U.S. patent application number 12/075197 was filed with the patent office on 2009-07-09 for stabilized anti-respiratory syncytial virus (rsv) antibody formulations.
This patent application is currently assigned to MedImmune, Inc.. Invention is credited to Christian B. Allan, Stephen T. Chang, Cynthia N. Oliver.
Application Number | 20090175883 12/075197 |
Document ID | / |
Family ID | 29736563 |
Filed Date | 2009-07-09 |
United States Patent
Application |
20090175883 |
Kind Code |
A1 |
Oliver; Cynthia N. ; et
al. |
July 9, 2009 |
Stabilized anti-respiratory syncytial virus (RSV) antibody
formulations
Abstract
The present invention provides liquid formulations of antibodies
or fragments thereof that immunospecifically bind to a respiratory
syncytial virus (RSV) antigen, which formulations exhibit
stability, low to undetectable levels of aggregation, and very
little to no loss of the biological activities of the antibodies or
antibody fragments, even during long periods of storage. In
particular, the present invention provides liquid formulations of
antibodies or fragments thereof that immunospecifically bind to a
RSV antigen, which formulations are substantially free of
surfactant, inorganic salts, and/or other common excipients.
Furthermore, the invention provides methods of preventing, treating
or ameliorating one or more symptoms associated with RSV infection
utilizing the liquid formulations of the present invention.
Inventors: |
Oliver; Cynthia N.; (N.
Potomac, MD) ; Allan; Christian B.; (Brookeville,
MD) ; Chang; Stephen T.; (Frederick, MD) |
Correspondence
Address: |
JONES DAY
222 EAST 41ST ST
NEW YORK
NY
10017
US
|
Assignee: |
MedImmune, Inc.
|
Family ID: |
29736563 |
Appl. No.: |
12/075197 |
Filed: |
March 10, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10461863 |
Jun 13, 2003 |
7425618 |
|
|
12075197 |
|
|
|
|
60388920 |
Jun 14, 2002 |
|
|
|
Current U.S.
Class: |
424/159.1 ;
424/178.1 |
Current CPC
Class: |
A61K 39/39591 20130101;
C07K 2317/56 20130101; C07K 2317/565 20130101; A61K 39/42 20130101;
A61P 31/14 20180101; A61P 31/12 20180101; A61K 9/0019 20130101;
A61K 2039/505 20130101; A61P 11/00 20180101; C07K 16/1027 20130101;
A61K 47/183 20130101; A61K 9/0043 20130101; A61K 39/42 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/159.1 ;
424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 31/12 20060101 A61P031/12 |
Claims
1.-57. (canceled)
58. A liquid antibody formulation comprising, in an aqueous
carrier: (a) at least 40 mg/ml of antibody that immunospecifically
binds to a respiratory syncytial virus (RSV) F antigen, wherein the
antibody is not palivizumab or an antigen-binding fragment thereof;
and (b) histidine.
59. A liquid antibody formulation comprising at least 40 mg/ml of
an antibody that immunospecifically binds to a RSV antigen in an
aqueous carrier, the formulation (i) having been prepared by a
process in which, for each step of said process, said antibody is
in an aqueous phase; and (ii) being suitable for injection in a
human subject, and wherein the antibody is not palivizumab or an
antigen-binding fragment thereof.
60. The liquid formulation of claim 58, wherein histidine is at a
concentration of about 1 mM to about 100 mM.
61. The liquid antibody formulation of claim 58, wherein the
histidine is at a concentration of about 10 mM to about 50 mM.
62. The liquid antibody formulation of claim 58, wherein the
histidine is at a concentration of about 20 mM to about 30 mM.
63. The liquid antibody formulation of claim 58, wherein the
histidine is at a concentration of about 25 mM.
64. The liquid antibody formulation of claim 58, wherein the
antibody is at a concentration of at least 50 mg/ml, at least 55
mg/ml, at least 60 mg/ml, at least 65 mg/ml, at least 70 mg/ml, at
least 75 mg/ml, at least 80 mg/ml, at least 85 mg/ml, at least 90
mg/ml, at least 95 mg/ml or at least 100 mg/ml.
65. The liquid antibody formulation of claim 63, wherein the
antibody is at a concentration of at least 80 mg/ml.
66. The liquid antibody formulation of claim 58, wherein the
formulation has a pH of about 5.5 to about 7.0.
67. The liquid antibody formulation of claim 58, wherein the
formulation has a pH of about 6.0.
68. The liquid antibody formulation of claim 63, wherein the
formulation has a pH of about 6.0.
69. The liquid antibody formulation of claim 65, wherein the
formulation has a pH of about 6.0.
70. The liquid antibody formulation of claim 58, wherein the
formulation is substantially free of surfactants and inorganic
salts.
71. The liquid antibody formulation of claim 58, wherein the
formulation is substantially free of surfactants, inorganic salts
and other excipients.
72. The liquid antibody formulation of claim 58, wherein the
formulation further comprises an excipient.
73. The liquid antibody formulation of claim 72, wherein the
excipient is glycine, a saccharide, or a polyol.
74. The liquid antibody formulation of claim 58, wherein the
aqueous carrier is distilled water.
75. The liquid antibody formulation of claim 69, wherein the
aqueous carrier is distilled water.
76. The liquid antibody formulation of claim 58, wherein the
formulation is sterile.
77. The liquid antibody formulation of claim 69, wherein the
formulation is sterile.
78. The liquid antibody formulation of claim 75, wherein the
formulation is sterile.
79. The liquid antibody formulation of claim 58, wherein the
formulation is stable at 40.degree. C. for at least 100 days as
determined by high performance size exclusion chromatography
(HPSEC).
80. The liquid antibody formulation of claim 69, wherein the
formulation is stable at 40.degree. C. for at least 100 days as
determined by HPSEC.
81. The liquid antibody formulation of claim 58, wherein the
formulation is stable at 4.degree. C. for at least 3 years as
determined by HPSEC.
82. The liquid antibody formulation of claim 69, wherein the
formulation is stable at 4.degree. C. for at least 3 years as
determined by HPSEC.
83. The liquid antibody formulation of claim 58 which has been
prepared by a process in which, for each step of the process, the
antibody is in an aqueous phase.
84. The liquid antibody formulation of claim 69 which has been
prepared by a process in which, for each step of the process, the
antibody is in an aqueous phase.
85. The liquid antibody formulation of claim 58, wherein the
antibody comprises: a. a variable heavy (VH) domain having the
amino acid sequence of SEQ ID NO: 9, 17, 24, 28, 33, 36, 40, 44,
48, 51, 55, 67, 70 or 78; b. a variable light (VL) domain having
the amino acid sequence of SEQ ID NO: 11, 13, 21, 26, 30, 34, 38,
42, 46, 49, 52, 54, 56, 58, 60, 62, 64, 65, 68, 71, 74 or 76; c. a
VH domain having the amino acid sequence of SEQ ID NO: 9, 17, 24,
28, 33, 36, 40, 44, 48, 51, 55, 67, 70 or 78 and a VL domain having
the amino acid sequence of SEQ ID NO: 11, 13, 21, 26, 30, 34, 38,
42, 46, 49, 52, 54, 56, 58, 60, 62, 64, 65, 68, 71, 74 or 76; d. a
VH complementarity determining region (CDR) having the amino acid
sequence of SEQ ID NO: 1, 2, 3, 10, 12, 18, 19, 20, 25, 29, 37, 41,
45 or 79; e. a variable light (VL) CDR having the amino acid
sequence of SEQ ID NO: 4, 5, 6, 14, 15, 16, 22, 23, 26, 27, 31, 32,
35, 39, 43, 47, 53, 57, 59, 61, 63, 66, 69, 72, 73, 75 or 77; f. a
VH CDR having the amino acid sequence of SEQ ID NO: 1, 2, 3, 10,
12, 18, 19, 20, 25, 29, 37, 41, 45 or 79 and a VL CDR having the
amino acid sequence of SEQ ID NO: 4, 5, 6, 14, 15, 16, 22, 23, 26,
27, 31, 32, 35, 39, 43, 47, 53, 57, 59, 61, 63, 66, 69, 72, 73, 75
or 77; g. a VH CDR having the amino acid sequence of a VH CDR in
Table 1 or Table 2; h. a VL CDR having the amino acid sequence of a
VL CDR in Table 1 or Table 2; or i. a VH CDR having the amino acid
sequence of a VH CDR in Table 1 or Table 2 and a VL CDR having the
amino acid sequence of a VL CDR in Table 1 or Table 2.
86. The liquid antibody formulation of claim 69, wherein the
antibody comprises: a. a variable heavy (VH) domain having the
amino acid sequence of SEQ ID NO: 9, 17, 24, 28, 33, 36, 40, 44,
48, 51, 55, 67, 70 or 78; b. a variable light (VL) domain having
the amino acid sequence of SEQ ID NO: 11, 13, 21, 26, 30, 34, 38,
42, 46, 49, 52, 54, 56, 58, 60, 62, 64, 65, 68, 71, 74 or 76; c. a
VH domain having the amino acid sequence of SEQ ID NO: 9, 17, 24,
28, 33, 36, 40, 44, 48, 51, 55, 67, 70 or 78 and a VL domain having
the amino acid sequence of SEQ ID NO: 11, 13, 21, 26, 30, 34, 38,
42, 46, 49, 52, 54, 56, 58, 60, 62, 64, 65, 68, 71, 74 or 76; d. a
VH complementarity determining region (CDR) having the amino acid
sequence of SEQ ID NO: 1, 2, 3, 10, 12, 18, 19, 20, 25, 29, 37, 41,
45 or 79; e. a variable light (VL) CDR having the amino acid
sequence of SEQ ID NO: 4, 5, 6, 14, 15, 16, 22, 23, 26, 27, 31, 32,
35, 39, 43, 47, 53, 57, 59, 61, 63, 66, 69, 72, 73, 75 or 77; f. a
VH CDR having the amino acid sequence of SEQ ID NO: 1, 2, 3, 10,
12, 18, 19, 20, 25, 29, 37, 41, 45 or 79 and a VL CDR having the
amino acid sequence of SEQ ID NO: 4, 5, 6, 14, 15, 16, 22, 23, 26,
27, 31, 32, 35, 39, 43, 47, 53, 57, 59, 61, 63, 66, 69, 72, 73, 75
or 77; g. a VH CDR having the amino acid sequence of a VH CDR in
Table 1 or Table 2; h. a VL CDR having the amino acid sequence of a
VL CDR in Table 1 or Table 2; or i. a VH CDR having the amino acid
sequence of a VH CDR in Table 1 or Table 2 and a VL CDR having the
amino acid sequence of a VL CDR in Table 1 or Table 2.
87. The liquid antibody formulation of claim 58, wherein the
antibody is conjugated to a therapeutic moiety, a drug moiety, a
diagnostic agent or a detectable agent.
88. The liquid antibody formulation of claim 59, wherein the
formulation comprises histidine.
89. The liquid antibody formulation of claim 88, wherein the
histidine is at a concentration of about 10 mM to about 50 mM.
90. The liquid antibody formulation of claim 59, wherein the
antibody is at a concentration of at least 50 mg/ml, at least 55
mg/ml, at least 60 mg/ml, at least 65 mg/ml, at least 70 mg/ml, at
least 75 mg/ml, at least 80 mg/ml, at least 85 mg/ml, at least 90
mg/ml, at least 95 mg/ml or at least 100 mg/ml.
91. The liquid antibody formulation of claim 59, wherein the
formulation is at a pH of about 5.5 to about 7.0.
92. The liquid antibody formulation of claim 59, wherein the
formulation is substantially free of surfactants, inorganic salts
and other excipients.
93. The liquid antibody formulation of claim 59, wherein the
formulation is stable at 40.degree. C. for at least 100 days as
determined by HPSEC.
94. The liquid antibody formulation of claim 59, wherein the
formulation is stable at 4.degree. C. for at least 3 years as
determined by HPSEC.
95. The liquid antibody formulation of claim 59, wherein the
antibody comprises: a. a variable heavy (VH) domain having the
amino acid sequence of SEQ ID NO: 9, 17, 24, 28, 33, 36, 40, 44,
48, 51, 55, 67, 70 or 78; b. a variable light (VL) domain having
the amino acid sequence of SEQ ID NO: 11, 13, 21, 26, 30, 34, 38,
42, 46, 49, 52, 54, 56, 58, 60, 62, 64, 65, 68, 71, 74 or 76; c. a
VH domain having the amino acid sequence of SEQ ID NO: 9, 17, 24,
28, 33, 36, 40, 44, 48, 51, 55, 67, 70 or 78 and a VL domain having
the amino acid sequence of SEQ ID NO: 11, 13, 21, 26, 30, 34, 38,
42, 46, 49, 52, 54, 56, 58, 60, 62, 64, 65, 68, 71, 74 or 76; d. a
VH complementarity determining region (CDR) having the amino acid
sequence of SEQ ID NO: 1, 2, 3, 10, 12, 18, 19, 20, 25, 29, 37, 41,
45 or 79; e. a variable light (VL) CDR having the amino acid
sequence of SEQ ID NO: 4, 5, 6, 14, 15, 16, 22, 23, 26, 27, 31, 32,
35, 39, 43, 47, 53, 57, 59, 61, 63, 66, 69, 72, 73, 75 or 77; f. a
VH CDR having the amino acid sequence of SEQ ID NO: 1, 2, 3, 10,
12, 18, 19, 20, 25, 29, 37, 41, 45 or 79 and a VL CDR having the
amino acid sequence of SEQ ID NO: 4, 5, 6, 14, 15, 16, 22, 23, 26,
27, 31, 32, 35, 39, 43, 47, 53, 57, 59, 61, 63, 66, 69, 72, 73, 75
or 77; g. a VH CDR having the amino acid sequence of a VH CDR in
Table 1 or Table 2; h. a VL CDR having the amino acid sequence of a
VL CDR in Table 1 or Table 2; or i. a VH CDR having the amino acid
sequence of a VH CDR in Table 1 or Table 2 and a VL CDR having the
amino acid sequence of a VL CDR in Table 1 or Table 2.
96. The liquid antibody formulation of claim 59, wherein the
antibody is conjugated a therapeutic moiety, a drug moiety, a
diagnostic agent or a detectable agent.
97. A pharmaceutical unit dosage form comprising the formulation of
claim 58, which dosage form is suitable for administration to a
human and is in a suitable container.
98. A pharmaceutical unit dosage form comprising the formulation of
claim 69, which dosage form is suitable for administration to a
human and is in a suitable container.
99. A pharmaceutical unit dosage form comprising the formulation of
claim 59, which dosage form is suitable for administration to a
human and is in a suitable container.
100. The pharmaceutical unit dosage of claim 98, wherein the
antibody has a concentration of at least 80 mg/ml in a volume of
1.2 ml.
101. A method for preventing, treating or managing a RSV infection,
comprising administering to a subject a prophylactically or
therapeutically effective amount of the formulation of claim
58.
102. A method for preventing, treating or managing a RSV infection,
comprising administering to a subject a prophylactically or
therapeutically effective amount of the formulation of claim
69.
103. A method for preventing, treating or managing a RSV infection,
comprising administering to a subject a prophylactically or
therapeutically effective amount of the formulation of claim 59.
Description
[0001] This application is entitled to and claims priority benefit
to U.S. provisional application Ser. No. 60/388,920 filed Jun. 14,
2002, which is incorporated herein by reference in its
entirety.
1. INTRODUCTION
[0002] The present invention relates to liquid formulations of
antibodies or fragments thereof that immunospecifically bind to a
respiratory syncytial virus (RSV) antigen, which formulations
exhibit stability, low to undetectable levels of antibody
fragmentation, low to undetectable levels of aggregation, and very
little to no loss of the biological activity (e.g., therapeutic
efficacy) of the antibodies or antibody fragments, even during long
periods of storage. In particular, the present invention relates to
liquid formulations of antibodies or fragments thereof that
immunospecifically bind to a RSV antigen, which formulations are
substantially free of surfactant and/or inorganic salt. The present
invention also relates to methods of preventing, treating, managing
or ameliorating a RSV infection or one or more symptoms thereof
utilizing liquid formulations of antibodies or fragments thereof
that immunospecifically bind to a RSV antigen.
2. BACKGROUND OF THE INVENTION
[0003] Respiratory syncytial virus (RSV) is the leading cause of
serious lower respiratory tract disease in infants and children
(Feigen et al., eds., 1987, In: Textbook of Pediatric Infectious
Diseases, WB Saunders, Philadelphia at pages 1653-1675; New Vaccine
Development, Establishing Priorities, Vol. 1, 1985, National
Academy Press, Washington D.C. at pages 397-409; and Ruuskanen et
al., 1993. Curr. Probl. Pediatr. 23:50-79). The yearly epidemic
nature of RSV infection is evident worldwide, but the incidence and
severity of RSV disease in a given season vary by region (Hall, C.
B., 1993, Contemp. Pediatr. 10:92-110). In temperate regions of the
northern hemisphere, it usually begins in late fall and ends in
late spring (Hall, C. B., 1995, In: Mandell G. L., Bernnett J. E.,
Dolin R., eds., 1995, Principles and Practice of Infectious
Diseases. 4th ed., Churchill Livingstone, New York at pages
1501-1519). It is estimated that RSV illness results in 90,000
hospitalizations and causes 4,500 deaths annually in the United
States. Primary RSV infection occurs most often in children from 6
weeks to 2 years of age and uncommonly in the first 4 weeks of life
during nosocomial epidemics (Hall et al., 1979, New Engl. J. Med.
300:393-396). RSV is estimated to cause as much as 75% of all
childhood bronchiolitis and up to 40% of all pediatric pneumonias
(Cunningham, C. K. et al., 1991, Pediatrics 88:527-532). Children
at increased risk from RSV infection include preterm infants (Hall
et al., 1979, New Engl. J. Med. 300:393-396) and children with
bronchopulmonary dysplasia (Groothuis et al., 1988, Pediatrics
82:199-203), congenital heart disease (MacDonald et al., New Engl.
J. Med. 307:397-400), congenital or acquired immunodeficiency (Ogra
et al., 1988, Pediatr. Infect. Dis. J. 7:246-249; and Pohl et al.,
1992, J. Infect. Dis. 165:166-169), and cystic fibrosis (Abman et
al., 1988, J. Pediatr. 113:826-830). The fatality rate in infants
with heart or lung disease who are hospitalized with RSV infection
is 3%-4% (Navas et al., 1992, J. Pediatr. 121:348-354).
[0004] RSV infects adults as well as infants and children. In
healthy adults, RSV causes predominantly upper respiratory tract
disease. It has recently become evident that some adults,
especially the elderly, have symptomatic RSV infections more
frequently than had been previously reported (Evans, A. S., eds.,
1989, Viral Infections of Humans. Epidemiology and Control,
3.sup.rd ed., Plenum Medical Book, New York at pages 525-544).
Several epidemics also have been reported among nursing home
patients and institutionalized young adults (Falsey, A. R., 1991,
Infect. Control Hosp. Epidemiol. 12:602-608; and Garvie et al,
1980, Br. Med. J. 281:1253-1254). Finally, RSV may cause serious
disease in immunosuppressed persons, particularly bone marrow
transplant patients (Hertz et al., 1989, Medicine 68:269-281).
[0005] Treatment options for established RSV disease are limited.
Severe RSV disease of the lower respiratory tract often requires
considerable supportive care, including administration of
humidified oxygen and respiratory assistance (Fields et al., eds,
1990, Fields Virology, 2.sup.nd ed., Vol. 1, Raven Press, New York
at pages 1045-1072). The only drug approved for treatment of
infection is the antiviral agent ribavirin (American Academy of
Pediatrics Committee on Infectious Diseases, 1993, Pediatrics
92:501-504). It has been shown to be effective in the treatment of
RSV pneumonia and bronchiolitis, modifying the course of severe RSV
disease in immunocompetent children (Smith et al, 1991, New Engl.
J. Med. 325:24-29). However, ribavirin has a number of limitations
including high cost, need for prolonged aerosol administration and
potential risk to pregnant women as well as to exposed health care
personnel. The American Academy of Pediatrics Committee on
Infectious Diseases revised their recommendation for use of
ribavirin. The current recommendation is that the decision to use
ribavirin should be based on the particular clinical circumstances
and physician's experience (American Academy of Pediatrics.
Summaries of Infectious Diseases. In: Pickering L. K., ed., 2000
Red Book: Report of the Committee on Infectious Diseases. 25th ed.,
Elk Grove Village, Ill., American Academy of Pediatrics, 2000, pp.
483-487).
[0006] While a vaccine might prevent RSV infection, no vaccine is
yet licensed for this indication. A major obstacle to vaccine
development is safety. A formalin-inactivated vaccine, though
immunogenic, unexpectedly caused a higher and more severe incidence
of lower respiratory tract disease due to RSV in immunized infants
than in infants immunized with a similarly prepared trivalent
parainfluenza vaccine (Kim et al, 1969, Am. J. Epidemiol.
89:422-434; and Kapikian et al, 1969, Am. J. Epidemiol.
89:405-421). Several candidate RSV vaccines have been abandoned and
others are under development (Murphy et al., 1994, Virus Res.
32:13-36), but even if safety issues are resolved, vaccine efficacy
must also be improved. A number of problems remain to be solved.
Immunization would be required in the immediate neonatal period
since the peak incidence of lower respiratory tract disease occurs
at 2-5 months of age. The immaturity of the neonatal immune
response together with high titers of maternally acquired RSV
antibody may be expected to reduce vaccine immunogenicity in the
neonatal period (Murphy et al, 1988, J. Virol. 62:3907-3910; and
Murphy et al., 1991, Vaccine 9:185-189). Finally, primary RSV
infection and disease do not protect well against subsequent RSV
disease (Henderson et al, 1979, New Engl. J. Med. 300:530-534).
[0007] Currently, the only approved approach to prophylaxis of RSV
disease is passive immunization. Initial evidence suggesting a
protective role for IgG was obtained from observations involving
maternal antibody in ferrets (Prince, G. A., Ph.D. diss.,
University of California, Los Angeles, 1975) and humans (Lambrecht
et al, 1976, J. Infect. Dis. 134:211-217; and Glezen et al., 1981,
J. Pediatr. 98:708-715). Hemming et al. (Morell et al., eds., 1986,
Clinical Use of Intravenous Immunoglobulins, Academic Press, London
at pages 285-294) recognized the possible utility of RSV antibody
in the treatment or prevention of RSV infection during studies
involving the pharmacokinetics of an intravenous immune globulin
(IVIG) in newborns suspected of having neonatal sepsis. They noted
that one infant, whose respiratory secretions yielded RSV,
recovered rapidly after IVIG infusion. Subsequent analysis of the
IVIG lot revealed an unusually high titer of RSV neutralizing
antibody. This same group of investigators then examined the
ability of hyperimmune serum or immune globulin, enriched for RSV
neutralizing antibody, to protect cotton rats and primates against
RSV infection (Prince et al, 1985, Virus Res. 3:193-206; Prince et
al, 1990, J. Virol. 64:3091-3092; Hemming et al., 1985, J. Infect.
