2.1 0.02 20.6 75.3 4.1 0.3 99.5 0.1
5.9 8 0 t = 30.degree. C., 24 hrs before PFVP infusion 2.4 mg/mL in
PO bag, CL, CO, 2.1 0.01 20.5 75.3 4.2 0.4 99.5 0.1 5.9 48 0 t =
5.degree. C., 24 hrs, passing PFVP through infusion set without
in-line filter 2.4 mg/mL in PO bag, CL, CO, 2.1 0.02 21.0 74.8 4.3
0.4 99.5 0.1 5.9 1 0 t = 5.degree. C., 24 hrs, passing PFVP through
infusion set with in-line filter 2.4 mg/mL in PO bag, CL, CO, 2.1
0.01 18.7 76.9 4.4 0.3 99.5 0.1 5.9 22 0 t = 30.degree. C., 24 hrs
passing PFVP through infusion set without in-line filter 2.4 mg/mL
in PO bag, CL, CO, 2.1 0.01 21.2 73.9 4.9 0.4 99.5 0.1 6.0 0 0 t =
30.degree. C., 24 hrs passing PFVP through infusion set with in-
line filter 9.6 mg/mL in PVC bag, T0 CL, CO, 8.7 0.05 18.3 77.3 4.4
0.4 99.5 0.1 5.9 35 0 PFVP 9.6 mg/mL in PVC bag, CL, CO, 8.6 0.03
19.0 76.8 4.2 0.4 9.5 0.1 5.9 6 1 t = 5.degree. C., 24 hrs before
PFVP infusion 9.6 mg/mL in PVC bag, CL, CO, 8.5 0.05 18.9 77.0 4.1
0.4 99.5 0.2 5.9 10 0 t = 30.degree. C., 24 hrs before PFVP
infusion 9.6 mg/mL in PVC bag, CL, CO, 8.8 0.03 19.2 76.4 4.4 0.3
99.6 0.1 6.0 29 0 t = 5.degree. C., 24 hrs, passing PFVP through
infusion set without in-line filter 9.6 mg/mL in PVC bag, CL, CO,
8.7 0.06 19.0 77.1 3.9 0.3 99.6 0.1 5.9 18 0 t = 5.degree. C., 24
hrs, passing PFVP through infusion set with in- line filter 9.6
mg/mL in PVC bag, CL, CO, 8.1 0.04 19.1 76.6 4.3 0.4 99.5 0.2 6.0 8
0 t = 30.degree. C., 24 hrs passing PFVP through infusion set
without in-line filter 9.6 mg/mL in PVC bag, CL, CO, 8.8 0.04 19.6
76.4 4.0 0.3 99.6 0.1 5.9 19 2 t = 30.degree. C., 24 hrs passing
PFVP through infusion set with in- line filter 9.6 mg/mL in PO bag,
T0 CL, CO, 8.4 0.03 18.6 78.0 3.4 0.4 99.5 0.1 5.8 33 2 PFVP 9.6
mg/mL in PO bag, CL, CO, 8.6 0.04 19.2 76.4 4.4 0.4 99.5 0.1 5.9 32
0 t = 5.degree. C., 24 hrs before PFVP infusion 9.6 mg/mL in PO
bag, CL, CO, 8.7 0.04 19.3 76.7 4.0 0.4 99.5 0.1 5.9 18 0 t =
30.degree. C., 24 hrs before PFVP infusion 9.6 mg/mL in PO bag, CL,
CO, 8.5 0.05 19.8 75.8 4.5 0.4 99.5 0.1 5.9 38 1 t = 5.degree. C.,
24 hrs, passing PFVP through infusion set without in-line filter
9.6 mg/mL in PO bag, CL, CO, 8.2 0.04 18.6 77.2 4.3 0.3 99.5 0.1
5.8 8 0 t = 5.degree. C., 24 hrs, passing PFVP through infusion set
with in-line filter 9.6 mg/mL in PO bag, CL, CO, 8.5 0.03 19.4 76.0
4.6 0.4 99.5 0.1 5.9 48 7 t = 30.degree. C., 24 hrs passing PFVP
through infusion set without in-line filter 9.6 mg/mL in PO bag,
CL, CO, 8.0 0.05 19.7 76.1 4.2 0.3 99.5 0.1 5.8 10 0 t = 30.degree.