Dis. 152:1083-1087; Prince et al., 1983, Infect. Immun. 42:81-87;
and Prince et al., 1985, J. Virol. 55:517-520). Results of these
studies suggested that RSV neutralizing antibody given
prophylactically inhibited respiratory tract replication of RSV in
cotton rats. When given therapeutically, RSV antibody reduced
pulmonary viral replication both in cotton rats and in a nonhuman
primate model. Furthermore, passive infusion of immune serum or
immune globulin did not produce enhanced pulmonary pathology in
cotton rats subsequently challenged with RSV.
[0008] A humanized antibody directed to an epitope in the A
antigenic site of the F protein of RSV, SYNAGIS.RTM., comprising
variable heavy (VH) complementarity determining regions (CDRs)
having the amino acid sequence of SEQ ID. NO:7 and variable light
(VL) CDRs having the amino acid sequence of SEQ ID. NO:8, is
approved for intramuscular administration to pediatric patients for
prevention of serious lower respiratory tract disease caused by RSV
at recommended monthly doses of 15 mg/kg of body weight throughout
the RSV season (November through April in the northern hemisphere).
SYNAGIS.RTM. is a composite of human (95%) and murine (5%) antibody
sequences. See, Johnson et al., 1997, J. Infect. Diseases
176:1215-1224 and U.S. Pat. No. 5,824,307, the entire contents of
which are incorporated herein by reference. The human heavy chain
sequence was derived from the constant domains of human IgG.sub.1
and the variable framework regions of the VH genes of Cor (Press et
al., 1970, Biochem. J. 117:641-660) and Cess (Takashi et al, 1984,
Proc. Natl. Acad. Sci. USA 81:194-198). The human light chain
sequence was derived from the constant domain of C.kappa. and the
variable framework regions of the VL gene K104 with J.kappa.-4
(Bentley et al, 1980, Nature 288:5194-5198). The murine sequences
were derived from a murine monoclonal antibody, Mab 1129 (Beeler et
al., 1989, J. Virology 63:2941-2950), in a process which involved
the grafting of the murine complementarity determining regions into
the human antibody frameworks.
[0009] SYNAGIS.RTM. has high specific activity against RSV in vitro
(approximately 50-100 times that of RespiGam.RTM.) and is known to
neutralize a broad range of RSV isolates. Since it is not derived
from human plasma, prophylactic treatment with SYNAGIS.RTM. does
not carry potential risk of transmission of blood borne
pathogens.
[0010] SYNAGIS.RTM. was initially formulated as a liquid for IV
use, at a concentration of 10 mg/ml SYNAGIS.RTM. in phosphate
buffered saline. A lyophilizcd formulation of SYNAGIS.RTM., which
allows a higher concentration (100 mg/ml after reconstitution, in
50 mM histidine and 3.2 mM glycine buffer with 6% (w/v) mannitol at
pH 6.0) of the antibody than this initial liquid formulation, was
produced later to allow intramuscular use. The lyophilized
formulation of SYNAGIS.RTM. is prepared by lyophilizing
SYNAGIS.RTM. at 54 mg/ml in an aqueous solution containing 25 mM
histidine, 1.6 mM glycine, and 3% (w/v) mannitol at pH 6.0. The
initial liquid formulation in PBS and the lyophilized formulation
of SYNAGIS.RTM. have been tested in phase I clinical studies in
healthy adults. The lyophilized formulation was tested in phase I
through phase IV studies in pediatric patients. SYNAGIS.RTM., at
doses of 15 mg/kg to 30 mg/kg for adults is found to be well
tolerated, and 15 mg/kg for children is found to be safe and
efficacious for RSV prophylaxis. The lyophilized formulation was
approved in 1998 by the FDA for use in the prevention of serious
lower respiratory tract disease caused by RSV in children at high
risk of RSV disease.
[0011] However, the lyophilized formulation has a number of
limitations, including a prolonged process for lyophilization and
resulting high cost for manufacturing. In addition, the lyophilized
formulation has to be reconstituted aseptically and accurately by
healthcare practitioners prior to administering to patients. The
reconstitution step itself requires certain specific procedures:
(1) a sterile diluent (i.e., water or 5% dextrose in water for
intravenous administration and water for intramuscular
administration) is added to the vial containing lyophilized
SYNAGIS.RTM., slowly and aseptically, and the vial must be swirled
very gently for 30 seconds to avoid foaming; (2) the reconstituted
SYNAGIS0 needs to stand at room temperature for a minimum of 20
minutes until the solution clarifies; and (3) the reconstituted
preparation must be administered within six (6) hours after the
reconstitution. Such reconstitution procedure is cumbersome and the
time limitation after the reconstitution can cause a great
inconvenience in administering the formulation to patients, leading
to significant waste, if not reconstituted properly or if the
reconstituted dose is not used within six (6) hours and must be
discarded.
[0012] Thus, a need exists for a liquid formulation of anti-RSV
antibodies, in general, at a concentration comparable to or higher
than the reconstituted lyophilized formulation so that there is no
need to reconstitute the formulation prior to administration. This
allows healthcare practitioners much quicker and easier
administration of anti-RSV antibodies to a patient.
[0013] Prior liquid antibody preparations have short shelf lives
and may lose biological activity of the antibodies resulting from
chemical and physical instabilities during the storage. Chemical
instability may be caused by deamidation, racemization, hydrolysis,
oxidation, beta elimination or disulfide exchange, and physical
instability may be caused by antibody denaturation, aggregation,
precipitation or adsorption. Among those, aggregation, deamidation
and oxidation are known to be the most common causes of the
antibody degradation (Wang et al., 1988, J. of Parenteral Science
& Technology 42 (Suppl):S4-S26; Cleland et al., 1993, Critical
Reviews in Therapeutic Drug Carrier Systems 10(4):307-377). Thus,
there is a need for a stable liquid formulation of an anti-RSV
antibody effective to prevent RSV infection.
3. SUMMARY OF INVENTION
[0014] The present invention is based, in part, on the development
of high concentration liquid formulations of antibodies or
fragments thereof that immunospecifically bind to a RSV antigen,
which formulations exhibit, in the absence of surfactant, inorganic
salts, and/or other excipients, stability and low to undetectable
levels of antibody fragmentation and/or aggregation, and very
little to no loss of biological activities of the antibody or
antibody fragment during manufacture, preparation, transportation,
and storage. In particular, the present invention provides liquid
formulation of antibodies or fragments thereof immunospecifically
bind to a RSV antigen, which antibodies are highly potent, have an
improved pharmacokinetic profile and, thus, have an overall
improved therapeutic profile, compared to SYNAGIS.RTM.. The liquid
formulations of the present invention facilitate the administration
of antibodies or fragments thereof that immunospecifically bind to
a RSV antigen for the prevention, treatment, management and/or
amelioration of a RSV infection, one or more symptoms thereof, and
other respiratory disorders that is associated with, potentiated by
or potentiates a RSV infection. In particular, the liquid
formulations of the present invention enable a healthcare
professional to quickly administer a sterile dosage of antibodies
or fragments thereof that immunospecifically bind to a RSV antigen
without having to accurately and aseptically reconstitute the
antibody or antibody fragment prior to administration as required
for the lyophilized dosage form. Such liquid formulations can be
manufactured more easily and cost effectively than lyophilized
formulations since liquid formulations do not require a prolonged
drying step, such as lyophilization, freeze-drying, etc. The liquid
formulations are made by a process in which the antibody being
formulated is in an aqueous phase throughout the purification and
formulation process. Preferably, the liquid formulations are made
by a process that does not include a drying step, for example, but
not by way of limitation, a lyophilization, freeze-drying,
spray-drying, or air-drying step.
[0015] The present invention provides liquid formulations of
anti-RSV antibodies or fragments thereof substantially free of
surfactant, inorganic salts, sugars, and/or other common
excipients, said formulations comprising histidine and a
concentration of about 15 mg/ml or higher of an antibody or a
fragment thereof that immunospecifically binds to a RSV antigen.
Optionally, the formulation may further comprise glycine.
Alternatively, the formulation of the present invention may further
comprise other common excipients, such as saccharides, polyols and
amino acids, including, but not limited to, arginine, lysine, and
methionine. The present invention also provides liquid formulations
substantially free of surfactant, inorganic salts, sugars, and/or
other commonly-known excipients, with pH ranges of about 5.0 to
about 7.0, preferably about 5.5 to 6.5, more preferably about 5.8
to about 6.2, and most preferably about 6.0, said formulations
comprising histidine and a concentration of about 15 mg/ml or
higher of an antibody or a fragment thereof that immunospecifically
binds to a RSV antigen.
[0016] The present invention encompasses stable liquid formulations
of an antibody or a fragment thereof that immunospecifically binds
to a RSV antigen, which formulations exhibit low to undetectable
levels of antibody aggregation and/or fragmentation with very
little to no loss of the biological activities of the antibody or
antibody fragment during manufacture, preparation, transportation,
and long periods of storage. The present invention also encompasses
stable liquid formulations of an antibody or a fragment thereof
that immunospecifically binds to a RSV antigen and have increased
in vivo half-lives relative to known antibodies such as, e.g.,
SYNAGIS.RTM., said formulations exhibiting low to undetectable
levels of antibody aggregation and/or fragmentation and very little
to no loss of biological activities of the antibodies or antibody
fragments. The present invention also encompasses stable liquid
formulations of an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen, said antibody or
antibody fragment comprising a variable heavy (VH) and/or variable
light (VL) domain having the amino acid sequence of any VH and/or
VL domain listed in Table 1, infra, and said formulations
exhibiting low to undetectable levels of antibody aggregation
and/or fragmentation, and very little to no loss of the biological
activities of the antibodies or antibody fragments. The present
invention further encompasses stable liquid formulations of an
antibody or a fragment thereof that immunospecifically binds to a
RSV antigen, said antibody or antibody fragment comprising one or
more VH complementarity determining regions (CDRs) and/or one or
more VL CDRs having the amino acid sequence of one or more VH CDRs
and/or VL CDRS listed in Table 1 and/or Table 2, infra, and said
formulations exhibiting low to undetectable levels of antibody
aggregation and/or fragmentation, and very little to no loss of the
biological activities of the antibodies or antibody fragments.
TABLE-US-00001 TABLE 1 Antibodies and Fragments Thereof Antibody VH
VL Name Domain VH CDR1 VH CDR2 VH CDR3 Domain VL CDR1 VL CDR2 VL
CDR3 *SYNAGIS SEQ ID TSGMSVG DIWWDDKK SMITNWYFDV SEQ ID KCQLSVGYMH
DTSKLAS FQGSGYPFT NO:7 (SEQ ID DYNPSLKS (SEQ ID NO:3) NO:8 (SEQ ID
NO:4) (SEQ ID (SEQ ID NO:1) (SEQ ID NO:2) NO:5) NO:6) AFFF SEQ ID
TAGMSVG DIWWDDKK SMITNFYFDV SEQ ID SASSSVGYMH DTFKLAS FQFSGYPFT
NO:9 (SEQ ID DYNPSLKS (SEQ ID NO:12) NO:13 (SEQ ID NO:14) (SEQ ID
(SEQ ID NO:10) (SEQ ID NO:2) NO:15) NO:16) p12f2 SEQ ID TPGMSVG
DIWWDDKK DMIFNFYFDV SEQ ID SLSSRVGYMH DTFYLSS FQGSGYPFT NO:17 (SEQ
ID HYNPSLKD (SEQ ID NO:20) NO:21 (SEQ ID NO:22) (SEQ ID (SEQ ID
NO:18) (SEQ ID NO:19) NO:23) NO:6) p12f4 SEQ ID TPGMSVG DIWWDGKK
DMIFNFYFDV SEQ ID SLSSRVGYMH DTRGLPS FQGSGYPFT NO:24 (SEQ ID
HYNPSLKD (SEQ ID NO:20) NO:26 (SEQ ID NO:22) (SEQ ID (SEQ ID NO:18)
(SEQ ID NO:25) NO:27) NO:6) p11d4 SEQ ID TPGMSVG DIWWDGKK
DMIFNWYFDV SEQ ID SPSSRVGYMH DTMRLAS FQGSGYPFT NO:28 (SEQ ID
HYNPSLKD (SEQ ID NO:29) NO:30 (SEQ ID NO:31) (SEQ ID (SEQ ID NO:18)
(SEQ ID NO:25) NO:32) NO:6) A1e109 SEQ ID TAGMSVG DIWWDGKK
DMIFNWYFDV SEQ ID SLSSRVGYMH DTFKLSS FQGSGYPFT NO:33 (SEQ ID
HYNPSLKD (SEQ ID NO:29) NO:34 (SEQ ID NO:22) (SEQ ID (SEQ ID NO:10)
(SEQ ID NO:25) NO:35) NO:6) A12a6 SEQ ID TAGMSVG DIWWDGKK
DMIFNFYFDV SEQ ID SASSRVGYMH DTFKLSS FQGSGYPFT NO:36 (SEQ ID
DYNPSLKD (SEQ ID NO:20) NO:38 (SEQ ID NO:39) (SEQ ID (SEQ ID NO:10)
(SEQ ID NO:37) NO:35) NO:6) A13c4 SEQ ID TAGMSVG DIWWDGKKS
DMIFNFYFDV SEQ ID SLSSRVGYMH DTMYQSS FQGSGYPFT NO:40 (SEQ ID
YNPSLKD (SEQ ID NO:20) NO:42 (SEQ ID NO:22) (SEQ ID (SEQ ID NO:10)
(SEQ ID NO:41) NO:43) NO:6) A17d4 SEQ ID TAGMSVG DIWWDDKKS
DMIFNFYFDV SEQ ID LPSSRVGYMH DTMYQSS FQGSGYPFT NO:44 (SEQ ID
YNPSLKD (SEQ ID NO:20) NO:46 (SEQ ID NO:47) (SEQ ID (SEQ ID NO:10)
(SEQ ID NO:45) NO:43) NO:6) A4B4 SEQ ID TAGMSVG DIWWDDKK DMIFNFYFDV
SEQ ID SASSRVGYMH DTFFLDS FQGSGYPFT NO:48 (SEQ ID HYNPSLKD (SEQ ID
NO:20) NO:49 (SEQ ID NO:39) (SEQ ID (SEQ ID NO:10) (SEQ ID NO:19)
NO:50) NO:6) A8C7 SEQ ID TAGMSVG DIWWDDKKS DMIFNWYFDV SEQ ID
SPSSRVGYMH DTRYQSS FQGSGYPFT NO:51 (SEQ ID YNPSLKD (SEQ ID NO:29)
NO:52 (SEQ ID NO:31) (SEQ ID (SEQ ID NO:10) (SEQ ID NO:45) NO:53)
NO:6) 1X-493L1FR SEQ ID TSGMSVG DIWWDDKK SMITNWYFDV SEQ ID
SASSSVGYMH DTSKLAS FQGSGYPFT NO:7 (SEQ ID DYNPSLKS (SEQ ID NO:3)
NO:54 (SEQ ID NO:14) (SEQ ID (SEQ ID NO:1) (SEQ ID NO:2) NO:5)
NO:6) H3-3F4 SEQ ID TAGMSVG DIWWDDKK DMIFNWYFDV SEQ ID SASSSVGYMH
DTFKLAS FQGSGYPFT NO:55 (SEQ ID DYNPSLKS (SEQ ID NO:29) NO:56 (SEQ
ID NO:14) (SEQ ID (SEQ ID NO: 10) (SEQ ID NO:2) NO:15) NO:6) M3H9
SEQ ID TAGMSVG DIWWDDKK DMIFNWYFDV SEQ ID SASSSVGYMH DTYKQTS
FQGSGYPFT NO:55 (SEQ ID DYNPSLKS (SEQ ID NO:29) NO:56 (SEQ ID
NO:14) (SEQ ID (SEQ ID NO:10) (SEQ ID NO:2) NO:57) NO:6) Y10H6 SEQ
ID TAGMSVG DIWWDDKK DMIFNWYFDV SEQ ID SASSSVGYMH DTRYLSS FQGSGYPFT
NO:55 (SEQ ID DYNPSLKS (SEQ ID NO:29) NO:58 (SEQ ID NO:14) (SEQ ID
(SEQ ID NO:10) (SEQ ID NO:2) NO:59) NO:6) DG SEQ ID TAGMSVG
DIWWDDKK DMITNFYFDV SEQ ID SASSSVGYMH DTFKLAS FQGSGYPFT NO:78 (SEQ
ID DYNPSLKS (SEQ ID NO:79) NO:56 (SEQ ID NO:14) (SEQ ID (SEQ ID
NO:10) (SEQ ID NO:2) NO:15) NO:6) AFFF (1) SEQ ID TAGMSVG DLWWDDKK
SMITNFYFDV SEQ ID SASSSVGYMH DTFKLAS FQGSFYPFT NO:9 (SEQ ID
DYNPSLKS (SEQ ID NO:12) NO:60 (SEQ ID NO:14) (SEQ ID (SEQ ID NO:10)
(SEQ ID NO:2) NO:15) NO:61) 6H8 SEQ ID TAGMSVG DIWWDDKK DMITNFYFDV
SEQ ID SASSSVGYMH DTFKLTS FQGSGYPFT NO:78 (SEQ ID DYNPSLKS (SEQ ID
NO:79) NO:62 (SEQ ID NO:14) (SEQ ID (SEQ ID NO:10) (SEQ ID NO:2)
NO:63) NO:6) L1-7E5 SEQ ID TAGMSVG DIWWDDKK DMITNFYFDV SEQ ID
SASSRVGYMH DTFKLAS FQGSGYPFT NO:78 (SEQ ID DYNPSLKS (SEQ ID NO:79)
NO:64 (SEQ ID NO:39) (SEQ ID (SEQ ID NO:10) (SEQ ID NO:2) NO:15)
NO:6) L215B10 SEQ ID TAGMSVG DIWWDDKK DMITNFYFDV SEQ ID SASSSVGYMH
DTFRLAS FQGSGYPFT NO:78 (SEQ ID DYNPSLKS (SEQ ID NO:79) NO:65 (SEQ
ID NO:14) (SEQ ID (SEQ ID NO:10) (SEQ ID NO:2) NO:66) NO:6) A13A11
SEQ ID TAGMSVG DIWWDDKK DMIFNWYFDV SEQ ID SPSSRVGYMH DTYRHSS
FQGSGYPFT NO:67 (SEQ ID HYNPSLKD (SEQ ID NO:29) NO:68 (SEQ ID
NO:31) (SEQ ID (SEQ ID NO:10) (SEQ ID NO:19) NO:69) NO:6) A1H5 SEQ
ID TAGMSVG DIWWDGKK DMIFNWYFDV SEQ ID SLSSSVGYMH DTFFHRS FQGSGYPFT
NO:70 (SEQ ID HYNPSLKD (SEQ ID NO:29) NO:71 (SEQ ID NO:72) (SEQ ID
(SEQ ID NO:10) (SEQ ID NO:25) NO:73) NO:6) A4B4 (1) SEQ ID TAGMSVG
DIWWDDKK DMIFNFYFDV SEQ ID SASSRVGYMH DTLLLDS FQGSGYPFT NO:48 (SEQ
ID HYNPSLKD (SEQ ID NO:20) NO:74 (SEQ ID NO:39) (SEQ ID (SEQ ID
NO:10) (SEQ ID NO:19) NO:75) NO:6) A4B4L1FR-S28R SEQ ID TAGMSVG
DIWWDDKK DMIFNFYFDV SEQ ID SASSRVGYMH DTSKLAS FQGSGYPFT NO:48 (SEQ
ID HYNPSLKD (SEQ ID NO:20) NO:11 (SEQ ID NO:39) (SEQ ID (SEQ ID
NO:10) (SEQ ID NO:19) NO:5) NO:6) A4B4-F52S SEQ ID TAGMSVG DIWWDDKK
DMIFNFYFDV SEQ ID SASSRVGYMH DTSFLDS FQGSGYPFT NO:48 (SEQ ID
HYNPSLKD (SEQ ID NO:20) NO:76 (SEQ ID NO:39) (SEQ ID (SEQ ID NO:10)
(SEQ ID NO:19) NO:77) NO:6) *Bold faced residues are preferred
residues for substitution to obtain antibodies with improved
affinity for a RSV antigen. Underlined residues are the amino acid
residues which are distinct from those residues in SYNAGIS
.RTM..
TABLE-US-00002 TABLE 2 CDR Sequences VH CDR1 VH CDR2 VH CDR3 VL
CDR1 VL CDR2 VL CDR3 TSGMSVG DIWWDDKKDYNPSLKS SMITNWYFDV KCQLSVGYMH
DTSKLAS FQGSGYPFT (SEQ ID NO:1) (SEQ ID NO:2) (SEQ ID NO:3) (SEQ ID
NO:4) (SEQ ID NO:5) (SEQ ID NO:6) TPGMSVG DIWWDDKKHYNPSLKD
DMITNFYFDV KCQSSVGYMH DTSYLAS FQFSGYPFT (SEQ ID NO:18) (SEQ ID
NO:19) (SEQ ID NO:208) (SEQ ID NO:80) (SEQ ID NO:81) (SEQ ID
NO:209) TAGMSVG DIWWDDKKHYNPSLKS DMITNWYFDV KCQSRVGYMH DTSYLSS
FQGSFYPFT (SEQ ID NO:10) (SEQ ID NO:82) (SEQ ID NO:83) (SEQ ID
NO:84) (SEQ ID NO:85) (SEQ ID NO:61) DIWWDDKKDYNPSLKD DMIFNWYFDV
KCQLRVGYMH DTKKLSS (SEQ ID NO:86) (SEQ ID NO:29) (SEQ ID NO:87)
(SEQ ID NO:88) DIWWDDKKHYNPSLKS DMIFNFYFDV KLQLSVGYMH DTFYLSS (SEQ
ID NO:91) (SEQ ID NO:20) (SEQ ID NO:89) (SEQ ID NO:90)
DIWWDDKKDYNPSLKD SMITNFYFDV KLQSSVGYMH DTFKLAS (SEQ ID NO:93) (SEQ
ID NO:12) (SEQ ID NO:92) (SEQ ID NO:15) DIWWDGKKHYNPSLKD SMIFNWYFDV
KLQSRVGYMH DTFKLSS (SEQ ID NO:25) (SEQ ID NO:94) (SEQ ID NO:95)
(SEQ ID NO:96) DIWWDGKKDYNPSLKS SMIFNFYFDV KLQLRVGYMH DTFYLAS (SEQ
ID NO:100) (SEQ ID NO:97) (SEQ ID NO:98) (SEQ ID NO:99)
DIWWDGKKDYNPSLKD KLSLSVGYMH DTSKLPS (SEQ ID NO:103) (SEQ ID NO:101)
(SEQ ID NO:102) DIWWDGKKHYNPSLKS KLSSSVGYMH DTSGLAS (SEQ ID NO:106)
(SEQ ID NO:104) (SEQ ID NO:105) DIWWDDKKSYNPSLKS KLSSRVGYMH DTSGLPS
(SEQ ID NO:109) **(SEQ ID NO:107) (SEQ ID NO:108) DIWWDDKKSYNPSLKD
KLSLRVGYMH DTRGLPS (SEQ ID NO:111) (SEQ ID NO:110) (SEQ ID NO:27)
DIWWDGKKSYNPSLKS KCSLSVGYMH DTRKLAS (SEQ ID NO:114) (SEQ ID NO:112)
(SEQ ID NO:113) DIWWDGKKSYNPSLKD KCSSSVGYMH DTRGLAS (SEQ ID NO:41)
(SEQ ID NO:115) (SEQ ID NO:116) KCSSRVGYMH DTRKLPS (SEQ ID NO:117)
(SEQ ID NO:118) KCSLRVGYMH DTMRLAS (SEQ ID NO:119) (SEQ ID NO:32)
SLSLSVGYMH DTMKLAS (SEQ ID NO:120) (SEQ ID NO:121) SLSSSVGYMH
DTSRLAS (SEQ ID NO:122) (SEQ ID NO:123) SLSSRVGYMH DTSLLAS (SEQ ID
NO:22) (SEQ ID NO:124) SLSLRVGYMH DTSLLDS (SEQ ID NO:125) (SEQ ID
NO:126) SCQLSVGYMH DTSKLDS (SEQ ID NO:127) (SEQ ID NO:128)
SCQSSVGYMH DTLLLDS (SEQ ID NO:129) (SEQ ID NO:75) SCQSRVGYMH
DTLKLDS (SEQ ID NO:130) (SEQ ID NO:131) SCQLRVGYMH DTLLLAS (SEQ ID
NO:132) (SEQ ID NO:133) SLQLSVGYMH DTLKLAS (SEQ ID NO:134) (SEQ ID
NO:135) SLQSSVGYMH DTSKLSS (SEQ ID NO:136) (SEQ ID NO:137)
SLQSRVGYMH DTSKQAS (SEQ ID NO:138) (SEQ ID NO:139) SLQLRVGYMH
DTSKQSS (SEQ ID NO:140) (SEQ ID NO:141) SCSLSVGYMH DTSYLAS (SEQ ID
NO:142) (SEQ ID NO:143) SCSSSVGYMH DTSYLSS (SEQ ID NO:144) (SEQ ID
NO:145) SCSSRVGYMH DTSYQAS (SEQ ID NO:146) (SEQ ID NO:147)
SCSLRVGYMH DTSYQSS (SEQ ID NO:148) (SEQ ID NO:149) KPSSRVGYMH
DTMYQAS (SEQ ID NO:150) (SEQ ID NO:151) KPSLRVGYMH DTMYQSS (SEQ ID
NO:152) (SEQ ID NO:43) KPSSSVGYMH DTMKQAS (SEQ ID NO:153) (SEQ ID
NO:154) KPSLSVGYMH DTMKQSS (SEQ ID NO:155) (SEQ ID NO:156)
KPQSRVGYMH DTMYLAS (SEQ ID NO:157) (SEQ ID NO:158) KPQLRVGYMH
DTMYLSS (SEQ ID NO:159) (SEQ ID NO:160) KPQSSVGYMH DTMKLAS (SEQ ID
NO:161) (SEQ ID NO:162) KPQLSVGYMH DTMKLSS (SEQ ID NO:163) (SEQ ID
NO:164) SPSSRVGYMH DTSKLSS (SEQ ID NO:31) (SEQ ID NO:165)
SPSLRVGYMH DTRYQAS (SEQ ID NO:166) (SEQ ID NO:167) SPSSSVGYMH
DTRYQSS (SEQ ID NO:168) (SEQ ID NO:53) SPSLSVGYMH DTRKQAS (SEQ ID
NO:169) (SEQ ID NO:170) SPQSRVGYMH DTRKQSS (SEQ ID NO:171) (SEQ ID
NO:172) SPQLRVGYMH DTRKLAS (SEQ ID NO:173) (SEQ ID NO:174)
SPQSSVGYMH DTRKLSS (SEQ ID NO:175) (SEQ ID NO:176) SPQLSVGYMH
DTRYLAS (SEQ ID NO:177) (SEQ ID NO:178) KAQSRVGYMH DTRYLSS (SEQ ID
NO:179) (SEQ ID NO:59) KAQLRVGYMH DTFFLDS (SEQ ID NO:180) (SEQ ID
NO:50) KAQSSVGYMH DTSFLDS (SEQ ID NO:181) (SEQ ID NO:77) KAQLSVGYMH
(SEQ ID NO:182) KASSRVGYMH (SEQ ID NO:183) KASLRVGYMH (SEQ ID
NO:184) KASSSVGYMH (SEQ ID NO:185) KASLSVGYMH (SEQ ID NO:186)
SASSRVGYMH (SEQ ID NO:39) SASLRVGYMH (SEQ ID NO:187) SASSSVGYMH
(SEQ ID NO:14) SASLSVGYMH (SEQ ID NO:188) SAQSRVGYMH (SEQ ID
NO:189) SAQLRVGYMH (SEQ ID NO:190) SAQSSVGYMH (SEQ ID NO:191)
LPSSRVGYMH (SEQ ID NO:47) LPSLSVGYMH (SEQ ID NO:192) LPSSSVGYMH
(SEQ ID NO:193) LPSLRVGYMH (SEQ ID NO:194) LCSSRVGYMH (SEQ ID
NO:195) LCSLSVGYMH (SEQ ID NO:196) LCSSSVGYMH (SEQ ID NO:197)
LCSLRVGYMH (SEQ ID NO:198) LPQSRVGYMH (SEQ ID NO:199) LPQLSVGYMH
(SEQ ID NO:200) LPQSSVGYMH (SEQ ID NO:201) LPQLRVGYMH (SEQ ID
NO:202) LCQSRVGYMH (SEQ ID NO:203) LCQLSVGYMH (SEQ ID NO:204)
LCQSSVGYMH (SEQ ID NO:205) LCQLRVGYMH (SEQ ID NO:206) SAQLSVGYMH
(SEQ ID NO:207) Bold faced and underlined residues are the amino
acid residues which are distinct from those residues in SYNAGIS
.RTM..
[0017] The present invention encompasses liquid formulations of
antibodies or fragments thereof that immunospecifically bind to a
RSV antigen, said formulations having stability at 38-42.degree. C.
as assessed by high performance size exclusion chromatography
(HPSEC). The present invention encompasses liquid formulations of
an antibody or fragment thereof that immunospecifically binds to a
RSV antigen, said formulations having stability, as assessed by
HPSEC, at the temperature ranges of 38.degree. C.-42.degree. C. for
at least 60 days (in specific embodiments, not more than 120 days),
of 20.degree. C.-24.degree. C. for at least 1 year, and of
2.degree. C.-8.degree. C. for at least 3 years. The present
invention also encompasses liquid formulations of antibodies or
fragments thereof that immunospecifically bind to a RSV antigen,
said formulations having low to undetectable levels of antibody
aggregation as measured by HPSEC, and further, exhibit very little
to no loss of the biological activity(ies) of the antibodies or
antibody fragments of the formulation compared to the reference
antibodies as measured by antibody binding assays such as, e.g.,
ELISAs.
[0018] The present invention provides methods for preparing liquid
formulations of an antibody or fragment thereof that
immunospecifically binds to a RSV antigen, said methods comprising
concentrating a fraction containing the purified antibody or
antibody fragment to a final concentration about 15 mg/ml, about 20
mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60
mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100
mg/ml, about 200 mg/ml, about 250 mg/ml, or about 300 mg/ml using a
semi-permeable membrane with an appropriate molecular weight (mw)
cutoff (e.g., a 30 kD cutoff for whole antibody molecules and
F(ab').sub.2 fragments, and a 10 kD cutoff for antibody fragments
such as a Fab fragments and diafiltering the concentrated antibody
or antibody fragment fraction into the formulation buffer using the
same membrane. The formulation buffer of the present invention
comprises histidine at a concentration ranging from about 1 mM to
about 100 mM, about 10 mM to about 50 mM, about 20 mM to about 30
mM, or about 23 mM to about 27 mM, and is most preferably about 25
mM. To obtain an appropriate pH for a particular antibody or
antibody fragment, it is preferable that histidine (and glycine, if
added) is first dissolved in water to obtain a buffer solution with
higher pH than the desired pH and then the pH is brought down to
the desired level by the addition of HCl. This way, the formation
of inorganic salts (e.g., the formation of NaCl when, e.g.,
histidine hydrochloride is used as the source of histidine and the
pH is raised to the desired level by the addition of NaOH) can be
avoided.
[0019] The liquid formulations of the present invention are
prepared by maintaining the antibodies in an aqueous solution at
any time during the preparation. In other words, the liquid
formulations are prepared without involving any step of drying the
antibodies or the formulations themselves by, for example,
lyophilization, vacuum drying, etc.
[0020] The liquid formulations of the present invention may be
sterilized by sterile filtration using a 0.2 or 0.22 micron filter.
Sterilized liquid formulations of the present invention may be
administered to a subject to prevent, treat, manage or ameliorate a
RSV infection or one or more symptoms thereof, or other respiratory
conditions associated with, potentiated by, or potentiates a RSV
infection.
[0021] The present invention also provides kits comprising the
liquid formulations of antibodies or fragments thereof that
immunospecifically bind to a RSV antigen for use by, e.g., a
healthcare professional. The present invention further provides
methods of preventing, treating, managing or ameliorating a RSV
infection or one or more symptoms thereof, or other respiratory
conditions associated with, potentiated by, or potentiates a RSV
infection by administering the liquid formulations of the present
invention. The liquid formulations of the present invention can
also be used to diagnose, detect or monitor a RSV infection.
[0022] 3.1 Terminology
[0023] All liquid formulations of antibodies and/or fragments
thereof that immunospecifically bind to a RSV antigen described
above are herein collectively referred to as "liquid formulations
of the invention," "antibody liquid formulations of the invention,"
"liquid formulations of antibodies or fragments thereof that
immunospecifically bind to a RSV antigen," or "liquid formulations
of anti-RSV antibodies."
[0024] As used herein, the term "analogue" in the context of
proteinaceous agent (e.g., proteins, polypeptides, peptides, and
antibodies) refers to a proteinaceous agent that possesses a
similar or identical function as a second proteinaceous agent but
does not necessarily comprise a similar or identical amino acid
sequence of the second proteinaceous agent, or possess a similar or
identical structure of the second proteinaceous agent. The
analogues herein referred to do not include SYNAGIS.RTM.. In a
specific embodiment, antibody analogues immunospecifically bind to
the same epitope as the original antibodies from which the
analogues were derived. In an alternative embodiment, antibody
analogues immunospecifically bind to different epitopes than the
original antibodies from which the analogues were derived. In
another embodiment, antibody analogues compete with antibodies for
binding to the epitope that SYNAGIS.RTM. binds to or compete for
binding to an epitope with SYNAGIS.RTM.. A proteinaceous agent that
has a similar amino acid sequence refers to a second proteinaceous
agent that satisfies at least one of the following: (a) a
proteinaceous agent having an amino acid sequence that is at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95% or at least
99% identical to the amino acid sequence of a second proteinaceous
agent; (b) a proteinaceous agent encoded by a nucleotide sequence
that hybridizes under stringent conditions to a nucleotide sequence
encoding a second proteinaceous agent of at least 5 contiguous
amino, acid residues, at least 10 contiguous amino acid residues,
at least 15 contiguous amino acid residues, at least 20 contiguous
amino acid residues, at least 25 contiguous amino acid residues, at
least 40 contiguous amino, acid residues, at least 50 contiguous
amino acid residues, at least 60 contiguous amino residues, at
least 70 contiguous amino acid residues, at least 80 contiguous
amino acid residues, at least 90 contiguous amino acid residues, at
least 100 contiguous amino acid residues, at least 125 contiguous
amino acid residues, or at least 150 contiguous amino acid
residues; and (c) a proteinaceous agent encoded by a nucleotide
sequence that is at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95% or at least 99% identical to the nucleotide sequence
encoding a second proteinaceous agent. A proteinaceous agent with
similar structure to a second proteinaceous agent refers to a
proteinaceous agent that has a similar secondary, tertiary or
quaternary structure to the second proteinaceous agent. The
structure of a proteinaceous agent can be determined by methods
known to those skilled in the art, including but not limited to,
peptide sequencing, X ray crystallography, nuclear magnetic
resonance, circular dichroism, and crystallographic electron
microscopy.
[0025] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in the sequence of a first amino acid or nucleic acid
sequence for optimal alignment with a second amino acid or nucleic
acid sequence). The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity number of identical overlapping
positions/total number of positions.times.100%). In one embodiment,
the two sequences are the same length.
[0026] The determination of percent identity between two sequences
can also be accomplished using a mathematical algorithm. A
preferred, non limiting example of a mathematical algorithm
utilized for the comparison of two sequences is the algorithm of
Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264
2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad.
Sci. U.S.A. 90:5873 5877. Such an algorithm is incorporated into
the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol.
Biol. 215:403. BLAST nucleotide searches can be performed with the
NBLAST nucleotide program parameters set, e.g., for score=100,
wordlength=12 to obtain nucleotide sequences homologous to a
nucleic acid molecules of the present invention. BLAST protein
searches can be performed with the XBLAST program parameters set,
e.g., to score 50, wordlength=3 to obtain amino acid sequences
homologous to a protein molecule of the present invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., 1997, Nucleic Acids
Res. 25:3389 3402. Alternatively, PSI BLAST can be used to perform
an iterated search which detects distant relationships between
molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast
programs, the default parameters of the respective programs (e.g.,
of XBLAST and NBLAST) can be used (see, e.g., the NCBI website).
Another preferred, non limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller, 1988, CABIOS 4:11 17. Such an algorithm is incorporated
in the ALIGN program (version 2.0) which is part of the GCG
sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4 can be
used.
[0027] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, typically only
exact matches are counted.
[0028] As used herein, the term "analogue" in the context of a
non-proteinaceous analog refers to a second organic or inorganic
molecule which possess a similar or identical function as a first
organic or inorganic molecule and is structurally similar to the
first organic or inorganic molecule.
[0029] The term "antibody fragment" as used herein refers to a
fragment of an antibody that immunospecifically binds to a RSV
antigen. Antibody fragments may be generated by any technique known
to one of skill in the art. For example, Fab and F(ab').sub.2
fragments may be produced by proteolytic cleavage of immunoglobulin
molecules, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab').sub.2 fragments). F(ab').sub.2
fragments contain the complete light chain, and the variable
region, the CH 1 region and the hinge region of the heavy chain.
Antibody fragments can be also produced by recombinant DNA
technologies. Antibody fragments may be one or more complementarity
determining regions (CDRs) of antibodies.
[0030] The terms "antibody" and "antibodies" as used herein refer
to monoclonal antibodies, bispecific antibodies, multispecific
antibodies, human antibodies, humanized antibodies, chimeric
antibodies, camelised antibodies, single domain antibodies,
single-chain Fvs (scFv), single chain antibodies, Fab fragments,
F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic
(anti-Id) antibodies (including, e.g., anti-Id antibodies to
antibodies of the invention), and epitope-binding fragments of any
of the above. In particular, antibodies include immunoglobulin
molecules and immunologically active fragments of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site.
Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM,
IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and
IgA2) or subclass.
[0031] As used herein, the term "cytokine receptor modulator"
refers to an agent which modulates the phosphorylation of a
cytokine receptor, the activation of a signal transduction pathway
associated with a cytokine receptor, and/or the expression of a
particular protein such as a cytokine. Such an agent may directly
or indirectly modulate the phosphorylation of a cytokine receptor,
the activation of a signal transduction pathway associated with a
cytokine receptor, and/or the expression of a particular protein
such as a cytokine. Thus, examples of cytokine receptor modulators
include, but are not limited to, cytokines, fragments of cytokines,
fusion proteins and antibodies that immunospecifically binds to a
cytokine receptor or a fragment thereof. Further, examples of
cytokine receptor modulators include, but are not limited to,
peptides, polypeptides (e.g., soluble cytokine receptors), fusion
proteins and antibodies that immunospecifically binds to a cytokine
or a fragment thereof.
[0032] As used herein, the term "derivative" in the context of
proteinaceous agent (e.g., proteins, polypeptides, peptides, and
antibodies) refers to a proteinaceous agent that comprises an amino
acid sequence which has been altered by the introduction of amino
acid residue substitutions, deletions, and/or additions. The term
"derivative" as used herein also refers to a proteinaceous agent
which has been modified, i.e, by the covalent attachment of any
type of molecule to the proteinaceous agent. For example, but not
by way of limitation, an antibody may be modified, e.g., by
glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular ligand or other protein, etc. A
derivative of a proteinaceous agent may be produced by chemical
modifications using techniques known to those of skill in the art,
including, but not limited to specific chemical cleavage,
acetylation, formylation, metabolic synthesis of tunicamycin, etc.
Further, a derivative of a proteinaceous agent may contain one or
more non-classical amino acids. A derivative of a proteinaceous
agent possesses a similar or identical function as the
proteinaceous agent from which it was derived.
[0033] As used herein, the term "derivative" in the context of a
non-proteinaceous derivative refers to a second organic or
inorganic molecule that is formed based upon the structure of a
first organic or inorganic molecule. A derivative of an organic
molecule includes, but is not limited to, a molecule modified,
e.g., by the addition or deletion of a hydroxyl, methyl, ethyl,
carboxyl or amine group. An organic molecule may also be
esterified, alkylated and/or phosphorylated.
[0034] The term "epitope" as used herein refers to a fragment of a
RSV polypeptide having antigenic or immunogenic activity in an
animal, preferably a mammal, and most preferably in a human. An
epitope having immunogenic activity is a fragment of a RSV
polypeptide that elicits an antibody response in an animal. An
epitope having antigenic activity is a fragment of a RSV
polypeptide to which an antibody immunospecifically binds as
determined by any method well known in the art, for example, by the
immunoassays described herein. Antigenic epitopes need not
necessarily be immunogenic.
[0035] The term "excipients" as used herein refers to inert
substances which are commonly used as a diluent, vehicle,
preservatives, binders, or stabilizing agent for drugs and
includes, but not limited to, proteins (e.g., serum albumin, etc.),
amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine,
glycine, histidine, etc.), fatty acids and phospholipids (e.g.,
alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS,
polysorbate, nonionic surfactant, etc.), saccharides (e.g.,
sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol,
sorbitol, etc.). Also see Remington's Pharmaceutical Sciences (by
Joseph P. Remington, 18th ed., Mack Publishing Co., Easton, Pa.),
which is hereby incorporated in its entirety.
[0036] The term "fragment" as used herein refers to a peptide,
polypeptide, or protein (including an antibody) comprising an amino
acid sequence of at least 5 contiguous amino acid residues, at
least 10 contiguous amino acid residues, at least 15 contiguous
amino acid residues, at least 20 contiguous amino acid residues, at
least 25 contiguous amino acid residues, at least 40 contiguous
amino acid residues, at least 50 contiguous amino acid residues, at
least 60 contiguous amino residues, at least 70 contiguous amino
acid residues, at least contiguous 80 amino acid residues, at least
contiguous 90 amino acid residues, at least contiguous 100 amino
acid residues, at least contiguous 125 amino acid residues, at
least 150 contiguous amino acid residues, at least contiguous 175
amino acid residues, at least contiguous 200 amino acid residues,
or at least contiguous 250 amino acid residues of the amino acid
sequence of another polypeptide or protein. In a specific
embodiment, a fragment of a protein or polypeptide retains at least
one function of the protein or polypeptide. In another embodiment,
a fragment of a protein or polypeptide retains at least two, three
or four functions of the protein or polypeptide. Preferably a
fragment of an antibody that immunospecifically binds to a RSV
antigen retains the ability to bind to a RSV antigen.
[0037] The term "fusion protein" as used refers to a polypeptide or
protein that comprises an amino acid sequence of a first protein,
polypeptide or fragment, analogue or derivative thereof, and an
amino acid sequence of a heterologous protein or polypeptide (i.e.,
a second protein, polypeptide or fragment, analogue or derivative
thereof different than the first protein or fragment, analogue or
derivative thereof). In one embodiment, a fusion protein comprises
a prophylactic or therapeutic agent fused to a heterologous
protein, polypeptide or peptide. In accordance with this
embodiment, the heterologous protein, polypeptide or peptide may or
may not be a different type of prophylactic or therapeutic
agent.
[0038] The terms "high concentration" and "concentrated antibody"
as used herein refer to a concentration of 50 mg/ml or higher,
preferably 95 mg/ml or higher of an antibody or fragment thereof
that immunospecifically binds to a RSV antigen, in an antibody
formulation.
[0039] The term "host cell" as used herein includes a subject cell
transfected or transformed with a nucleic acid molecule and the
progeny or potential progeny of such a cell. Progeny of such a cell
may not be identical to the parent cell transfected with the
nucleic acid molecule due to mutations or environmental influences
that may occur in succeeding generations or integration of the
nucleic acid molecule into the host cell genome.
[0040] The term "hybridizes under stringent conditions" as used
herein describes conditions for hybridization and washing under
which nucleotide sequences at least 30% (preferably at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, or at least 95%) identical to each other
typically remain hybridized to each other. Such stringent
conditions are known to those skilled in the art and can be found
in Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y. (1989), 6.3.1-6.3.6. In one, non limiting example stringent
hybridization conditions are hybridization at 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.1.times.SSC, 0.2% SDS at about 68.degree.
C. In a preferred, non-limiting example stringent hybridization
conditions are hybridization in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
50-65.degree. C. (i.e., one or more washes at 50.degree. C.,
55.degree. C., 60.degree. C. or 65.degree. C.). It is understood
that the nucleic acids of the invention do not include nucleic acid
molecules that hybridize under these conditions solely to a
nucleotide sequence consisting of only A or T nucleotides.
[0041] The term "human infant" as used herein refers to a human
less than 24 months, preferably less than 16 months, less than 12
months, less than 6 months, less than 3 months, less than 2 months,
or less than 1 month of age.
[0042] The term "human infant born prematurely" as used herein
refers to a human born at less than 40 weeks gestational age,
preferably less than 35 weeks gestational age, who is less than 6
months old, preferably less than 3 months old, more preferably less
than 2 months old, and most preferably less than 1 month old.
[0043] As used herein, the term "immunospecifically bind to a RSV
antigen", "anti-RSV antibodies" and analogous terms refer to
antibodies or fragments thereof that specifically bind to a RSV
antigen and do not specifically bind to other polypeptides. An
antibody or fragment thereof that immunospecifically binds to a RSV
antigen may bind to other peptides or polypeptides with lower
affinity as determined by, e.g., immunoassays, BIAcore, isothermal
titration calorimetry, or other assays known in the art. An
antibody or a fragment thereof that immunospecifically binds to a
RSV antigen may be cross-reactive with related antigens.