C., 24 hrs passing PFVP through infusion set with in-line filter CO
= Colorless, CL = Clear, PFVP = Practically Free of Visible
Particulates, A.sub.350 = absorbance at 350 nm
TABLE-US-00041 TABLE 16 Agitation Stability of .alpha.-PDL1 diluted
in 0.9% NaCl infusion bags at 5.degree. C. for up to 6 hours
SE-HPLC Particulates Strength Turbidity ICIEF % % (counts/mL)
Sample CAC (mg/mL) A.sub.350 % Acidic % Main Peak % Basic % HMWS
Monomer LMWS pH .gtoreq.10 um .gtoreq.25 um 2.4 mg/mL in PO CL, CO,
2.13 0.02 17.5 79.1 3.4 0.8 99.1 0.1 5.9 3 0 bag, T0 PFVP 2.4 mg/mL
in PO bag, CL, CO, 2.09 0.01 17.1 79.8 3.1 0.8 99.1 0.1 5.9 113 2 2
hr agitation PFVP 2.4 mg/mL in PO bag, CL, CO, 2.12 0.02 17.3 79.6
3.1 0.8 99.1 0.1 5.9 31 0 4 hr agitation PFVP 2.4 mg/mL in PO bag,
CL, CO, 2.02 0.02 16.8 79.6 3.6 0.8 99.1 0.1 5.9 4 1 6 hr agitation
PFVP 2.4 mg/mL in PVC CL, CO, 2.42 0.02 17.9 78.6 3.5 0.8 99.1 0.1
5.9 6 0 bag, T0 PFVP 2.4 mg/mL in PVC CL, CO, 2.04 0.02 17.6 79.2
3.2 0.8 99.1 0.1 5.9 22 1 bag, 2 hr agitation PFVP 2.4 mg/mL in PVC
CL, CO, 2.10 0.03 18.5 78.0 3.6 0.8 99.1 0.1 5.9 22 1 bag, 4 hr
agitation PFVP 2.4 mg/mL in PVC CL, CO, 2.05 0.01 18.6 78.2 3.3 0.8
99.1 0.1 5.9 10 0 bag, 6 hr agitation PFVP CO = Colorless, CL =
Clear, PFVP = Practically Free of Visible Particulates, A.sub.350 =
absorbance at 350 nm
[0322] The product tested in simulated administration studies as
described above was physically and chemically stable under the
tested conditions. Infusion bags, infusion sets, filters, and/or IV
administration aids composed of different product-contacting
materials are added upon successful qualification.
[0323] In addition to the static stability, an IV bag agitation
study is performed with .alpha.-PDL1 formulated in 20 mM histidine
acetate, 120 mM sucrose, pH 5.8 with 0.02% PS20, which is
potentially the lowest PS20 level that could be observed in drug
product over shelf life. The agitation is performed at 2-8.degree.
C. with orbital shaker at speed of 100 rpm. The data suggests that
with 0.02% PS20 in drug product, .alpha.-PDL1 is stable upon
agitation at 5.degree. C. after diluting in IV bags (Table 16).