Preferably, an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen does not cross-react with
other antigens. An antibody or a fragment thereof that
immunospecifically binds to a RSV antigen can be identified, for
example, by immunoassays, BIAcore, or other techniques known to
those of skill in the art. An antibody or a fragment thereof binds
specifically to a RSV antigen when it binds to a RSV antigen with
higher affinity than to any cross-reactive antigen as determined
using experimental techniques, such as radioimmunoassays (RIA) and
enzyme-linked immunosorbent assays (ELISAs). See, e.g., Paul, ed.,
1989, Fundamental Immunology Second Edition, Raven Press, New York
at pages 332-336 for a discussion regarding antibody
specificity.
[0044] The term "immunomodulatory agent" and variations thereof
including, but not limited to, immunomodulatory agents, as used
herein refer to an agent that modulates a host's immune system. In
certain embodiments, an immunomodulatory agent is an
immunosuppressant agent. In certain other embodiments, an
immunomodulatory agent is an immunostimulatory agent. In accordance
with the invention, an immunomodulatory agent used in the
combination therapies of the invention does not include an anti-RSV
antibody or fragment thereof. Immunomodulatory agents include, but
are not limited to, small molecules, peptides, polypeptides,
proteins, fusion proteins, antibodies, inorganic molecules, mimetic
agents, and organic molecules.
[0045] The term "in combination" as used herein refers to the use
of more than one therapies (e.g., prophylactic and/or therapeutic
agents). The use of the term "in combination" does not restrict the
order in which therapies (e.g., prophylactic and/or therapeutic
agents) are administered to a subject with a RSV infection or a
respiratory condition associated with, potentiated by or
potentiating a RSV infection. A first therapy (e.g., a prophylactic
or therapeutic agent) can be administered prior to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks before), concomitantly with, or subsequent to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1
week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12
weeks after) the administration of a second therapy (e.g., a
prophylactic or therapeutic agent) to a subject with a RSV
infection or a respiratory condition associated with, potentiated
by or potentiating a RSV infection.
[0046] The term "inorganic salt" as used herein refers to any
compounds containing no carbon that result from replacement of part
or all of the acid hydrogen or an acid by a metal or a group acting
like a metal and are often used as a tonicity adjusting compound in
pharmaceutical compositions and preparations of biological
materials. The most common inorganic salts are NaCl, KCl,
NaH.sub.2PO.sub.4, etc.
[0047] As used herein, the term "isolated" in the context of a
proteinaceous agent (e.g., a peptide, polypeptide, fusion protein,
or antibody) refers to a proteinaceous agent which is substantially
free of cellular material or contaminating proteins from the cell
or tissue source from which it is derived, or substantially free of
chemical precursors or other chemicals when chemically synthesized.
The language "substantially free of cellular material" includes
preparations of a proteinaceous agent in which the proteinaceous
agent is separated from cellular components of the cells from which
it is isolated or recombinantly produced. Thus, a proteinaceous
agent that is substantially free of cellular material includes
preparations of a proteinaceous agent having less than about 30%,
20%, 10%, or 5% (by dry weight) of heterologous protein,
polypeptide, peptide, or antibody (also referred to as a
"contaminating protein"). When the proteinaceous agent is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about
20%, 10%, or 5% of the volume of the protein preparation. When the
proteinaceous agent is produced by chemical synthesis, it is
preferably substantially free of chemical precursors or other
chemicals, i.e., it is separated from chemical precursors or other
chemicals which are involved in the synthesis of the proteinaceous
agent. Accordingly, such preparations of a proteinaceous agent have
less than about 30%, 20%, 10%, 5% (by dry weight) of chemical
precursors or compounds other than the proteinaceous agent of
interest. In a preferred embodiment, an antibody of the invention
is isolated.
[0048] As used herein, the term "isolated" in the context of
nucleic acid molecules refers to a nucleic acid molecule which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized.
In a preferred embodiment, a nucleic acid molecule encoding an
antibody of the invention is isolated.
[0049] The phrase "low to undetectable levels of aggregation" as
used herein refers to samples containing no more than 5%, no more
than 4%, no more than 3%, no more than 2%, no more than 1% and most
preferably no more than 0.5% aggregation by weight of protein as
measured by high performance size exclusion chromatography
(HPSEC).
[0050] The term "low to undetectable levels of fragmentation" as
used herein refers to samples containing equal to or more than 80%,
85%, 90%, 95%, 98% or 99% of the total protein, for example, in a
single peak as determined by HPSEC, or in two peaks (heavy- and
light-chains) by reduced Capillary Gel Electrophoresis (rCGE),
representing the non-degraded antibody or a non-degraded fragment
thereof, and containing no other single peaks having more than 5%,
more than 4%, more than 3%, more than 2%, more than 1%, or more
than 0.5% of the total protein in each. The term "reduced Capillary
Gel Electrophoresis" as used herein refers to capillary gel
electrophoresis under reducing conditions sufficient to reduce
disulfide bonds in an antibody or fragment thereof.
[0051] As used herein, the terms "manage", "managing" and
"management" refer to the beneficial effects that a subject derives
from a therapy (e.g., a prophylactic or therapeutic agent), which
does not result in a cure of the infection. In certain embodiments,
a subject is administered one or more therapies (e.g., prophylactic
or therapeutic agents) to "manage" a infection, one or more
symptoms thereof, or a respiratory condition associated with,
potentiated by, or potentiating a RSV infection, so as to prevent
the progression or worsening of the infection.
[0052] As used herein, the term "mast cell modulator" refers to an
agent which modulates the activation of a mast cell, mast cell
degranulation, and/or expression of a particular protein such as a
cytokine. Such an agent may directly or indirectly modulate the
activation of a mast cell, degranulation of the mast cell, and/or
the expression of a particular protein such as a cytokine.
Non-limiting examples of mast cell modulators include, but are not
limited to, small molecules, peptides, polypeptides, proteins,
nucleic acids (e.g., DNA and RNA nucleotides including, but not
limited to, antisense nucleotide sequences, triple helices, RNAi,
and nucleotide sequences encoding biologically active proteins,
polypeptides, or peptides), fusion proteins, antibodies, synthetic
or natural inorganic molecules, synthetic or natural organic
molecule, or mimetic agents which inhibit and/or reduce the
expression, function, and/or activity of a stem cell factor, a mast
cell protease, a cytokine (such as IL-3, IL-4, and IL-9), a
cytokine receptor (such as IL-3R, IL-4R, and IL-9R), and a stem
cell receptor. Other non-limiting examples of mast cell modulators
include, but are not limited to small molecules, peptides,
polypeptides, proteins, nucleic acids (e.g., DNA and RNA
nucleotides including, but not limited to, antisense nucleotide
sequences, triple helices, RNAi, and nucleotide sequences encoding
biologically active proteins, polypeptides, or peptides), fusion
proteins, antibodies, synthetic or natural inorganic molecules,
synthetic or natural organic molecule, or mimetic agents which
inhibit and/or reduce the expression, function and/or activity of
IgE. In certain embodiments, a mast cell modulator is an agent that
prevents or reduces the activation of additional mast cells
following degranulation of mast cells. In other embodiments, a mast
cell modulator is an agent that inhibits or reduces mast cell
degranulation.
[0053] The terms "non-responsive" and "refractory" as used herein
describe patients treated with a currently available therapy (such
as but not limited to, a prophylactic or therapeutic agent) for a
RSV infection, one or more symptoms thereof, or a respiratory
condition associated with, potentiated by, or potentiating a RSV
infection, which is not clinically adequate to relieve one or more
symptoms associated with the infection. Typically, such patients
suffer from severe, persistently active infection and require
additional therapy to ameliorate the symptoms associated with their
infection or respiratory condition.
[0054] As used herein, the terms "nucleic acids" and "nucleotide
sequences" include DNA molecules (e.g., cDNA or genomic DNA), RNA
molecules (e.g., mRNA), combinations of DNA and RNA molecules or
hybrid DNA/RNA molecules, and analogues of DNA or RNA molecules.
Such analogues can be generated using, for example, nucleotide
analogues, which include, but are not limited to, inosine or
tritylated bases. Such analogues can also comprise DNA or RNA
molecules comprising modified backbones that lend beneficial
attributes to the molecules such as, for example, nuclease
resistance or an increased ability to cross cellular membranes. The
nucleic acids or nucleotide sequences can be single-stranded,
double-stranded, may contain both single-stranded and
double-stranded portions, and may contain triple-stranded portions,
but preferably is double-stranded DNA.
[0055] The term "pharmaceutically acceptable" as used herein means
being approved by a regulatory agency of the Federal or a state
government, or listed in the U.S. Pharmacopia, European Pharmacopia
or other generally recognized pharmacopia for use in animals, and
more particularly in humans.
[0056] The term "polyol" as used herein refers to a sugar that
contains many --OH groups compared to a normal saccharide.
[0057] The terms "prophylactic agent" and "prophylactic agents" as
used refer to any agent(s) which can be used in the prevention of a
RSV infection, one or more symptoms thereof, or a respiratory
condition associated with, potentiated by, or potentiating a RSV
infection. In certain embodiments, the term "prophylactic agent"
refers to an antibody or fragment thereof that immunospecifically
binds to a RSV antigen. In accordance with these embodiments, the
antibody or antibody fragment may be a component of a liquid
formulation of the invention. In certain other embodiments, the
term "prophylactic agent" does not refer to, an antibody or
fragment thereof that immunospecifically binds to a RSV antigen or
a formulation comprising such an antibody or antibody fragment. In
certain other embodiments, the term "prophylactic agent" does not
refer to SYNAGIS.RTM. or a antigen-binding fragment thereof.
[0058] The terms "prevent", "preventing" and "prevention" as used
herein refer to the prevention of the recurrence, onset, or
development of a RSV infection, one or more symptoms thereof, or a
respiratory condition associated with, potentiated by, or
potentiating a RSV infection in a subject resulting from the
administration of a therapy (e.g., a prophylactic or therapeutic
agent), or combination therapies (e.g., the administration of a
combination of prophylactic agents).
[0059] The phrase "prophylactically effective amount" as used
herein refers to the amount of a therapy (e.g., a prophylactic
agent (e.g., an antibody or fragment thereof that
immunospecifically binds to a RSV antigen or the amount of a liquid
formulation of the invention comprising said antibody or antibody
fragment)), which is sufficient to result in the prevention of the
development, recurrence, onset, or progression of a RSV infection,
one or more symptoms thereof, or a respiratory condition associated
with, potentiated by, or potentiating a RSV infection, or to
enhance or improve the prophylactic effect(s) of another therapy
(e.g., a prophylactic agent). In a specific embodiment, a
prophylactically effective amount of a prophylactic agent reduces
one or more of the following steps of a RSV life cycle: the docking
of the virus particle to a cell, the introduction of viral genetic
information into a cell, the expression of viral proteins, the
production of new virus particles and the release of virus
particles from a cell by at least 5%, preferably at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%,
at least 40%, at least 45%, at least 50%, at least 55%, at least
60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, or at least 100%. In another
specific embodiment, a prophylactically effective amount of a
prophylactic agent reduces the replication, multiplication or
spread of a virus by at least 5%, preferably at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, or at least 100% compared to the
same in the absence of the agent or the presence of a negative
control (e.g., control IgG or phosphate buffered saline (PBS)).
[0060] The term "RSV antigen" as used herein refers to a RSV
protein, polypeptide, peptide or fragment thereof to which an
antibody or antibody fragment immunospecifically binds. A RSV
antigen also refers to a derivative of a RSV protein, polypeptide,
peptide or a fragment thereof to which an antibody or antibody
fragment immunospecifically binds.
[0061] The term "saccharide" as used herein refers to a class of
molecules that are derivatives of polyhydric alcohols. Saccharides
are commonly referred to as carbohydrates and may contain different
amounts of sugar (saccharide) units, e.g., monosaccharides,
disaccharides and polysaccharides.
[0062] The phrase "side effects" as used herein encompasses
unwanted and adverse effects of a prophylactic or therapeutic
agent. Adverse effects are always unwanted, but unwanted effects
are not necessarily adverse. An adverse effect from a prophylactic
or therapeutic agent might be harmful or uncomfortable or
risky.
[0063] The term "small molecule" and analogous terms include, but
are not limited to, peptides, peptidomimetics, amino acids, amino
acid analogues, polynucleotides, polynucleotide analogues,
nucleotides, nucleotide analogues, organic or inorganic compounds
(i.e., including heterorganic and/or ganometallic compounds) having
a molecular weight less than about 10,000 grams per mole, organic
or inorganic compounds having a molecular weight less than about
5,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 1,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 500
grams per mole, and salts, esters, and other pharmaceutically
acceptable forms of such compounds.
[0064] The terms "stability" and "stable" as used herein in the
context of a liquid formulation comprising an antibody or fragment
thereof that immunospecifically binds to a RSV antigen refer to the
resistance of the antibody or antibody fragment in the formulation
to thermal and chemical unfolding, aggregation, degradation or
fragmentation under given manufacture, preparation, transportation
and storage conditions. The "stable" formulations of the invention
retain biological activity equal to or more than 80%, 85%, 90%,
95%, 98%, 99%, or 99.5% under given manufacture, preparation,
transportation and storage conditions. The stability of the
antibody or antibody fragment can be assessed by degrees of
aggregation, degradation or fragmentation by methods known to those
skilled in the art, including but not limited to reduced Capillary
Gel Electrophoresis (rCGE), Sodium Dodecyl Sulfate Polyacrylamide
Gel Electrophoresis (SDS-PAGE) and HPSEC, compared to a reference,
that is, a commercially available lyophilized SYNAGIS.RTM.
reconstituted to 100 mg/ml in 50 mM histidine/3.2 mM glycine buffer
with 6% mannitol at pH 6.0. The reference regularly gives a single
peak (.gtoreq.97% area) by HPSEC. The overall stability of a
formulation comprising an antibody or fragment thereof that
immunospecifically binds to a RSV antigen can be assessed by
various immunological assays including, for example, ELISA and
radioimmunoassay using the specific epitope of RSV.
[0065] As used herein, the term "SYNAGIS.RTM. standard reference"
or analogous terms refer to commercially available lyophilized
SYNAGIS.RTM., as described in the Physicians' Desk Reference,
56.sup.th edition, 2002. Reconstituted SYNAGIS.RTM. may contain,
e.g., the following excipients: 47 mM histidine, 3.0 .mu.M glycine
and 5.6% manitol and the active ingredient, the antibody, at a
concentration of 100 milligrams per ml solution.
[0066] As used herein, the terms "subject" and "patient" are used
interchangeably. As used herein, the terms "subject" and "subjects"
refer to an animal, preferably a mammal including a non-primate
(e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a
non-primate (e.g., a monkey such as a cynomolgous monkey and a
human), and more preferably a human.
[0067] The term "substantially free of surfactant" as used herein
refers to a formulation of an antibody or fragment thereof that
immunospecifically binds to a RSV antigen, said formulation
containing less than 0.0005%, less than 0.0003%, or less than
0.0001% of surfactants and/or less than 0.0005%, less than 0.0003%,
or less than 0.0001% of surfactants.
[0068] The term "substantially free of salt" as used herein refers
to a formulation of an antibody or fragment thereof that
immunospecifically binds to a RSV antigen, said formulation
containing less than 0.0005%, less than 0.0003%, or less than
0.0001% of inorganic salts.
[0069] The term "surfactant" as used herein refers to organic
substances having amphipathic structures; namely, they are composed
of groups of opposing solubility tendencies, typically an
oil-soluble hydrocarbon chain and a water-soluble ionic group.
Surfactants can be classified, depending on the charge of the
surface-active moiety, into anionic, cationic, and nonionic
surfactants. Surfactants are often used as wetting, emulsifying,
solubilizing, and dispersing agents for various pharmaceutical
compositions and preparations of biological materials.
[0070] The term "synergistic" as used herein refers to a
combination of therapies (e.g., use of prophylactic or therapeutic
agents) which is more effective than the additive effects of any
two or more single therapy. For example, a synergistic effect of a
combination of prophylactic or therapeutic agents permits the use
of lower dosages of one or more of the agents and/or less frequent
administration of said agents to a subject with a RSV infection.
The ability to utilize lower dosages of prophylactic or therapeutic
therapies and/or to administer said therapies less frequently
reduces the toxicity associated with the administration of said
therapies to a subject without reducing the efficacy of said
therapies in the prevention, management or treatment of a RSV
infection. In addition, a synergistic effect can result in improved
efficacy of therapies in the prevention or treatment of a RSV
infection. Finally, synergistic effect of a combination of
therapies (e.g., prophylactic or therapeutic agents) may avoid or
reduce adverse or unwanted side effects associated with the use of
any single therapy.
[0071] The terms "therapeutic agent" and "therapeutic agents" as
used herein refer to any agent(s) which can be used in the
treatment, management or amelioration a RSV infection, one or more
symptoms thereof, or a respiratory condition associated with,
potentiated by, or potentiating a RSV infection. In certain
embodiments, the term "therapeutic agent" refers to an antibody or
fragment thereof that immunospecifically binds to a RSV antigen. In
accordance with these embodiments, the antibody or antibody
fragment may be a component of a liquid formulation of the
invention. In certain other embodiments, the term "therapeutic
agent" does not refer to an antibody or fragment thereof that
immunospecifically binds to a RSV antigen or a liquid formulation
comprising such an antibody or antibody fragment. In certain other
embodiments, the term "therapeutic agent" does not refer to
SYNAGIS.RTM. or an antigen-binding fragment thereof.
[0072] The term "therapeutically effective amount" as used herein
refers to the amount of a therapy (e.g., a therapeutic agent (e.g.,
an antibody or a fragment thereof, which immunospecifically binds
to a RSV antigen or a liquid formulation of the invention
comprising said antibody or antibody fragment)), which is
sufficient to (i) reduce the severity, and/or duration of a RSV
infection, or a respiratory condition associated with, potentiated
by, or potentiating a RSV infection; (ii) ameliorate one or more
symptoms associated with a RSV infection, or a respiratory
condition associated with, potentiated by, or potentiating a RSV
infection; (iii) prevent the advancement of a RSV infection, or a
respiratory condition associated with, potentiated by, or
potentiating a RSV infection; (iv) cause regression of a RSV
infection, or a respiratory condition associated with, potentiated
by, or potentiating a RSV infection; or (v) enhance or improve the
therapeutic effect(s) of another therapy (e.g., another therapeutic
agent). With respect to the treatment of a RSV infection, a
therapeutically effective amount refers to the amount of a
therapeutic agent sufficient to reduce or inhibit the replication
of a virus, inhibit or reduce the infection of cell with the virus,
inhibit or reduce the production of the viral particles, inhibit or
reduce the release of viral particles, inhibit or reduce the spread
of the virus to other tissues or subjects, or ameliorate one or
more symptoms associated with the infection. In a specific
embodiment, a therapeutically effective amount of a therapeutic
agent reduces one or more of the following steps of a RSV life
cycle: the docking of the virus particle to a cell, the
introduction of viral genetic information into a cell, the
expression of viral proteins, the production of new virus particles
and the release of virus particles from a cell by at least 5%,
preferably at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
or at least 100%. In another specific embodiment, a therapeutically
effective amount of a therapeutic agent reduces the replication,
multiplication or spread of a virus by at least 5%, preferably at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, or at least
100% compared to the absence of the agent or the presence of a
negative control (e.g., a control IgG or PBS).
[0073] As used herein, the terms "therapies" and "therapy" can
refer to any protocol(s), method(s) and/or agent(s) that can be
used in the prevention, treatment, management or amelioration of a
RSV infection, one or more symptoms thereof, or a respiratory
condition associated with, potentiated by, or potentiating a RSV
infection. In certain embodiments, the terms "therapy" and
"therapies" refer to hormonal therapy, biological therapy, and/or
other therapies useful for the treatment of a RSV infection, one or
more symptoms thereof, or a respiratory condition associated with,
potentiated by or potentiating a RSV infection known to medical
professionals.
[0074] The terms "treat", "treatment" and "treating" as used herein
refer to the reduction or amelioration of the progression,
severity, and/or duration of a RSV infection, one or more symptoms
thereof, or a respiratory condition associated with, potentiated
by, or potentiating a RSV infection resulting from the
administration of one or more therapies (including but not limited
to, the administration of one or more prophylactic or therapeutic
agents, and any other methods that can be used). In specific
embodiments, such terms refer to the reduction or inhibition of the
replication of a respiratory syncytial virus (RSV), the inhibition
or reduction in the spread of a respiratory syncytial virus (RSV)
to other tissues or subjects, the inhibition or reduction of
infection of a cell with a respiratory syncytial virus (RSV), or
the amelioration of one or more symptoms associated with a
respiratory syncytial virus (RSV) infection.
[0075] As used herein, the term "T cell receptor modulator" refers
to an agent which modulates the phosphorylation of a T cell
receptor, the activation of a signal transduction pathway
associated with a T cell receptor and/or the expression of a
particular protein such as a cytokine. Such an agent may directly
or indirectly modulate the phosphorylation of a T cell receptor,
the activation of a signal transduction pathway associated with a T
cell receptor, and/or the expression of a particular protein such
as a cytokine. Examples of T cell receptor modulators include, but
are not limited to, peptides, polypeptides, proteins, fusion
proteins and antibodies which immunospecifically bind to a T cell
receptor or a fragment thereof. Further, examples of T cell
receptor modulators include, but are not limited to, proteins,
peptides, polypeptides (e.g., soluble T cell receptors), fusion
proteins and antibodies that immunospecifically binds to a ligand
for a T cell receptor or a fragment thereof.
[0076] The term "very little to no loss of the biological
activities" as used herein refers to antibody activities, including
specific binding abilities of antibodies or antibody fragments to a
RSV antigen as measured by various immunological assays, including,
but not limited to ELISAs and radioimmunoassays. In one embodiment,
the antibodies or antibody fragments of the formulations of the
invention retain approximately 50%, preferably 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95% or 98% of the ability to immunospecifically
bind to a RSV antigen as compared to a reference antibody or
antibody fragment (e.g., SYNAGIS.RTM.) as measured by an
immunological assay known to one of skill in the art or described
herein. For example, an ELISA based assay may be used to compare
the ability of an antibody or fragment thereof to
immunospecifically bind to a RSV antigen to a SYNAGIS.RTM.
reference standard. In this assay, plates are coated with a RSV
antigen and the binding signal of a set concentration of a
SYNAGIS.RTM. reference standard is compared to the binding signal
of the same concentration of a test antibody or antibody
fragment.
4. BRIEF DESCRIPTION OF THE FIGURES
[0077] FIG. 1 is a schematic diagram showing the outline for
preparing purified antibodies that immunospecifically bind to a RSV
antigen.