[0324] Sequences of the Antibody Used in the Examples
TABLE-US-00042 .alpha.-PDL1 Light Chain Variable Region (SEQ ID NO:
7) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR
.alpha.-PDL1 Heavy Chain Variable Region (SEQ ID NO: 8)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSSASTK .alpha.-PDL1 Full Light Chain (SEQ ID NO: 9)
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
.alpha.-PDL1 Full Heavy Chain (SEQ ID NO: 10)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAW
ISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH
WPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
Sequence CWU 1
1
36111PRTArtificial SequenceSynthetic Construct 1Arg Ala Ser Gln Asp
Val Ser Thr Ala Val Ala1 5 1027PRTArtificial SequenceSynthetic
Construct 2Ser Ala Ser Phe Leu Tyr Ser1 539PRTArtificial
SequenceSynthetic Construct 3Gln Gln Tyr Leu Tyr His Pro Ala Thr1
5410PRTArtificial SequenceSynthetic Construct 4Gly Phe Thr Phe Ser
Asp Ser Trp Ile His1 5 10518PRTArtificial SequenceSynthetic
Construct 5Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val1 5 10 15Lys Gly69PRTArtificial SequenceSynthetic Construct
6Arg His Trp Pro Gly Gly Phe Asp Tyr1 57108PRTArtificial
SequenceSynthetic Construct 7Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu
Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 1058122PRTArtificial
SequenceSynthetic Construct 8Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 1209214PRTArtificial
SequenceSynthetic Construct 9Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu
Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21010447PRTArtificial SequenceSynthetic Construct 10Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295
300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410
415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 440 4451110PRTArtificial SequenceSynthetic
ConstructVARIANT6Xaa = D or G 11Gly Phe Thr Phe Ser Xaa Ser Trp Ile
His1 5 101218PRTArtificial SequenceSynthetic ConstructVARIANT4Xaa =
S or LVARIANT10Xaa = T or S 12Ala Trp Ile Xaa Pro Tyr Gly Gly Ser
Xaa Tyr Tyr Ala Asp Ser Val1 5 10 15Lys Gly139PRTArtificial
SequenceSynthetic Construct 13Arg His Trp Pro Gly Gly Phe Asp Tyr1
51425PRTArtificial SequenceSynthetic Construct 14Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser 20 251513PRTArtificial SequenceSynthetic
Construct 15Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val1 5
101632PRTArtificial SequenceSynthetic Construct 16Arg Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln1 5 10 15Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25
301711PRTArtificial SequenceSynthetic Construct 17Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ala1 5 101811PRTArtificial
SequenceSynthetic ConstructVARIANT5Xaa = D or VVARIANT6Xaa = V or
IVARIANT7Xaa = S or NVARIANT9Xaa = A or FVARIANT10Xaa = V or L
18Arg Ala Ser Gln Xaa Xaa Xaa Thr Xaa Xaa Ala1 5 10197PRTArtificial
SequenceSynthetic ConstructVARIANT4Xaa = F or TVARIANT6Xaa = Y or A
19Ser Ala Ser Xaa Leu Xaa Ser1 5209PRTArtificial SequenceSynthetic
ConstructVARIANT3Xaa = Y, G, F, or SVARIANT4Xaa = L, Y, F or
WVARIANT5Xaa = Y, N, A, T, G, F or IVARIANT6Xaa = H, V, P, T or
IVARIANT8Xaa = A, W, R, P or T 20Gln Gln Xaa Xaa Xaa Xaa Pro Xaa
Thr1 52123PRTArtificial SequenceSynthetic Construct 21Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys 202215PRTArtificial SequenceSynthetic Construct
22Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr1 5 10
152332PRTArtificial SequenceSynthetic Construct 23Gly Val Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25
302411PRTArtificial SequenceSynthetic Construct 24Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg1 5 102510PRTArtificial
SequenceSynthetic Construct 25Gly Phe Thr Phe Ser Asp Ser Trp Ile
His1 5 102618PRTArtificial SequenceSynthetic Construct 26Ala Trp
Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val1 5 10 15Lys
Gly2711PRTArtificial SequenceSynthetic Construct 27Arg Ala Ser Gln
Asp Val Ser Thr Ala Val Ala1 5 10287PRTArtificial SequenceSynthetic
Construct 28Ser Ala Ser Phe Leu Tyr Ser1 5299PRTArtificial
SequenceSynthetic Construct 29Gln Gln Tyr Leu Tyr His Pro Ala Thr1
530118PRTArtificial SequenceSynthetic Construct 30Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln
Gly Thr 100 105 110Leu Val Thr Val Ser Ala 11531108PRTArtificial
SequenceSynthetic Construct 31Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Ser Ala Ser Phe Leu
Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala 85 90 95Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 10532118PRTArtificial
SequenceSynthetic Construct 32Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Ser 20 25 30Trp Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Trp Ile Ser Pro Tyr
Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser 1153311PRTArtificial SequenceSynthetic
Construct 33Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5
103430PRTArtificial SequenceSynthetic Construct 34Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25
303514PRTArtificial SequenceSynthetic Construct 35Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala1 5 103610PRTArtificial
SequenceSynthetic Construct 36Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys1 5 10
* * * * *