5. DETAILED DESCRIPTION OF THE INVENTION
[0078] The liquid formulations of the present invention provide a
ready-to-use preparation of an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen for administering to a
subject without having to reconstitute the preparation accurately
and aseptically and waiting for a period of time until the solution
clarifies before administering the formulation to the subject. It
simplifies the procedure of administering the formulation to a
subject for a healthcare professional. Furthermore, due to its high
stability during the storage, the formulations of the present
invention can contain an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen at concentrations in the
range of about 15 mg/ml to about 300 mg/ml without causing an
adverse effect on the biological activities of the antibody or a
fragment thereof due to protein aggregation and/or fragmentation
during a prolonged storage. Such stability not only ensures the
efficacy of the antibodies or antibody fragments but also reduces
possible risks of causing adverse effects on a subject.
Furthermore, the use of fewer components in the formulation results
in fewer risks of introducing contamination. In addition, the
manufacturing process of the liquid formulations of the present
invention is simplified and more efficient than the manufacturing
process for the lyophilized version because all stages of the
manufacturing of the liquid formulations are carried out in an
aqueous solution, involving no drying process, such as
lyophilization and freeze-drying. Accordingly, it is more cost
effective as well.
[0079] 5.1. Liquid Formulations of Anti-RSV Antibodies
[0080] The liquid formulations of the present invention provide
antibody formulations which are substantially free of surfactant,
inorganic salts, and/or other excipients and yet exhibit high
stability during long periods of storage. In a specific embodiment,
such antibody formulations are homogeneous. The formulations of the
present invention comprise histidine at concentrations between 1
and 100 mM and an antibody or a fragment thereof which
immunospecifically binds to a RSV antigen at concentrations of
about 15 mg/ml to about 300 mg/ml. In one embodiment, the
formulations of the invention do not comprise other ingredients
except for water or suitable solvents. In a specific embodiment,
the antibody or antibody fragment that immunospecifically binds to
a RSV antigen which is included in the liquid formulations of the
invention is not SYNAGIS.RTM. or a fragment thereof. In an
alternative embodiment, at least one of the antibodies or antibody
fragments that is included in the liquid formulations of the
invention comprises two or more antibodies or antibody fragments
that immunospecifically bind to a RSV antigen which is included in
the liquid formulations of the invention is SYNAGIS.RTM. or a
fragment thereof.
[0081] In one embodiment, the antibody or antibody fragment that
immunospecifically binds to a RSV antigen which is included in the
liquid formulations of the invention is an antibody or antibody
fragment comprising a VH domain and/or VL domain listed in Table 1,
supra. In another embodiment, the antibody or antibody fragment
that immunospecifically binds to a RSV antigen which is included in
the liquid formulations of the invention is an antibody or antibody
fragment comprising one or more VH CDRs and/or one or more VL CDRs
in Table 1, supra. In another embodiment, the antibody or antibody
fragment that immunospecifically binds to a RSV antigen which is
included in the liquid formulations of the invention is an antibody
or antibody fragment conjugated to another moiety, including, but
not limited to, a heterologous polypeptide, another antibody or
another fragment, a marker sequence, a diagnostic agent, a
therapeutic agent, a radioactive metal ion, a polymer, albumin, and
a solid support. In yet another embodiment, liquid formulations of
the invention comprise two or more antibodies or antibody fragments
that immunospecifically binds to a RSV antigen, wherein at least
one of the antibodies or antibody fragments is SYNAGIS.RTM. or a
fragment thereof.
[0082] The concentration of an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen which is included in the
liquid formulations of the invention is at least 15 mg/ml, at least
20 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 35 mg/ml,
at least 40 mg/ml, at least 45 mg/ml, at least 50 mg/ml, at least
55 mg/ml, at least 60 mg/ml, at least 65 mg/ml, at least 70 mg/ml,
at least 75 mg/ml, at least 80 mg/ml, at least 85 mg/ml, at least
90 mg/ml, at least 95 mg/ml, at least 100 mg/ml, at least 105
mg/ml, at least 110 mg/ml, at least 115 mg/ml, at least 120 mg/ml,
at least 125 mg/ml, at least 130 mg/ml, at least 135 mg/ml, at
least 140 mg/ml, at least 150 mg/ml, at least 200 mg/ml, at least
250 mg/ml, or at least 300 mg/ml.
[0083] The concentration of histidine which is included in the
liquid formulations of the invention ranges from about 1 mM to
about 100 mM, about 10 mM to about 50 mM, about 20 mM to about 30
mM, or about 23 mM to about 27 mM, and is most preferably about 25
mM. Histidine can be in the form of L-histidine, D-histidine, or a
mixture thereof, but L-histidine is the most preferable. Histidine
can be also in the form of hydrates. Histidine may be used in a
form of pharmaceutically acceptable salt, such as hydrochloride
(e.g., monohydrochloride and dihydrochloride), hydrobromide,
sulfate, acetate, etc. The purity of histidine should be at least
98%, preferably at least 99%, and most preferably at least
99.5%.
[0084] The pH of the formulation should not be equal to the
isoelectric point of the particular antibody to be used in the
formulation and may range from about 5.0 to about 7, preferably
about 5.5 to about 6.5, more preferably about 5.8 to about 6.2, and
most preferably about 6.0.
[0085] In addition to histidine and an antibody or a fragment
thereof that immunospecifically binds to a RSV antigen, the
formulations of the present invention may further comprise glycine
at a concentration of less than 100 mM, less than 50 mM, less than
3.0 mM, less than 2.0 mM, or less than 1.8 mM, and most preferably
1.6 mM. The amount of glycine in the formulation should not cause a
significant buffering effect so that antibody precipitation at its
isoelectric point can be avoided. Glycine may be also used in a
form of pharmaceutically acceptable salt, such as hydrochloride,
hydrobromide, sulfate, acetate, etc. The purity of glycine should
be at least 98%, preferably at least 99%, and most preferably
99.5%. In a specific embodiment, glycine is included in the
formulations of the present invention.
[0086] Optionally, the formulations of the present invention may
further comprise other excipients, such as saccharides (e.g.,
sucrose, mannose, trehalose, etc.) and polyols (e.g., mannitol,
sorbitol, etc.). In one embodiment, the other excipient is a
saccharide. In a specific embodiment, the saccharide is sucrose,
which is at a concentration ranging from between about 1% to about
20%, preferably about 5% to about 15%, and more preferably about 8%
to 10%. In another embodiment, the other excipient is a polyol.
Preferably, however, the liquid formulations of the present
invention do not contain mannitol. In a specific embodiment, the
polyol is polysorbate (e.g., Tween 20), which is at a concentration
ranging from between about 0.001% to about 1%, preferably, about
0.01 to about 0.1.
[0087] The liquid formulations of the present invention exhibit
stability at the temperature ranges of 38.degree. C.-42.degree. C.
for at least 60 days and, in some embodiments, not more than 120
days, of 20.degree. C.-24.degree. C. for at least 1 year, of
2.degree. C.-8.degree. C. (in particular, at 4.degree. C.) for at
least 3 years, at least 4 years, or at least 5 years and at
-20.degree. C. for at least 3 years, at least 4 years, or at least
5 years, as assessed by high performance size exclusion
chromatography (HPSEC). Namely, the liquid formulations of the
present invention have low to undetectable levels of aggregation
and/or fragmentation, as defined herein, after the storage for the
defined periods as set forth above. Preferably, no more than 5%, no
more than 4%, no more than 3%, no more than 2%, no more than 1%,
and most preferably no more than 0.5% of the antibody or antibody
fragment forms an aggregate as measured by HPSEC, after the storage
for the defined periods as set forth above. Furthermore, liquid
formulations of the present invention exhibit almost no loss in
biological activities of the antibody or antibody fragment during
the prolonged storage under the condition described above, as
assessed by various immunological assays including, but not limited
to, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay
to measure the ability of the antibody or antibody fragment to
immunospecifically bind to a RSV antigen, and by a C3a/C4a assay to
measure the complement activating ability of the antibody. The
liquid formulations of the present invention retain after the
storage for the above-defined periods more than 80%, more than 85%,
more than 90%, more than 95%, more than 98%, more than 99%, or more
than 99.5% of the initial biological activities of the formulation
prior to the storage.
[0088] The liquid formulations of the present invention can be
prepared as unit dosage forms. For example, a unit dosage per vial
may contain 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml,
10 ml, 15 ml, or 20 ml of different concentrations of an antibody
or a fragment thereof that immunospecifically binds to a RSV
antigen ranging from about 15 mg/ml to about 300 mg/ml. If
necessary, these preparations can be adjusted to a desired
concentration by adding a sterile diluent to each vial.
[0089] The invention encompasses stable liquid formulations
comprising a single antibody or fragment thereof that
immunospecifically binds to a RSV antigen, with the proviso that
said antibody is not SYNAGIS.RTM.. The invention also encompasses
stable liquid formulations comprising two or more antibodies or
fragments thereof that immunospecifically bind to a RSV antigen. In
one embodiment, a stable liquid formulation of the invention
comprises two or more antibodies or fragments thereof that
immunospecifically bind to a RSV antigen, wherein one of the
antibodies or antibody fragments is SYNAGIS.RTM. or a fragment
thereof. In an alternative embodiment, a stable liquid formulation
of the invention comprises two or more antibodies or fragments
thereof that immunospecifically bind to a RSV antigen, with the
proviso that the antibodies or antibody fragments do not include
SYNAGIS.RTM. or a fragment thereof.
5.1.1 Antibodies Immunospecific for a RSV Antigen
[0090] The invention relates to liquid formulations comprising
antibodies that immunospecifically bind to a RSV antigen. In a
preferred embodiment, the invention provides liquid formulations
comprising one or more of the antibodies listed in Table 1,
supra.
[0091] The present invention encompasses stable liquid formulations
comprising one or more analogues or derivatives of one or more
antibodies recited in Table 1, supra. Such antibodies or fragments
thereof having increased affinity for a RSV antigen will result in
increased efficacy for prophylactic or therapeutic uses such that
lower serum titers are prophylactically or therapeutically
effective, thus allowing administration of lower dosages and/or
reduced frequency of administration. Such antibodies or fragments
thereof which have increased affinity for a RSV antigen may be
obtained by introducing one or more amino acid residue
modifications, such as amino acid substitutions, in the light-chain
variable domain (VL) and/or heavy-chain variable domain (VH), of
the antibodies listed in Table 1, supra. Furthermore, antibodies or
fragments having improved affinity for a RSV antigen may be
obtained by introducing one or more amino acid residue
modifications, such as amino acid substitutions, in one or more
complementarity determining regions (CDRs) of the VL and/or VH of
the antibodies listed in Table 1, supra.
[0092] Standard techniques known to those of skill in the art can
be used to introduce mutations (e.g., additions, deletions, and/or
substitutions) in the nucleotide sequence encoding an antibody of
the invention, including, for example, site-directed mutagenesis
and PCR-mediated mutagenesis which results in amino acid
substitutions. Preferably, the derivatives include less than 25
amino acid substitutions, less than 20 amino acid substitutions,
less than 15 amino acid substitutions, less than 10 amino acid
substitutions, less than 5 amino acid substitutions, less than 4
amino acid substitutions, less than 3 amino acid substitutions, or
less than 2 amino acid substitutions relative to the original
molecule. In a preferred embodiment, the derivatives have
conservative amino acid substitutions are made at one or more
predicted non-essential amino acid residues (i.e., amino acid
residues which are not critical for the antibody to
immunospecifically bind to a RSV antigen). A "conservative amino
acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a side chain with a
similar charge. Families of amino acid residues having side chains
with similar charges have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for biological activity to
identify mutants that retain activity. Following mutagenesis, the
encoded antibody can be expressed and the activity of the antibody
can be determined.
[0093] The antibody generated by introducing substitutions in the
VH domain, VH CDRs, VL domain and/or VL CDRs of an antibody listed
in Table 1 can be tested in vitro and in vivo, for example, for its
ability to bind to a RSV antigen (by, e.g., immunoassays including,
but not limited to ELISAs and BLAcore), or for its ability to
prevent, treat, manage or ameliorate a RSV infection or a symptom
thereof.
[0094] In a specific embodiment, an antibody that
immunospecifically binds to a RSV infection comprises a nucleotide
sequence that hybridizes to the nucleotide sequence encoding an
antibody listed in Table 1 under stringent conditions, e.g.,
hybridization to filter-bound DNA in 6.times. sodium
chloride/sodium citrate (SSC) at about 45 .sub.1C followed by one
or more washes in 0.2.times.SSC/0.1% SDS at about 50-65 .sub.1C,
under highly stringent conditions, e.g., hybridization to
filter-bound nucleic acid in 6.times.SSC at about 45.degree. C.
followed by one or more washes in 0.1.times.SSC/0.2% SDS at about
68 .sub.1C, or under other stringent hybridization conditions which
are known to those of skill in the art (see, for example, Ausubel,
F. M. et al., eds., 1989, Current Protocols in Molecular Biology,
Vol. I, Green Publishing Associates, Inc. and John Wiley &
Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).
[0095] In a specific embodiment, an antibody that
immunospecifically binds to a RSV antigen comprises an amino acid
sequence of a VH domain or an amino acid sequence a VL domain
encoded by a nucleotide sequence that hybridizes to the nucleotide
sequence encoding the VH or VL domains of an antibody listed in
Table 1 under stringent conditions, e.g., hybridization to
filter-bound DNA in 6.times. sodium chloride/sodium citrate (SSC)
at about 45 .sub.1C followed by one or more washes in
0.2.times.SSC/0.1% SDS at about 50-65 .sub.1C, under highly
stringent conditions, e.g., hybridization to filter-bound nucleic
acid in 6.times.SSC at about 45 .sub.1C followed by one or more
washes in 0.1.times.SSC/0.2% SDS at about 68 .sub.1C, or under
other stringent hybridization conditions which are known to those
of skill in the art (see, for example, Ausubel, F. M. et al., eds.,
1989, Current Protocols in Molecular Biology, Vol. I, Green
Publishing Associates, Inc. and John Wiley & Sons, Inc., New
York at pages 6.3.1-6.3.6 and 2.10.3).
[0096] In another embodiment, an antibody that immunospecifically
binds to a RSV antigen comprises an amino acid sequence of a VH CDR
and/or an amino acid sequence of a VL CDR encoded by a nucleotide
sequence that hybridizes to the nucleotide sequence encoding any
one of the VH CDRs or VL CDRs listed in Table 1 or Table 2 under
stringent conditions e.g., hybridization to filter-bound DNA in
6.times. sodium chloride/sodium citrate (SSC) at about 45 .sub.1C
followed by one or more washes in 0.2.times.SSC/0.1% SDS at about
50-65 .sub.1C, under highly stringent conditions, e.g.,
hybridization to filter-bound nucleic acid in 6.times.SSC at about
45 .sub.1C followed by one or more washes in 0.1.times.SSC/0.2% SDS
at about 68 .sub.1C, or under other stringent hybridization
conditions which are known to those of skill in the art.
[0097] In a specific embodiment, an antibody that
immunospecifically binds to a RSV antigen comprises an amino acid
sequence of a VH domain and/or VL domain that is at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, or at least 99% identical to the
amino acid sequence of a VH domain and/or a VL domain of an
antibody listed in Table 1, and/or an amino acid sequence of one or
more VL CDRs. The determination of percent identity of two amino
acid sequences can be determined by any method known to one skilled
in the art, including BLAST protein searches.
[0098] In another embodiment, an antibody that immunospecifically
binds to a RSV antigen comprises an amino acid sequence of one or
more VH CDRs and/or an amino acid sequence of one or more VL CDRs
that are at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, or at least
99% identical to any of one of the VH CDRs and/or any of the VL
CDRs of an antibody listed in Table 1 or Table 2.
[0099] In a specific embodiment, an antibody or fragment thereof
that immunospecifically binds to a RSV antigen comprises one or
more amino acid residue substitutions of the amino acid residues
indicated in bold face and underlining in Table 2 (see Section 3,
supra). In another specific embodiment, an antibody or a fragment
thereof that immunospecifically binds to a RSV antigen comprises a
VH domain having an amino acid sequence of any one of the VH
domains listed in Table 1 (see Section 3) and/or a VL domain having
an amino acid sequence of any one of the VL domains listed in Table
1. In another embodiment, an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen comprises one, two or
more of the VH CDRs listed in Table 1 and/or Table 2, and/or one or
more of the VL CDRs listed in Table 1 and/or Table 2. In yet
another embodiment, an antibody or fragment thereof that
immunospecifically binds to a RSV antigen comprises a VH CDR1 and a
VL CDR1; a VH CDR1 and a VL CDR2; a VH CDR1 and a VL CDR3; a VH
CDR2 and a VL CDR1; VH CDR2 and VL CDR2; a VH CDR2 and a VL CDR3; a
VH CDR3 and a VH CDR1; a VH CDR3 and a VL CDR2; a VH CDR3 and a VL
CDR3; a VH 1. CDR1, a VH CDR2 and a VL CDR1; a VH CDR1, a VH CDR2
and a VL CDR2; a VH CDR1, a VH CDR2 and a VL CDR3; a VH CDR2, a VH
CDR3 and a VL CDR1, a VH CDR2, a VH CDR3 and a VL CDR2; a VH CDR2,
a VH CDR2 and a VL CDR3; a VH CDR1, a VL CDR1 and a VL CDR2; a VH
CDR1, a VL CDR1 and a VL CDR3; a VH CDR2, a VL CDR1 and a VL CDR2;
a VH CDR2, a VL CDR1 and a VL CDR3; a VH CDR3, a VL CDR1 and a VL
CDR2; a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, a VH CDR2, a
VH CDR3 and a VL CDR1; a VH CDR1, a VH CDR2, a VH CDR3 and a VL
CDR2; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR3; a VH CDR1, a
VH CDR2, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VL CDR1
and a VL CDR3; a VH CDR1, a VH CDR3, a VL CDR1 and a VL CDR2; a VH
CDR1, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a
VL CDR1 and a VL CDR2; a VH CDR2, a VH CDR3, a VL CDR1 and a VL
CDR3; a VH CDR2, a VH CDR3, a VL CDR2 and a VL CDR3; a VH CDR1, a
VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2,
a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR1, a VH CDR2, a VL
CDR1, a VL CDR2, and a VL CDR3; a VH CDR1, a VH CDR3, a VL CDR1, a
VL CDR, and a VL CDR3; a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2,
and a VL CDR3; or any combination thereof of the VH CDRs and VL
CDRs listed in Table 1, and/or Table 2, supra. Such antibodies and
methods for preparing them are disclosed in copending U.S. patent
application Ser. Nos. 09/724,396 and 09/724,531, both filed Nov.
28, 2000, entitled "Methods of Administering/Dosing Anti-RSV
Antibodies for Prophylaxis and Treatment" and by J. Young et al.;
continuation-in-part application Ser. Nos. 09/996,288 and
09/996,265, both filed Nov. 28, 2001, also entitled "Methods of
Administering/Dosing Anti-RSV Antibodies for Prophylaxis and
Treatment" by Young et al.; and continuation-in-part application
Ser. No. 10/403,180, filed Mar. 31, 2003, all of which are hereby
incorporated by reference in their entireties. In certain
embodiments, the antibodies or fragments thereof that
immunospecifically bind to a RSV antigen do not comprise a VH CDR,
a VL CDR, the VH domain or the VL domain of SYNAGIS.RTM..
[0100] The present invention also encompasses the liquid
formulations comprising antibodies that are not those listed in
Table 1, which immunospecifically bind to a RSV antigen. In other
words, the invention encompasses liquid formulations of any
antibodies and fragments thereof which immunospecifically bind to
one or more RSV antigens. Further, the invention encompasses liquid
formulations comprising one or more of the novel antibodies,
fragments and other biological or macromolecules that
immunospecifically bind to one or more RSV antigens. These novel
agents are disclosed in detail in pending U.S. patent application
Ser. No. 09/865,499 filed May 25, 2001, which is hereby
incorporated by reference in its entirety.
[0101] Preferably, the antibodies or fragments thereof contained in
the formulations of the invention immunospecifically bind to a RSV
antigen regardless of the strain of RSV. Alternatively, the
antibodies or fragments thereof may differentially or
preferentially bind to RSV antigens from one strain of RSV versus
another RSV strain, as assessed, for example, by competitive
immunoassays. In a specific embodiment, the antibodies or fragments
thereof contained in the formulations of the present invention
immunospecifically bind to the RSV F glycoprotein, G glycoprotein
or SH protein. In a preferred embodiment, the antibodies or
fragments thereof immunospecifically bind to the RSV F
glycoprotein. In another preferred embodiment, the antibodies or
fragments thereof contained in the formulations of the present
invention immunospecifically bind to the A, B, or C antigenic sites
of the RSV F glycoprotein. In certain embodiments, such antibodies
are not SYNAGIS.RTM..
[0102] Antibodies contained in the formulations of the invention
include, but are not limited to, monoclonal antibodies,
multispecific antibodies, human antibodies, humanized antibodies,
chimeric antibodies, single-chain. Fvs (scFv), single domain
antibodies, single chain antibodies, Fab fragments, F(ab).sub.2
fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic
(anti-Id) antibodies (including, e.g., anti-Id antibodies to
antibodies of the invention), and epitope-binding fragments of any
of the above. In particular, antibodies of the present invention
include immunoglobulin molecules and immunologically active
fragments of immunoglobulin molecules, i.e., molecules that contain
an antigen binding site that immunospecifically binds to a RSV
antigen. The immunoglobulin molecules of the invention can be of
any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g.,
IgG.sub.1, IgG.sub.2, IgG3, IgG.sub.4, IgA.sub.1 and IgA2) or
subclass of immunoglobulin molecule.
[0103] The antibodies contained in the formulations of the present
invention may be from any animal origin including birds and mammals
(e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig,
camel, horse, or chicken). Preferably, the antibodies of the
invention are human or humanized monoclonal antibodies. As used
herein, "human antibodies" include antibodies having the amino acid
sequence of a human immunoglobulin and include antibodies isolated
from human immunoglobulin libraries or from mice that express
antibodies from human genes.
[0104] The antibodies contained in the formulations of the present
invention may be monospecific, bispecific, trispecific or of
greater multi specificity. Multispecific antibodies may be specific
for different epitopes of a RSV protein or polypeptide or may be
specific for both a RSV protein or polypeptide as well as for a
heterologous epitope, such as a heterologous polypeptide or solid
support material. See, e.g., International Publication Nos: WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.
Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;
4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.
148:1547-1553 (1992).
[0105] The antibodies or fragments thereof contained in the
formulations of the present invention may exhibit a high potency as
described in copending U.S. patent application Ser. Nos.
09/724,396, 60/168,426, 60/186,252. High potency antibodies or
fragments thereof can be produced by methods disclosed in copending
U.S. patent application Ser. Nos. 60/168,426 and 60/186,252, filed
Jan. 27, 2000 and Mar. 1, 2000, respectively, both entitled "High
Potency Recombinant Antibodies and Methods for Producing Them," and
methods described in U.S. patent application Ser. No. 09/724,396,
each of which is hereby incorporated by reference by its entirety.
For example, high potency antibodies can be produced by genetically
engineering appropriate antibody gene sequences and expressing the
antibody sequences in a suitable host. The antibodies produced can
be screened to identify antibodies having, for example, a high
association constant (k.sub.on) in a BIAcore assay. The antibodies
or fragments thereof contained in the formulations of the present
invention may also exhibit ultra high affinity as described in
copending U.S. application Ser. No. 09/771,415, filed Jan. 26,
2001, which is hereby incorporated by reference in its
entirety.
[0106] The present invention encompasses antibodies that compete
with an antibody described herein for binding to a RSV antigen. In
a specific embodiment, the present invention encompasses antibodies
that compete with SYNAGIS.RTM. or an antigen-binding fragment
thereof for binding to a RSV antigen. In a particular embodiment,
the present invention encompasses antibodies that compete with
SYNAGIS.RTM. or an antigen-binding fragment thereof for binding to
the same epitope of a RSV antigen (in particular RSV F antigen) and
does not just sterically inhibit the binding of SYNAGIS.RTM. or
antigen-binding fragment thereof to its epitope. Techniques
well-known in the art (e.g., competitive binding assays) can be
used to identify antibodies or fragments thereof that compete with
SYNAGIS.RTM. or an antigen-binding fragment thereof for binding to
its epitope. The binding affinity of an antibody to an antigen and
the off-rate of an antibody-antigen interaction can be determined
by competitive binding assays. See U.S. patent Ser. No. 09/996,228
filed Nov. 28, 2001, which is incorporated herein by reference in
its entirety. One example of a competitive binding assay is a
radioimmunoassay comprising the incubation of labeled antigen
(e.g., .sup.3H or .sup.125I) with the antibody of interest in the
presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of the present invention or a fragment
thereof for a RSV antigen and the binding off-rates can be
determined from the data by scatchard plot analysis. Competition
with a second antibody can also be determined using
radioimmunoassays. In this case, a RSV antigen is incubated with an
antibody of the present invention or a fragment thereof conjugated
to a labeled compound (e.g., .sup.3H or .sup.125I) in the presence
of increasing amounts of an unlabeled second antibody.
[0107] In a preferred embodiment, BIAcore kinetic analysis is used
to determine the binding on and off rates of antibodies or
fragments thereof to a RSV antigen. BIAcore kinetic analysis
comprises analyzing the binding and dissociation of a RSV antigen
from chips with immobilized antibodies or fragments thereof on
their surface.
[0108] Antibodies that immunospecifically bind to a RSV antigen
include derivatives that are modified, i.e, by the covalent
attachment of any type of molecule to the antibody such that
covalent attachment. For example, but not by way of limitation, the
antibody derivatives include antibodies that have been modified,
e.g., by glycosylation, acetylation, pegylation, phosphorylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to a cellular ligand or other
protein, etc. Any of numerous chemical modifications may be carried
out by known techniques, including, but not limited to, specific
chemical cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc. Additionally, the derivative may contain one or
more non-classical amino acids.
[0109] The present invention also encompasses antibodies that
immunospecifically bind to a RSV antigen, said antibodies
comprising a framework region known to those of skill in the art.
Preferably, the fragment region of an antibody of the invention is
human. In a specific embodiment, an antibody that
immunospecifically binds to a RSV antigen comprises the framework
region of SYNAGIS.RTM..
[0110] 5.1.2 Antibodies Having Increased Half-Lives that
Immunospecifically Bind to a RSV Antigen
[0111] The present invention encompasses stable liquid formulations
comprising one or more antibodies or fragments thereof that
immunospecifically bind to a RSV antigen and have improved
half-lives compared to other known anti-RSV antibodies, e.g.,
SYNAGIS.RTM.. In particular, the present invention provides liquid
formulations comprising one or more antibodies or fragments thereof
that immunospecifically bind to a RSV antigen which have a
half-life in an animal, preferably a mammal and most preferably a
human, of greater than 3 days, greater than 7 days, greater than 10
days, preferably greater than 15 days, greater than 25 days,
greater than 30 days, greater than 35 days, greater than 40 days,
greater than 45 days, greater than 2 months, greater than 3 months,
greater than 4 months, or greater than 5 months. By prolonging the
half-lives of antibodies, it is possible to reduce the amount
and/or frequency of dosing of the antibodies.
[0112] Antibodies that immunospecifically bind to a RSV antigen and
have increased half-lives in vivo relative to the anti-RSV
antibodies may be produced by, for example, introducing
modifications (e.g., by amino acid substitution, deletion, or
insertion) into the constant domain or FcRn (Fc Receptor-neonate)
binding domain of an IgG molecule. This increases the affinity of
the constant domain or FcRn binding domain for the FcRn which, in
turn, increases the in vivo half-life of the IgG molecule.
Antibodies and fragments thereof with improved in vivo half-lives
and methods for preparing them are disclosed in U.S. Pat. No.
6,277,375; International Publication Nos. WO 98/23289 and WO
97/3461; and copending U.S. patent application Ser. No. 10/020,354
filed Dec. 12, 2001, which claims priority to U.S. provisional
application Nos. 60/254,884 filed Dec. 12, 2000 and 60/289,760
filed May 9, 2001, all entitled "Molecules with Extended
Half-Lives, Compositions and Uses" and by L. Johnson et al.; each
of which is incorporated herein by reference in its entirety.
[0113] The serum circulation of antibodies (e.g., monoclonal
antibodies, single chain antibodies and Fab fragments) in vivo may
also be prolonged by attaching inert polymer molecules such as high
molecular weight polyethyleneglycol (PEG) to the antibodies with or
without a multifunctional linker either through site-specific
conjugation of the PEG to the N- or C-terminus of the antibodies or
via epsilon-amino groups present on lysine residues. Linear or
branched polymer derivatization that results in minimal loss of
biological activity will be used. The degree of conjugation can be
closely monitored by SDS-PAGE and mass spectrometry to ensure
proper conjugation of PEG molecules to the antibodies. Unreacted
PEG can be separated from antibody-PEG conjugates by size-exclusion
or by ion-exchange chromatography. PEG-derivatized antibodies can
be tested for binding activity as well as for in vivo efficacy
using methods known to those of skill in the art, for example, by
immunoassays described herein.
[0114] Further, antibodies or antibody fragments that
immunospecific bind to a RSV antigen can be conjugated to albumin
in order to make the antibody or antibody fragment more stable in
vivo or have a longer half life in vivo. The techniques are well
known in the art, see e.g., International Publication Nos. WO
93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP
413, 622, all of which are incorporated herein by reference.
[0115] 5.1.3 Antibody Conjugates
[0116] The present invention encompasses the use of liquid
formulations of antibodies or fragments thereof that
immunospecifically bind to a RSV antigen that conjugated to one or
more moieties, including but not limited to, peptide's,
polypeptides, proteins, fusion proteins, nucleic acid molecules,
small molecules, mimetic agents, synthetic drugs, inorganic
molecules, and organic molecules.
[0117] The present invention encompasses the use of liquid
formulations of an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen recombinantly fused or
chemically conjugated (including both covalent and non-covalent
conjugations) to one or more moieties including, but not limited
to, peptides, polypeptides, proteins, fusion proteins, nucleic acid
molecules, small molecules, mimetic agents, synthetic drugs,
inorganic molecules and organic molecules. The present invention
encompasses the use of antibodies or fragments thereof
recombinantly fused or chemically conjugated to heterologous
protein or polypeptide (or fragment thereof, preferably to a
polypepetide of at least 10, at least 20, at least 30, at least 40,
at least 50, at least 60, at least 70, at least 80, at least 90 or
at least 100 amino acids) to generate fusion proteins. The fusion
does not necessarily need to be direct, but may occur through
linker sequences. For example, an antibody may be used to target a
heterologous polypeptide to a particular cell type, either in vitro
or in vivo, by fusing or conjugating the antibody to another
antibody specific for particular cell surface receptors. An
antibody fused or conjugated to a heterologous polypeptide may also
be used in in vitro immunoassays and purification methods using
methods known in the art. See e.g., International publication No.
WO 93/21232; European Patent No. EP 439,095; Naramura et al., 1994,
Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al.,
1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol.
146:2446-2452, which are incorporated by reference in their
entireties.
[0118] The present invention further includes compositions
comprising a heterologous protein, peptide or polypeptide fused or
conjugated to an antibody fragments. For example, a heterologous
polypeptides may be fused or conjugated to a Fab fragment, Fd
fragment, Fv fragment, F(ab).sub.2 fragment, a VH domain, a VL
domain, a VH CDR, a VL CDR, or fragment thereof. Methods for fusing
or conjugating a polypeptide to an antibody fragment are known in
the art. See, e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,
5,349,053, 5,447,851, and 5,112,946; European Patent Nos. EP
307,434 and EP 367,166; International publication Nos. WO 96/04388
and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA
88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and
Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341 (all
references are incorporated herein by reference in their
entireties).
[0119] Additional fusion proteins may be generated through the
techniques of gene-shuffling, motif-shuffling, exon-shuffling,
and/or codon-shuffling (collectively referred to as "DNA
shuffling"). DNA shuffling may be employed to alter the activities
of SYNAGIS.RTM. or fragments thereof (e.g., an antibody or a
fragment thereof with higher affinities and lower dissociation
rates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;
5,830,721; 5,834,252; and 5,837,458, and Patten et al., 1997, Curr.
Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol.
16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and
Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of
these patents and publications are hereby incorporated by reference
in its entirety). An antibody or a fragment thereof that
immunospecifically binds to a RSV antigen, or the nucleic acid
encoding an antibody or a fragment thereof that immunospecifically
binds to a RSV antigen, may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or
other methods prior to recombination. One or more portions of a
polynucleotide encoding an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen or a fragment thereof,
which portions immunospecifically bind a RSV antigen, may be
recombined with one or more components, motifs, sections, parts,
domains, fragments, etc. of one or more heterologous molecules.
[0120] Moreover, an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen or a fragment thereof can
be fused to a marker sequence, such as a peptide to facilitate
purification. In preferred embodiments, the marker amino acid
sequence is a hexa-histidine peptide, such as the tag provided in a
pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,
91311), among others, many of which are commercially available. As
described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA
86:821-824, for instance, hexa-histidine provides for convenient
purification of the fusion protein. Other peptide tags useful for
purification include, but are not limited to, the hemagglutinin
"HA" tag, which corresponds to an epitope derived from the
influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767)
and the "flag" tag.
[0121] The present invention also encompasses the liquid
formulations of an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen or a variant thereof
conjugated to a diagnostic or detectable agent or any other
molecule for which serum half-life is desired to be increased. Such
an antibody can be useful for monitoring or prognosing the
development or progression of a disease, disorder or infection as
part of a clinical testing procedure, such as determining the
efficacy of a particular therapy. Such diagnosis and detection can
be accomplished by coupling an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen or a fragment thereof to
a detectable substance including, but not limited to, various
enzymes, such as but not limited to, horseradish peroxidase,
alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
prosthetic groups, such as but not limited to, streptavidin/biotin
and avidin/biotin; fluorescent materials, such as but not limited
to, umbelliferone, fluorescein, fluorescein isothiocynate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; luminescent materials, such as but not limited to,
luminol; bioluminescent materials, such as but not limited to,
luciferase, luciferin, and aequorin; radioactive materials, such as
but not limited to iodine (.sup.131I, .sup.125I, .sup.123I,
.sup.121I,), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115In, .sup.113In, .sup.112In,
.sup.111In,), and technetium (.sup.99Tc), thallium (.sup.201Ti),
gallium (.sup.68Ga, .sup.67Ga), palladium (.sup.103Pd), molybdenum
(.sup.99Mo), xenon (.sup.133Xe), fluorine (.sup.18F), .sup.153Sm,
.sup.177Lu, .sup.159Gd, .sup.149 Pm, .sup.140La, .sup.175Yb,
.sup.166Ho, .sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re,
.sup.142Pr, .sup.105Rh, .sup.97Ru, .sup.68Ge, .sup.57Co, .sup.65Zn,
.sup.85Sr, .sup.32P, .sup.153Gd, .sup.169Yb, .sup.51Cr, .sup.54Mn,
.sup.75Se, .sup.113Sn, and .sup.117Tin; positron emitting metals
using various positron emission tomographies, noradioactive
paramagnetic metal ions, and molecules that are radiolabelled or
conjugated to specific radioisotopes. The detectable substance may
be coupled or conjugated either directly to an antibody or
indirectly, through an intermediate (such as, for example, a linker
known in the art) using techniques known in the art. See, e.g.,
U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to
antibodies for use as a diagnostics according to the present
invention.
[0122] The present invention further encompasses uses of an
antibody or a fragment thereof that immunospecifically binds to a
RSV antigen conjugated to a therapeutic moiety in the liquid
formulations of the invention. An antibody or antigen-binding
fragment may be conjugated to a therapeutic moiety such as a
cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic
agent or a radioactive metal ion, e.g., alpha-emitters. A cytotoxin
or cytotoxic agent includes any agent that is detrimental to cells.
Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogues or homologs
thereof. Therapeutic moieties include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BCNU) and lomustine (CCNU)), cyclothosphamide,
busulfan, dibromomannitol, streptozotocin, mitomycin C, and
cisdichlorodiamine platinum (II) (DDP) cisplatin)); anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin);
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)); Auristatin molecules (e.g.,
auristatin PHE, bryostatin 1, solastatin 10, see Woyke et al.,
Antimicrob. Agents Chemother. 46:3802-8 (2002), Woyke et al.,
Antimicrob. Agents Chemother. 45:3580-4 (2001), Mohammad et al.,
Anticancer Drugs 12:735-40 (2001), Wall et al., Biochcm. Biophys.
Res. Commun. 266:76-80 (1999), Mohammad et al., Int. J. Oncol.
15:367-72 (1999), all of which are incorporated herein by
reference); anti-mitotic agents (e.g., vincristine and
vinblastine); hormones (e.g., glucocorticoids, progestatins,
androgens, and estrogens); DNA repair enzyme inhibitors (e.g.,
etoposide or topotecan); kinase inhibitors (e.g., compound ST1571,
imatinib mesylate (Kantarjian et al., Clin Cancer Res. 8(7):2167 76
(2002)), and those compounds disclosed in U.S. Pat. Nos. 6,245,759,
6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196, 6,218,410,
6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769, 5,925,376,
5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868,
5,648,239, and 5,587,459); farnesyl transferase inhibitors (e.g.,
R115777, BMS 214662, and those disclosed by, for example, U.S. Pat.
Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959,
6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615,
6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487,
6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338,
6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786,
6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465,
6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853,
6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574,
and 6,040,305); topoisomerase inhibitors (e.g., camptothecin,
irinotecan, SN 38, topotecan, 9 aminocamptothecin, GG 211 (GI
147211), DX 8951f; IST 622, rubitecan, pyrazoloacridine, XR 5000,
saintopin, UCE6, UCE1022, TAN 1518A, TAN 1518B, KT6006, KT6528, ED
110, NB 506, ED 110, NB 506, rebeccamycin, and bulgarein); DNA
minor groove binders such as Hoescht dye 33342 and Hoechst dye
33258; nitidine; fagaronine; epiberberine; coralyne; beta
lapachone; BC 4 1; and pharmaceutically acceptable salts, solvates,
clathrates, and prodrugs thereof (See, e.g., Rothenberg, M. L.,
Annals of Oncology 8:837 855 (1997); and Moreau et al., J. Med.
Chem. 41:1631 1640 (1998)). Therapeutic moieties may also be
antisense oligonucleotides (e.g., those disclosed in the U.S. Pat.
Nos. 6,277,832, 5,998,596, 5,885,834, 5,734,033, and 5,618,709);
immunomodulators (e.g., antibodies and cytokines); antibodies
(e.g., rituximab (Rituxan.RTM.), calicheamycin (Mylotarg.RTM.),
ibritumomab tiuxetan (Zevalin.RTM.), and tositumomab
(Bexxar.RTM.)); and adnosine deaminase inhibitors (e.g.,
Fludarabine phosphate and 2 Chlorodeoxyadenosine).
[0123] Further, an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen or a fragment thereof may
be conjugated to a therapeutic moiety or drug moiety that modifies
a given biological response. Therapeutic moiety or drug moieties
are not to be construed as limited to classical chemical
therapeutic agents. For example, the drug moiety may be a protein
or polypeptide possessing a desired biological activity. Such
proteins may include, for example, a toxin such as abrin, ricin A,
pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein
such as tumor necrosis factor, .alpha.-interferon,
.beta.-interferon, nerve growth factor, platelet derived growth
factor, tissue plasminogen activator, an apoptotic agent, e.g.,
TNF-.alpha., TNF-.beta., AIM I (see, International publication No.
WO 97/33899), AIM II (see, International Publication No. WO
97/34911), Fas Ligand (Takahashi et al., 1994, J. Immunol.,
6:1567-1574), and VEGF (see, International publication No. WO
99/23105); or, a biological response modifier such as, for example,
a lymphokine (e.g., interferon .alpha., .beta., or .gamma.,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-4
("IL-4"), interleukin-6 ("IL-6"), interleukin-9 ("IL-9"),
interleukin-12 ("IL-12"), granulocyte macrophage colony stimulating
factor ("GM-CSF"), and granulocyte colony stimulating factor
("G-CSF")), a growth factor (e.g., growth hormone ("GH")).
[0124] Moreover, an antibody can be conjugated to therapeutic
moieties such as a radioactive metal ion, such as alpha-emitters
such as .sup.213Bi or macrocyclic chelators useful for conjugating
radiometal ions, including but not limited to, .sup.131In,
.sup.131LU, .sup.131Y, .sup.131Ho, .sup.131Sm, to polypeptides. In
certain embodiments, the macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid
(DOTA) which can be attached to the antibody via a linker molecule.
Such linker molecules are commonly known in the art and described
in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson
et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al.,
1999, Nucl. Med. Biol. 26(8):943-50, each incorporated by reference
in their entireties.
[0125] Techniques for conjugating therapeutic moieties to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies 84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol.
Rev. 62:119-58.
[0126] Alternatively, an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen or a fragment thereof can
be conjugated to a second antibody to form an antibody
heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980,
which is incorporated herein by reference in its entirety.
[0127] An antibody or a fragment thereof that immunospecifically
binds to a RSV antigen or a fragment thereof may also be attached
to solid supports, which are particularly useful for immunoassays
or purification of the target antigen. Such solid supports include,
but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene, polyvinyl chloride or polypropylene.
[0128] The therapeutic moiety or drug conjugated to an antibody or
a fragment thereof that immunospecifically binds to a RSV antigen
or a fragment thereof should be chosen to achieve the desired
prophylactic or therapeutic effect(s) for a particular disorder in
a subject. A clinician or other medical personnel should consider
the following when deciding on which therapeutic moiety or drug to
conjugate to an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen or a fragment thereof:
the nature of the infection, the severity of the infection, and the
condition of the subject.
[0129] An antibody or a fragment thereof that immunospecifically
binds to a RSV antigen or a fragment thereof, with or without a
therapeutic moiety conjugated to it, administered alone or in
combination with cytotoxic factor(s) and/or cytokine(s) can be used
as a therapeutic.
[0130] In a specific embodiment, antibodies of the invention are
bispecific T cell engagers (BiTE). Bispecific T cell engagers
(BiTE) are bispecific antibodies that can redirect T cells for
antigen-specific elimination of targets. A BiTE molecule has an
antigen-binding domain that binds to a T cell antigen (e.g., CD3)
at one end of the molecule and an antigen binding domain that will
bind to an antigen on the target cell. A BiTE molecule was recently
described in International Publication No. WO 99/54440, which is
herein incorporated by reference. This publication describes a
novel single-chain multifunctional polypeptide that comprises
binding sites for the CD19 and CD3 antigens (CD19.times.CD3). This
molecule was derived from two antibodies, one that binds to CD19 on
the B cell and an antibody that binds to CD3 on the T cells. The
variable regions of these different antibodies are linked by a
polypeptide sequence, thus creating a single molecule. Also
described, is the linking of the variable heavy chain (VH) and
light chain (VL) of a specific binding domain with a flexible
linker to create a single chain, bispecific antibody.
[0131] In one embodiment, an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen comprises a portion of
the BITE molecule. For example, the VH and/or VL (preferably a
scFV) of an antibody that binds a RSV antigen can be fused to an
anti-CD3 binding portion such as that of the molecule described
above, thus creating a BiTE molecule that targets a RSV antigen. In
addition to the variable heavy and/or light chain of antibody
against a RSV antigen, other molecules that bind a RSV antigen can
comprise the BiTE molecule. In another embodiment, the BiTE
molecule comprises a molecule that binds to other T cell antigens
(other than CD3). For example, antibodies or fragments thereof that
immunospecifically bind to T-cell antigens like CD2, CD4, CD8,
CD11a, TCR, and CD28 are contemplated to be part of this invention.
This list is not meant to be exhaustive but only to illustrate that
other molecules that can immunospecifically bind to a T cell
antigen can be used as part of a BiTE molecule. These molecules can
include the VH and/or VL portion of the antibody or fragment
thereof.
[0132] The "binding domain" as used in accordance with the present
invention denotes a domain comprising a three-dimensional structure
capable of specifically binding to an epitope like native
antibodies, free scFv fragments or one of their corresponding
immunoglobulin chains, preferably the VH chain. Thus, said domain
can comprise the VH and/or VL domain of an antibody or an
immunoglobulin chain, preferably at least the VH domain or more
preferably the VH and VL domain linked by a flexible polypeptide
linker (scFv). On the other hand, said binding domain contained in
the polypeptide of the invention may comprise at least one
complementarity determining region (CDR) of an antibody or
immunoglobulin chain recognizing an antigen on the T cell or a
cellular antigen. In this respect, it is noted that the binding
domain present in the polypeptide of the invention may not only be
derived from antibodies but also from other T cell or cellular
antigen binding protein, such as naturally occurring surface
receptors or ligands. It is further contemplated that in an
embodiment of the invention, said first and or second domain of the
above-described polypeptide mimic or correspond to a VH and VL
region from a natural antibody. The antibody providing the binding
site for the polypeptide of the invention can be, e.g., a
monoclonal antibody, polyclonal antibody, chimeric antibody,
humanized antibody, bispecific antibody, synthetic antibody,
antibody fragment, such as Fab, Fv or scFv fragments etc., or a
chemically modified derivative of any of these.
[0133] 5.1.4 Method of Preparing the Antibody Formulations
[0134] The present invention provides methods for preparing liquid
formulations of antibodies, in particular, those listed in Table 2,
or derivatives, analogues, or fragments thereof that
immunospecifically bind to a RSV antigen. FIG. 1 is a schematic
diagram showing the outline for preparing purified anti-RSV
antibodies. The methods for preparing liquid formulations of the
present invention comprise: concentrating a fraction containing the
purified antibody or a fragment to a final antibody or fragment
concentration of from about 15 mg/ml, about 20 mg/ml, about 30
mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70
mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110
mg/ml, about 125 mg/ml, about 150 mg/ml, about 200 mg/ml, about 250
mg/ml, or about 300 mg/ml using a semipermeable membrane with an
appropriate molecular weight (MW) cutoff (e.g., 30 kD cutoff for
whole antibody molecules and F(ab').sub.2 fragments; and 10 kD
cutoff for antibody fragments, such as Fab fragments) and
difiltrating the concentrated antibody fraction into the
formulation buffer using the same membrane. Conditioned medium
containing antibody or a fragment thereof that immunospecifically
binds to a RSV antigen is subjected to CUNO filtration and the
filtered antibody is subjected to HS50 cation exchange
chromatography. The fraction from the HS50 cation exchange
chromatography is then subjected to rProtein A affinity
chromatography followed by low pH treatment. Following low pH
treatment, the antibody fraction is subject to super Q 650 anion
exchange chromatography and then nanofiltration. The fraction of
the antibody obtained after nanofiltration is then subjected to
diafiltration to concentrate the antibody fraction into the
formulation buffer using the same membrane.
[0135] The formulation buffer of the present invention comprises
histidine at a concentration ranging from about 1 mM to about 100
mM, about 10 mM to about 50 mM, about 20 mM to about 30 mM, or
about 23 mM to about 27 mM. Preferably, the formulation buffer of
the present invention comprises histidine at a concentration of
about 25 mM. The formulations may further comprise glycine at a
concentration of less than 100 mM, less than 50 mM, less than 3.0
mM, less than 2.0 mM, or less than 1.8 mM. Preferably, the
formulations comprise glycine at a concentration of 1.6 mM. The
amount of glycine in the formulation should not cause a significant
buffering in order to avoid antibody precipitation at its
isoelectric point. The pH of the formulation may range from about
5.0 to about 7.0, preferably about 5.5 to about 6.5, more
preferably about 5.8 to about 6.2, and most preferably about 6.0.
To obtain an appropriate pH for a particular antibody, it is
preferable that histidine (and glycine, if added) is first
dissolved in water to obtain a buffer solution with higher pH than
the desired pH and then the pH is brought down to the desired level
by adding HCl. This way, the formation of inorganic salts (e.g.,
formation of NaCl when, for example, histidine hydrochloride is
used as histidine and pH is raised to a desired level by adding
NaOH) can be avoided.
[0136] The liquid formulations of the present invention can be
prepared as unit dosage forms by preparing a vial containing an
aliquot of the liquid formulation for a one-time use. For example,
a unit dosage per vial may contain 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6
ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml of different
concentrations of an antibody or a fragment thereof that
immunospecifically binds to RSV ranging from about 15 mg/ml to
about 300 mg/ml. If necessary, these preparations can be adjusted
to a desired concentration by adding a sterile diluent to each
vial.
[0137] The liquid formulations of the present invention may be
sterilized by various sterilization methods, including sterile
filtration, radiation, etc. In a most preferred embodiment, the
difiltrated antibody formulation is filter-sterilized with a
presterilized 0.2 or 0.22-micron filter. Sterilized liquid
formulations of the present invention may be administered to a
subject to prevent, treat, manage or ameliorate a RSV infection,
one or more symptoms thereof, or a respiratory condition associated
with, potentiated by, potentiating a RSV infection.
[0138] The liquid formulations of the invention can also be used
for diagnostic purposes to detect, diagnose, or monitor a RSV
infection. In particular, the liquid formulations of the invention
comprising antibodies or fragments thereof that immunospecifically
bind to a RSV antigen conjugated or fused to a detectable agent or
label can be used to detect, diagnose, or monitor a RSV
infection.
[0139] Although the invention is directed to liquid non-lyophilized
formulations, it should be noted for the purpose of equivalents
that the formulations of the invention may be lyophilized if
desired. Thus, the invention encompasses lyophilized forms of the
formulations of the invention although such lyophilized
formulations are not necessary and, thus, not preferred.
[0140] 5.2 Methods of Preparing Antibodies
[0141] The antibodies that immunospecifically bind to a RSV antigen
can be produced by any method known in the art for the synthesis of
antibodies, in particular, by chemical synthesis or preferably, by
recombinant expression techniques.
[0142] Polyclonal antibodies immunospecific for a RSV antigen can
be produced by various procedures well-known in the art. For
example, a RSV antigen can be administered to various host animals
including, but not limited to, rabbits, mice, rats, etc. to induce
the production of sera containing polyclonal antibodies specific
for the human antigen. Various adjuvants may be used to increase
the immunological response, depending on the host species, and
include but are not limited to, Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille
Calmette-Guerin) and corynebacterium parvum. Such adjuvants are
also well known in the art.
[0143] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981) (said references incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0144] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
Briefly, mice can be immunized with a non-murine antigen and once
an immune response is detected, e.g., antibodies specific for the
antigen are detected in the mouse serum, the mouse spleen is
harvested and splenocytes isolated. The splenocytes are then fused
by well known techniques to any suitable myeloma cells, for example
cells from cell line SP20 available from the ATCC. Hybridomas are
selected and cloned by limited dilution. The hybridoma clones are
then assayed by methods known in the art for cells that secrete
antibodies capable of binding a polypeptide of the invention.
Ascites fluid, which generally contains high levels of antibodies,
can be generated by immunizing mice with positive hybridoma
clones.
[0145] The present invention provides methods of generating
monoclonal antibodies as well as antibodies produced by the method
comprising culturing a hybridoma cell secreting an antibody of the
invention wherein, preferably, the hybridoma is generated by fusing
splenocytes isolated from a mouse immunized with a non-murine
antigen with myeloma cells and then screening the hybridomas
resulting from the fusion for hybridoma clones that secrete an
antibody able to bind to the antigen.
[0146] Antibody fragments which recognize specific particular
epitopes may be generated by any technique known to those of skill
in the art. For example, Fab and F(ab').sub.2 fragments of the
invention may be produced by proteolytic cleavage of immunoglobulin
molecules, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab').sub.2 fragments). F(ab').sub.2
fragments contain the variable region, the light chain constant
region and the CH1 domain of the heavy chain. Further, the
antibodies of the present invention can also be generated using
various phage display methods known in the art.
[0147] In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. In particular, DNA
sequences encoding VH and VL domains are amplified from animal cDNA
libraries (e.g., human or murine cDNA libraries of affected
tissues). The DNA encoding the VH and VL domains are recombined
together with an scFv linker by PCR and cloned into a phagemid
vector. The vector is electroporated in E. coli and the E. coli is
infected with helper phage. Phage used in these methods are
typically filamentous phage including fd and M13 and the VH and VL
domains are usually recombinantly fused to either the phage gene
III or gene VIII. Phage expressing an antigen binding domain that
binds to a particular antigen can be selected or identified with
antigen, e.g., using labeled antigen or antigen bound or captured
to a solid surface or bead. Examples of phage display methods that
can be used to make the antibodies of the present invention include
those disclosed in Brinkman et al., 1995, J. Immunol. Methods
182:41-50; Ames et al, 1995, J. Immunol. Methods 184:177-186;
Kettleborough et al, 1994, Eur. J. Immunol. 24:952-958; Persic et
al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in
Immunology 57:191-280; International application No. PCT/GB91/O1
134; International publication Nos. WO 90/02809, WO 91/10737, WO
92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and
WO97/13844; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484,
5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908,
5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108; each of
which is incorporated herein by reference in its entirety.
[0148] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described below. Techniques to
recombinantly produce Fab, Fab' and F(ab').sub.2 fragments can also
be employed using methods known in the art such as those disclosed
in PCT publication No. WO 92/22324; Mullinax et al., 1992,
BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and
Better et al., 1988, Science 240:1041-1043 (said references
incorporated by reference in their entireties).
[0149] To generate whole antibodies, PCR primers including VH or VL
nucleotide sequences, a restriction site, and a flanking sequence
to protect the restriction site can be used to amplify the VH or VL
sequences in scFv clones. Utilizing cloning techniques known to
those of skill in the art, the PCR amplified VH domains can be
cloned into vectors expressing a VH constant region, e.g., the
human gamma 4 constant region, and the PCR amplified VL domains can
be cloned into vectors expressing a VL constant region, e.g., human
kappa or lambda constant regions. Preferably, the vectors for
expressing the VH or VL domains comprise an EF-1.alpha. promoter, a
secretion signal, a cloning site for the variable domain, constant
domains, and a selection marker such as neomycin. The VH and VL
domains may also cloned into one vector expressing the necessary
constant regions. The heavy chain conversion vectors and light
chain conversion vectors are then co-transfected into cell lines to
generate stable or transient cell lines that express full-length
antibodies, e.g., IgG, using techniques known to those of skill in
the art.
[0150] For some uses, including in vivo use of antibodies in humans
and in vitro detection assays, it may be preferable to use
humanized antibodies or chimeric antibodies. Completely human
antibodies and humanized antibodies are particularly desirable for
therapeutic treatment of human subjects. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also U.S. Pat. Nos.
4,444,887 and 4,716,111; and International publication Nos. WO
98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO
96/33735, and WO 91/10741; each of which is incorporated herein by
reference in its entirety.
[0151] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes . . . . The mouse heavy and light chain
immunoglobulin genes may be rendered non-functional separately or
simultaneously with the introduction of human immunoglobulin loci
by homologous recombination. In particular, homozygous deletion of
the JH region prevents endogenous antibody production. The modified
embryonic stem cells are expanded and microinjected into
blastocysts to produce chimeric mice. The chimeric mice are then be
bred to produce homozygous offspring which express human
antibodies. The transgenic mice are immunized in the normal fashion
with a selected antigen, e.g., all or a portion of a polypeptide of
the invention. Monoclonal antibodies directed against the antigen
can be obtained from the immunized, transgenic mice using
conventional hybridoma technology. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and
somatic mutation. Thus, using such a technique, it is possible to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
For an overview of this technology for producing human antibodies,
see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., International publication
Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos.
5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806,
5,814,318, and 5,939,598, which are incorporated by reference
herein in their entirety. In addition, companies such as Abgenix,
Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0152] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different immunoglobulin
molecules. Methods for producing chimeric antibodies are known in
the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al.,
1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol.
Methods 125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,
4,816,397, and 6,311,415, which are incorporated herein by
reference in their entirety.
[0153] A humanized antibody is an antibody or its variant or
fragment thereof which is capable of binding to a predetermined
antigen and which comprises a framework region having substantially
the amino acid sequence of a human immunoglobulin and a CDR having
substantially the amino acid sequence of a non-human immuoglobulin.
A humanized antibody comprises substantially all of at least one,
and typically two, variable domains (Fab, Fab', F(ab').sub.2, Fabc,
Fv) in which all or substantially all of the CDR regions correspond
to those of a non-human immunoglobulin (i.e., donor antibody) and
all or substantially all of the framework regions are those of a
human immunoglobulin consensus sequence. Preferably, a humanized
antibody also comprises at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin.
Ordinarily, the antibody will contain both the light chain as well
as at least the variable domain of a heavy chain. The antibody also
may include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy
chain. The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including IgG1, IgG2, IgG3 and IgG4. Usually the constant
domain is a complement fixing constant domain where it is desired
that the humanized antibody exhibit cytotoxic activity, and the
class is typically IgG.sub.1. Where such cytotoxic activity is not
desirable, the constant domain may be of the IgG.sub.2 class. The
humanized antibody may comprise sequences from more than one class
or isotype, and selecting particular constant domains to optimize
desired effector functions is within the ordinary skill in the art.
The framework and CDR regions of a humanized antibody need not
correspond precisely to the parental sequences, e.g. the donor CDR
or the consensus framework may be mutagenized by substitution,
insertion or deletion of at least one residue so that the CDR or
framework residue at that site docs not correspond to either the
consensus or the import antibody. Such mutations, however, will not
be extensive. Usually, at least 75% of the humanized antibody
residues will correspond to those of the parental framework and CDR
sequences, more often 90%, and most preferably greater than 95%. A
humanized antibody can be produced using variety of techniques
known in the art, including but not limited to, CDR-grafting (see
e.g., European Patent No. EP 239,400; International Publication No.
WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and
5,585,089, each of which is incorporated herein in its entirety by
reference), veneering or resurfacing (see e.g., European Patent
Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology
28(4/5):489-498; Studnicka et al., 1994, Protein Engineering
7(6):805-814; and Roguska et al., 1994, PNAS. 91:969-973, each of
which is incorporated herein by its entirety by reference), chain
shuffling (see e.g., U.S. Pat. No. 5,565,332, which is incorporated
herein in its entirety by reference), and techniques disclosed in,
e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886,
International Publication No. WO 9317105, Tan et al., J. Immunol.
169:1119-25 (2002), Caldas et al., Protein Eng. 13(5):353-60
(2000), Morea et al., Methods 20(3):267-79 (2000), Baca et al., J.
Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng.
9(10):895-904 (1996), Couto et al., Cancer Res. 55 (23.
Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22
(1995), Sandhu J S, Gene 150(2):409-10 (1994), and Pedersen et al,
J. Mol. Biol. 235(3):959-73 (1994), each of which is incorporated
herein in its entirety by reference. Often, framework residues in
the framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and
Riechmann et al., 1988, Nature 332:323, which are incorporated
herein by reference in their entireties.)
[0154] Further, the antibodies that immunospecifically bind to a
RSV antigen can, in turn, be utilized to generate anti-idiotype
antibodies that "mimic" an antigen using techniques well known to
those skilled in the art. (See, e.g., Greenspan & Bona, 1989,
FASEB J. 7(5):437-444; and Nissinoff, 1991, J. Immunol.
147(8):2429-2438).
[0155] 5.2.1 Polynucleotide Sequences Encoding an Antibody
[0156] The invention provides polynucleotides comprising a
nucleotide sequence encoding an antibody or fragment thereof that
immunospecifically binds to an antigen. The invention also
encompasses polynucleotides that hybridize under high stringency,
intermediate or lower stringency hybridization conditions, e.g., as
defined supra, to polynucleotides that encode an antibody of the
invention.
[0157] The polynucleotides may be obtained, and the nucleotide
sequence of the polynucleotides determined, by any method known in
the art. The nucleotide sequence of antibodies immunospecific for a
desired antigen can be obtained, e.g., from the literature or a
database such as GenBank. Nucleotide or alternatively, if the amino
acid sequence of an antibody or fragment thereof that
immunospecifically binds to a RSV antigen is known, the nucleotide
sequences encoding the antibody or a fragment thereof (e.g., a CDR)
can be determined using methods well known in the art, i.e.,
nucleotide codons known to encode particular amino acids are
assembled in such a way to generate a nucleic acid that encodes the
antibody. Such a polynucleotide encoding the antibody may be
assembled from chemically synthesized oligonucleotides (e.g., as
described in Kutmeier et al., 1994, BioTechniques 17:242), which,
briefly, involves the synthesis of overlapping oligonucleotides
containing portions of the sequence encoding the antibody,
annealing and ligating of those oligonucleotides, and then
amplification of the ligated oligonucleotides by PCR.
[0158] Alternatively, a polynucleotide encoding an antibody may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from, any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody of the invention) by PCR amplification using
synthetic primers hybridizable to the 3' and 5' ends of the
sequence or by cloning using an oligonucleotide probe specific for
the particular gene sequence to identify, e.g., a cDNA clone from a
cDNA library that encodes the antibody. Amplified nucleic acids
generated by PCR may then be cloned into replicable cloning vectors
using any method well known in the art.
[0159] Once the nucleotide sequence of the antibody is determined,
the nucleotide sequence of the antibody may be manipulated using
methods well known in the art for the manipulation of nucleotide
sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, for example, the techniques described
in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual,
2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and
Ausubel et al., eds., 1998, Current Protocols in Molecular Biology,
John Wiley & Sons, NY, which are both incorporated by reference
herein in their entireties), to generate antibodies having a
different amino acid sequence, for example to create amino acid
substitutions, deletions, and/or insertions.
[0160] In a specific embodiment, one or more of the CDRs is
inserted within framework regions using routine recombinant DNA
techniques. The framework regions may be naturally occurring or
consensus framework regions, and preferably human framework regions
(see, e.g., Chothia et al., 1998, J. Mol. Biol. 278: 457-479 for a
listing of human framework regions). Preferably, the polynucleotide
generated by the combination of the framework regions and CDRs
encodes an antibody that specifically binds to a particular
antigen. Preferably, as discussed supra, one or more amino acid
substitutions may be made within the framework regions, and,
preferably, the amino acid substitutions improve binding of the
antibody to its antigen. Additionally, such methods may be used to
make amino acid substitutions or deletions of one or more variable
region cysteine residues participating in an intrachain disulfide
bond to generate antibody molecules lacking one or more intrachain
disulfide bonds. Other alterations to the polynucleotide are
encompassed by the present invention and within the skill of the
art.
[0161] 5.2.2 Recombinant Expression of an Antibody
[0162] Recombinant expression of an antibody of the invention,
derivative, analogue or fragment thereof, (e.g., a heavy or light
chain of an antibody of the invention or a portion thereof or a
single chain antibody of the invention), requires construction of
an expression vector containing a polynucleotide that encodes the
antibody. Once a polynucleotide encoding an antibody molecule or a
heavy or light chain of an antibody, or portion thereof
(preferably, but not necessarily, containing the heavy or light
chain variable domain), of the invention has been obtained, the
vector for the production of the antibody molecule may be produced
by recombinant DNA technology using techniques well-known in the
art. See, e.g., U.S. Pat. No. 6,331,415, which is incorporated
herein by reference in its entirety. Thus, methods for preparing a
protein by expressing a polynucleotide containing an antibody
encoding nucleotide sequence are described herein. Methods which
are well known to those skilled in the art can be used to construct
expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, a heavy or light chain of an antibody, a heavy or
light chain variable domain of an antibody or a portion thereof, or
a heavy or light chain CDR, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., International
Publication No. WO 86/05807 and WO 89/01036; and U.S. Pat. No.
5,122,464) and the variable domain of the antibody may be cloned
into such a vector for expression of the entire heavy, the entire
light chain, or both the entire heavy and light chains.
[0163] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody of the invention.
Thus, the invention includes host cells containing a polynucleotide
encoding an antibody of the invention or fragments thereof, or a
heavy or light chain thereof, or portion thereof, or a single chain
antibody of the invention, operably linked to a heterologous
promoter. In preferred embodiments for the expression of
double-chained antibodies, vectors encoding both the heavy and
light chains may be co-expressed in the host cell for expression of
the entire immunoglobulin molecule, as detailed below.
[0164] A variety of host-expression vector systems may be utilized
to express the antibody molecules of the invention (see; e.g., U.S.
Pat. No. 5,807,715). Such host-expression systems represent
vehicles by which the coding sequences of interest may be produced
and subsequently purified, but also represent cells which may, when
transformed or transfected with the appropriate nucleotide coding
sequences, express an antibody molecule of the invention in situ.
These include but are not limited to microorganisms such as
bacteria (e.g., E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing antibody coding sequences; yeast (e.g.,
Saccharomyces Pichia) transformed with recombinant yeast expression
vectors containing antibody coding sequences; insect cell systems
infected with recombinant virus expression vectors (e.g.,
baculovirus) containing antibody coding sequences; plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing antibody coding sequences; or mammalian cell
systems (e.g., COS, CHO, BHK, 293, NSO, and 3T3 cells) harboring
recombinant expression constructs containing promoters derived from
the genome of mammalian cells (e.g., metallothionein promoter) or
from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter). Preferably, bacterial cells such as
Escherichia coli, and more preferably, eukaryotic cells, especially
for the expression of whole recombinant antibody molecule, are used
for the expression of a recombinant antibody molecule. For example,
mammalian cells such as Chinese hamster ovary cells (CHO), in
conjunction with a vector such as the major intermediate early gene
promoter element from human cytomegalovirus is an effective
expression system for antibodies (Foecking et al., 1986, Gene
45:101; and Cockett et al., 1990, Bio/Technology 8:2). In a
specific embodiment, the expression of nucleotide sequences
encoding antibodies which immunospecifically bind to a RSV antigen
is regulated by a constitutive promoter, inducible promoter or
tissue specific promoter.
[0165] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such an antibody is to be produced, for the generation
of pharmaceutical compositions of an antibody molecule, vectors
which direct the expression of high levels of fusion protein
products that are readily purified may be desirable. Such vectors
include, but are not limited to, the E. coli expression vector
pUR278 (Ruther et al., 1983, EMBO 12:1791), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids
Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
24:5503-5509); and the like. pGEX vectors may also be used to
express foreign polypeptides as fusion proteins with glutathione
5-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0166] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The antibody
coding sequence may be cloned individually into non-essential
regions (for example the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example the polyhedrin
promoter).
[0167] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the antibody coding sequence of interest may be
ligated to an adenovirus transcription/translation control complex,
e.g., the late promoter and tripartite leader sequence. This
chimeric gene may then be inserted in the adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region
of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the
antibody molecule in infected hosts (e.g., see Logan & Shenk,
1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see, e.g., Bittner et al., 1987, Methods in
Enzymol. 153:51-544).
[0168] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g. cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO
(a murine myeloma cell line that does not endogenously produce any
immunoglobulin chains), CRL7030 and HsS78Bst cells.
[0169] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker . . . . Following the introduction of the
foreign DNA, engineered cells may be allowed to grow for 1-2 days
in an enriched media, and then are switched to a selective media.
The selectable marker in the recombinant plasmid confers resistance
to the selection and allows cells to stably integrate the plasmid
into their chromosomes and grow to form foci which in turn can be
cloned and expanded into cell lines. This method may advantageously
be used to engineer cell lines which express the antibody molecule.
Such engineered cell lines may be particularly useful in screening
and evaluation of compositions that interact directly or indirectly
with the antibody molecule.
[0170] A number of selection systems may be used, including but not
limited to, the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthineguanine
phosphoribosyltransferase (Szybalska & Szybalski, 1992, Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase
(Lowy et al., 1980, Cell 22:8-17) genes can be employed in tk-,
hgprt- or aprt-cells, respectively. Also, antimetabolite resistance
can be used as the basis of selection for the following genes:
dhfr, which confers resistance to methotrexate (Wigler et al.,
1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl.
Acad. Sci. USA 78:1527); gpt, which confers resistance to
mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad.
Sci. USA 78:2072); neo, which confers resistance to the
aminoglycoside G-418 (Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;
Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993,
Ann. Rev. Biochem. 62: 191-217; May, 1993, TIB TECH 11(5):155-2
15); and hygro, which confers resistance to hygromycin (Santerre et
al., 1984, Gene 30:147). Methods commonly known in the art of
recombinant DNA technology may be routinely applied to select the
desired recombinant clone, and such methods are described, for
example, in Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer
and Expression, A laboratory Manual, Stockton Press, NY (1990); and
in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in
Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin
et al., 1981, J. Mol. Biol. 150:1, which are incorporated by
reference herein in their entireties.
[0171] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of
inhibitor present in culture of host cell will increase the number
of copies of the marker gene. Since the amplified region is
associated with the antibody gene, production of the antibody will
also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).
[0172] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes, and is capable of expressing, both heavy and light
chain polypeptides. In such situations, the light chain should be
placed before the heavy chain to avoid an excess of toxic free
heavy chain (Proudfoot, 1986, Nature 322:52; and Kohler, 1980,
Proc. Natl. Acad. Sci. USA 77:2 197). The coding sequences for the
heavy and light chains may comprise cDNA or genomic DNA.
[0173] Once an antibody molecule of the invention has been produced
by recombinant expression, it may be purified by any method known
in the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A,
and sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins. Further, the antibodies of the present invention or
fragments thereof may be fused to heterologous polypeptide
sequences described herein or otherwise known in the art to
facilitate purification.
[0174] 5.3 Methods of Monitoring the Stability and Aggregation of
Antibody Formulations
[0175] There are various methods available for assessing the
stability of the liquid formulations of the present invention,
based on the physical and chemical structures of the proteins
(e.g., antibodies or fragments thereof) as well as on their
biological activities. For example, to study denaturation of
proteins, methods such as charge-transfer absorption, thermal
analysis, fluorescence spectroscopy, circular dichroism, NMR, and
HPSEC, are available. See, for example, Wang et al., 1988, J. of
Parenteral Science & Technology 42(Suppl): S4-S26.
[0176] The rCGE and HPSEC are the most common and simplest methods
to assess the formation of protein aggregates, protein degradation,
and protein fragmentation. Accordingly, the stability of the liquid
formulations of the present invention may be assessed by these
methods.
[0177] For example, the stability of the liquid formulations of the
present invention may be evaluated by HPSEC or rCGE, wherein the
percent area of the peaks represents the non-degraded antibody or
non-degraded antibody fragments. In particular, approximately 250
.mu.g of the antibody or antibody fragment that immunospecifically
binds to a RSV antigen (approximately 25 .mu.l of a liquid
formulation comprising 10 mg/ml said antibody or antibody fragment)
is injected onto a TosoH Biosep TSK G3000SW.sub.XL column (7.8
mm.times.30 cm) fitted with a TSK SW .times.1 guard column (6.0 mm
CX 4.0 cm). The antibody or antibody fragment is eluted
isocratically with 0.1 M disodium phosphate containing 0.1 M sodium
sulfate and 0.05% sodium azide, at a flow rate of 0.8 to 1.0
ml/min. Eluted protein is detected using UV absorbance at 280 mm.
SYNAGIS.RTM. reference standard is run in the assay as a control,
and the results are reported as the area percent of the product
monomer peak compared to all other peaks excluding the included
volume peak observed at approximately 12 to 14 minutes. Peaks
eluting earlier than the monomer peak are recorded as percent
aggregate.
[0178] The liquid formulations of the present invention exhibit low
to undetectable levels of aggregation as measured by HPSEC or rCGE,
that is, no more than 5%, no more than 4%, no more than 3%, no more
than 2%, no more than 1%, and most preferably no more than 0.5%
aggregate by weight protein, and low to undetectable levels of
fragmentation, that is, 80% or higher, 85% or higher, 90% or
higher, 95% or higher, 98% or higher, or 99% or higher, or 99.5% or
higher of the total peak area in the peak(s) representing intact
antibodies or fragments thereof. In the case of SDS-PAGE, the
density or the radioactivity of each band stained or labeled with
radioisotope can be measured and the % density or % radioactivity
of the band representing non-degraded antibodies or fragments
thereof can be obtained.
[0179] The stability of the liquid formulations of the present
invention can be also assessed by any assays which measures the
biological activity of the antibody or fragments thereof in the
formulation. The biological activities of antibodies include, but
are not limited to, antigen-binding activity, complement-activation
activity, Fc-receptor binding activity, and so forth.
Antigen-binding activity of the antibodies can be measured by any
method known to those skilled in the art, including but not limited
to ELISA, radioimmunoassay, Western blot, and the like.
Complement-activation activity can be measured by a C3a/C4a assay
in the system where the antibody which imnunospecifically binds to
a RSV antigen is reacted in the presence of the complement
components with the cells expressing the RSV antigen. Also see
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988) (incorporated by reference herein
in its entirety). An ELISA based assay, e.g., may be used to
compare the ability of an antibody or fragment thereof to
immunospecifically bind to a RSV antigen to a SYNAGIS.RTM.
reference standard. In this assay, plates are coated with a RSV
antigen and the binding signal of a set concentration of a
SYNAGIS.RTM. reference standard is compared to the binding signal
of the same concentration of a test antibody or antibody
fragment.
[0180] The purity of the liquid antibody formulations of the
invention may be measured by any method well-known to one of skill
in the art such as, e.g., HPSEC. The sterility of the liquid
antibody formulations may be assessed as follows: sterile
soybean-casein digest medium and fluid thioglycollate medium are
inoculated with a test liquid antibody formulation by filtering the
liquid antibody formulation through a sterile filter having a
nominal porosity of 0.45 .mu.m. When using the Sterisure.TM. or
Steritest.TM. method, each filter device is aseptically filled with
approximately 100 ml of sterile soybean-casein digest medium or
fluid thioglycollate medium. When using the conventional method,
the challenged filter is aseptically transferred to 100 ml of
sterile soybean-casein digest medium or fluid thioglycollate
medium. The media are incubated at appropriate temperatures and
observed three times over a 14 day period for evidence of bacterial
or fungal growth.
[0181] 5.4 Prophylactic and Therapeutic Utility of the Antibody
Formulations
[0182] The present invention is also directed to antibody-based
therapies which involve administering to a subject, preferably a
human, the liquid antibody formulations of the present invention
for preventing, treating, managing or ameliorating a RSV infection,
one or more symptoms thereof, or a respiratory condition associated
with, potentiated by, or potentiating a RSV infection. The liquid
formulations of the invention comprise an antibody or a fragment
thereof at concentrations of from about 15 mg/ml to about 300 mg/ml
in a solution containing histidine, which antibody or a fragment
thereof immunospecifically binds to a RSV antigen. The liquid
formulations of the invention may comprise a single antibody or
fragment thereof that immunospecifically binds to a RSV antigen,
with the proviso that said antibody or antibody fragment is not
SYNAGIS.RTM. or a fragment thereof. The liquid formulations of the
invention may also comprise two or more antibodies or fragments
thereof that immunospecifically bind to a RSV antigen. In a
specific embodiment, one of the antibodies or antibody fragments
included in such liquid formulations is SYNAGIS.RTM. or a fragment
thereof. In an alternative embodiment, one of the antibodies or
antibody fragments included in such liquid formulations is not
SYNAGIS.RTM. or a fragment thereof.
[0183] The liquid formulations of the invention may comprise
antibodies or fragments thereof that immunospecifically bind to a
RSV antigen and exhibiting improved in vivo half-lives compared to
know antibodies that immunospecifically bind to a RSV antigen
(e.g., unmodified SYNAGIS.RTM.).
[0184] Antibodies or fragments thereof in the liquid formulations
of the present invention may function as antagonists of a RSV
infection and can be administered to a subject, preferably a human,
to treat, prevent, manage or ameliorate a RSV infection, one or
more symptoms thereof, or a respiratory condition associated with,
potentiated by, or potentiating a RSV infection. For example,
antibodies or fragments thereof which disrupt or prevent the
interaction between a RSV antigen and its host cell receptor may be
administered to a subject, preferably a human, in the liquid
formulations of the present invention to treat, prevent, manage or
ameliorate a RSV infection, one or more symptoms thereof, or a
respiratory condition associated with, potentiated by, or
potentiating a RSV infection.
[0185] In a specific embodiment, a liquid formulation of the
present invention comprises an antibody or a fragment thereof that
prevents RSV from binding to its host cell receptor by at least
99%, at least 95%, at least 90%, at least 85%, at least 80%, at
least 75%, at least 70%, at least 60%, at least 50%, at least 45%,
at least 40%, at least 45%, at least 35%, at least 30%, at least
25%, at least 20%, or at least 10% relative to RSV binding to its
host cell receptor in the absence of said antibodies or antibody
fragments or the presence of an negative control (e.g., an
unrelated IgG antibody or phosphate buffered saline). In another
embodiment, a liquid formulation of the present invention comprises
a combination of antibodies, a combination of antibody fragments,
or a combination of antibodies and antibody fragments that prevents
RSV from binding to its host cell receptor by at least 99%, at
least 95%, at least 90%, at least 85%, at least 80%, at least 75%,
at least 70%, at least 60%, at least 50%, at least 45%, at least
40%, at least 45%, at least 35%, at least 30%, at least 25%, at
least 20%, or at least 10% relative to RSV binding to its host cell
receptor in the absence of said antibodies and/or antibody
fragments or the presence of an negative control (e.g., an
unrelated IgG antibody or phosphate buffered saline). In a
preferred embodiment, one of antibodies in the combination of
antibodies and/or antibody fragments in the liquid formulations of
the present invention, is one of the antibodies listed in Table 1,
not including SYNAGIS.RTM. or a fragment thereof, which
immunospecifically binds to a RSV. In another embodiment, one of
the antibodies in the combination of antibodies and/or antibody
fragments in the liquid formulations of the present invention is
SYNAGIS.RTM. or a fragment thereof.
[0186] Antibodies or fragments thereof which do not prevent RSV
from binding its host cell receptor but inhibit or downregulate RSV
replication can also be administered in the liquid formulations of
the invention to a subject to treat, prevent, manage or ameliorate
a RSV infection, one or more symptoms thereof, or a respiratory
condition associated with, potentiated by or potentiating a RSV
infection. The ability of an antibody or a fragment thereof to
inhibit or downregulate RSV replication may be determined by
techniques described herein or otherwise known in the art. For
example, the inhibition or downregulation of RSV replication can be
determined by detecting the RSV titer in the lungs of a subject,
preferably a human.
[0187] In a specific embodiment, a liquid formulation of the
present invention comprises an antibody or a fragment thereof that
inhibits or downregulates RSV replication by at least 99%, at least
95%, at least 90%, at least 85%, at least 80%, at least 75%, at
least 70%, at least 60%, at least 50%, at least 45%, at least 40%,
at least 45%, at least 35%, at least 30%, at least 25%, at least
20%, or at least 10% relative to RSV replication in absence of said
antibodies or antibody fragments or the presence of an negative
control (e.g., an unrelated IgG antibody or phosphate buffered
saline). In another embodiment, a liquid formulation of the present
invention comprises a combination of antibodies, a combination of
antibody fragments, or a combination of antibodies and antibody
fragments that inhibit or downregulate RSV replication by at least
99%, at least 95%, at least 90%, at least 85%, at least 80%, at
least 75%, at least 70%, at least 60%, at least 50%, at least 45%,
at least 40%, at least 45%, at least 35%, at least 30%, at least
25%, at least 20%, or at least 10% relative to RSV replication in
absence of said antibodies and/or antibody fragments or the
presence of an negative control (e.g., an unrelated IgG antibody or
phosphate buffered saline). In another embodiment, the liquid
formulations of the invention are administered to a subject in an
institution or group home (e.g., a nursing home or orphanage).
[0188] The liquid formulations of the present invention may be used
locally or systemically in the body of a subject in need thereof as
prophylactic or therapeutic agent. The formulations of the present
invention may also be advantageously utilized in combination with
other agents (see Section 5.5) locally or systemically in the body
of a subject in need thereof to prevent or treat a RSV infection or
a respiratory condition that is associated with, potentiated by or
potentiates a RSV infection.
[0189] Generally, administration of products of a species origin or
species reactivity (in the case of antibodies) that is the same
species as that of the patient is preferred. Thus, in a preferred
embodiment, human or humanized antibodies, fragments derivatives,
or analogues, are administered to a human patient for therapy or
prophylaxis.
[0190] In a specific embodiment, a liquid formulation of the
invention is administered in combination with a IL-9 antagonist to
a subject in need thereof to prevent, treat, manage or ameliorating
wheezing associated with a RSV infection. In certain cases,
wheezing precedes the onset or development of a RSV infection. In a
specific embodiment, the invention provides methods of preventing,
treating, ameliorating, or managing wheezing associated with a RSV
infection, said methods comprising administering to a subject in
need thereof an effective amount of one or more IL-9 antagonists in
combination with a liquid formulation of the invention. In other
embodiments, the invention provides methods of preventing the onset
and/or development of asthma (which may associated with,
potentiated by or potentiates a RSV infection) in subjects with
wheezing, said method comprising administering to said subject an
effective amount of one or more IL-9 antagonists in combination
with an effective amount of a liquid formulation of the
invention.
[0191] In certain embodiments, a liquid formulation of the
invention and one or more other therapies (e.g., one or more other
prophylactic or therapeutic agents) useful for prevention,
treatment, management or amelioration of a RSV infection are
administered in a cycle of less than about 3 weeks, about once
every two weeks, about once every 10 days or about once every week.
One cycle can comprise the administration of a therapy (e.g., a
therapeutic or prophylactic agent) by infusion over about 90
minutes every cycle, about 1 hour every cycle, about 45 minutes
every cycle. Each cycle can comprise at least 1 week of rest, at
least 2 weeks of rest, at least 3 weeks of rest. The number of
cycles administered is from about 1 to about 12 cycles, more
typically from about 2 to about 10 cycles, and more typically from
about 2 to about 8 cycles.
[0192] It is preferred to use high affinity and/or potent in vivo
inhibiting antibodies and/or neutralizing antibodies that
immunospecifically bind to a RSV antigen (for prevention,
treatment, management or amelioration of a RSV infection or a
symptom thereof). It is also preferred to use antibodies that have
improved in vivo half-lives compared to known antibodies that
immunospecifically binds to a RSV antigen, for example,
SYNAGIS.RTM.. Such antibodies or fragments thereof will preferably
have an affinity for the RSV F glycoprotein and/or fragments of the
F glycoprotein.
[0193] In one embodiment, the liquid formulations of the present
invention are administered to a subject, preferably a human, to
treat, prevent or ameliorate one or more symptoms associated with
RSV infection. In another embodiment, the liquid formulations of
the invention are administered to a human with cystic fibrosis,
bronchopulmonary dysplasia, congenital heart disease, congenital
immunodeficiency or acquired immunodeficiency, or to a human who
has had a bone marrow transplant to treat, prevent or ameliorate
one or more symptoms associated with RSV infection. In another
embodiment, the liquid formulations of the invention are
administered to a human infant, preferably a human infant born
prematurely or a human infant at risk of hospitalization for RSV
infection to treat, prevent or ameliorate one or more symptoms
associated with RSV infection. In another embodiment, the liquid
formulations of the invention are administered to an elderly person
to prevent, treat, or ameliorate one or more symptoms associated
with RSV infection. In yet another embodiment, the liquid
formulations of the invention are administered to a subject in an
institution or group home (e.g., a hospital, nursing home, or
orphanage).
[0194] 5.5 Agent Useful in Combination with the Antibody
Formulations
[0195] The present invention provides methods for preventing,
managing, treating, or ameliorating a RSV infection, one or more
symptoms thereof, or a respiratory condition associated with,
potentiated by or potentiating a RSV infection comprising
administering to a subject in need thereof one or more antibody
liquid formulations of the invention alone or in combination with
one or more therapies (e.g., one or more prophylactic or
therapeutic agents) other than the antibody liquid formulations of
the invention. The present invention provides methods for
preventing, managing, treating, or ameliorating a RSV infection,
one or more symptoms thereof, or a respiratory condition (e.g.,
airway hyperresponsiveness, asthma, etc.) that is associated with,
potentiated by or potentiating a RSV infection comprising
administering to, a subject in need thereof one or more liquid
formulations of the invention alone or in combination with one or
more therapies (e.g., one or more prophylactic or therapeutic
agents) other than an antibody liquid formulation of the invention.
The present invention also provides compositions comprising a
liquid formulation of an antibody or a fragment thereof that
immunospecifically bind to a RSV antigen and one or more
prophylactic or therapeutic agents other than a liquid antibody
formulation of the invention and methods of preventing, managing,
treating, or ameliorating a RSV infection, one or more symptoms
thereof, or a respiratory condition associated with, potentiated by
or potentiating a RSV infection utilizing said compositions.
Therapeutic or prophylactic agents include, but are not limited to,
small molecules, synthetic drugs, peptides, polypeptides, proteins,
nucleic acids (e.g., DNA and RNA nucleotides including, but not
limited to, antisense nucleotide sequences, triple helices, RNA
interference (RNAi), and nucleotide sequences encoding biologically
active proteins, polypeptides or peptides) antibodies, synthetic or
natural inorganic molecules, mimetic agents, and synthetic or
natural organic molecules.
[0196] Any therapy which is known to be useful, or which has been
used or is currently being used for the prevention, management,
treatment, or amelioration of a RSV infection, one or more symptoms
thereof, or a respiratory condition associated with, potentiated by
or potentiating a RSV infection can be used in combination with an
antibody liquid formulation in accordance with the invention
described herein. See, e.g., Gilman et al., Goodman and Gilman's:
The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill,
New York, 2001; The Merck Manual of Diagnosis and Therapy, Berkow,
M. D. et al. (eds.), 17th Ed., Merck Sharp & Dohme Research
Laboratories, Rahway, N.J., 1999; Cecil Textbook of Medicine, 20th
Ed., Bennett and Plum (eds.), W.B. Saunders, Philadelphia, 1996,
for information regarding therapies (e.g., prophylactic or
therapeutic agents) which have been or are currently being used for
preventing, treating, managing, or ameliorating a RSV infection or
a respiratory condition associated with, potentiated by or
potentiating a RSV infection or one or more symptoms thereof.
Examples of such agents include, but are not limited to,
immunomodulatory agents, anti-inflammatory agents (e.g.,
adrenocorticoids, corticosteroids (e.g., beclomethasone,
budesonide, flunisolide, fluticasone, triamcinolone,
methlyprednisolone, prednisolone, prednisone, hydrocortisone),
glucocorticoids, steroids, non-steroidal anti-inflammatory drugs
(e.g., aspirin, ibuprofen, diclofenac, and COX-2 inhibitors), pain
relievers, leukotreine antagonists (e.g., montelukast, methyl
xanthines, zafirlukast, and zileuton), beta2-agonists (e.g.,
albuterol, biterol, fenoterol, isoetharie, metaproterenol,
pirbuterol, salbutamol, terbutalin formoterol, salmeterol, and
salbutamol terbutaline), anticholinergic agents (e.g., ipratropium
bromide and oxitropium bromide), sulphasalazine, penicillamine,
dapsone, antihistamines, anti-malarial agents (e.g.,
hydroxychloroquine)), anti-viral agents, and antibiotics (e.g.,
dactinomycin (formerly actinomycin), bleomycin, erythromycin,
penicillin, mithramycin, and anthramycin (AMC)).
[0197] In specific embodiments, a liquid formulation of the
invention is used in combination with a monoclonal or chimeric
antibody, or with a lymphokine or hematopoictic growth factor (such
as, e.g., IL-2, IL-3, IL-4, IL-7, IL-9, IL-10, IL12, and interferon
.alpha., .beta., and .gamma.), which, for example, serves to
increase the number or activity of effector cells which interact
with the antibody. A liquid formulation of the present invention
may also be advantageously utilized in combination with other
monoclonal or chimeric antibodies, or with lymphokines or
hematopoietic growth factors (such as, e.g., IL-2, IL-3, IL-4,
IL-7, IL-9, IL-10, IL12, and interferon .alpha., .beta., and
.gamma.), which, for example, serve to increase the immune
response. The liquid formulations of the present invention may also
be advantageously utilized in combination with one or more drugs
used to treat RSV infection such as, for example anti-viral agents.
Further, the liquid formulations of the present invention may be
used in combination with one or more of the following drugs:
NIH-351 (Gemini Technologies), recombinant RSV vaccine (MedImmune
Vaccines, Inc. U.S. Application Nos. 60/358,934 filed Feb. 21,
2002, 10/373,567 filed Feb. 21, 2003, 10/371,099 filed Feb. 21,
2003, 10/371,122 filed Feb. 21, 2003, 10/371,264 filed Feb. 21,
2003, 60/466,181 filed Apr. 25, 2003 and 60/465,811 filed Apr. 25,
2003, all of which are incorporated herein by reference), RSVf-2
(Intracel), F-50042 (Pierre Fabre), T-786 (Trimeris), VP-36676
(ViroPharma), RFI-641 (American Home Products), VP-14637
(ViroPharma), PFP-1 and PFP-2 (American Home Products), RSV vaccine
(Avant Immunotherapeutics), and F-50077 (Pierre Fabre).
[0198] 5.5.1 Immunomodulatory Agents
[0199] Any immunomodulatory agent well-known to one of skilled in
the art may be used in the methods and compositions of the
invention. Immunomodulatory agents can affect one or more or all
aspects of the immune response in a subject. Aspects of the immune
response include, but are not limited to, the inflammatory
response, the complement cascade, leukocyte and lymphocyte
differentiation, proliferation, and/or effector function, monocyte
and/or basophil counts, and the cellular communication among cells
of the immune system. In certain embodiments of the invention, an
immunomodulatory agent modulates one aspect of the immune response.
In other embodiments, an immunomodulatory agent modulates more than
one aspect of the immune response. In a preferred embodiment of the
invention, the administration of an immunomodulatory agent to a
subject inhibits or reduces one or more aspects of the subject's
immune response capabilities. In an alternative embodiment of the
invention, the immunomodulatory agent enhances one or more aspects
of a subject's immune response. In certain embodiments, an
immunomodulatory agent is not an anti-inflammatory agent. In other
embodiments, an immunomodulatory agent is an agent other than a
chemotherapeutic agent.
[0200] Examples of immunomodulatory agents include, but are not
limited to, proteinaceous agents such as cytokines, peptide
mimetics, and antibodies (e.g., human, humanized, chimeric,
monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab).sub.2 fragments or
epitope binding fragments), nucleic acid molecules (e.g., antisense
nucleic acid molecules and triple helices), small molecules,
organic compounds, and inorganic compounds. In particular,
immunomodulatory agents include, but are not limited to,
methotrexate, leflunomide, cyclophosphamide, cytoxan, Immuran,
cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506
(tacrolimus)), methylprednisolone (MP), corticosteroids, steroids,
mycophenolate mofetil, rapamycin (sirolimus), mizoribine,
deoxyspergualin, brequinar, malononitriloamindes (e.g.,
leflunamide), T cell receptor modulators, cytokine receptor
modulators, and modulators mast cell modulators.
[0201] Examples of T cell receptor modulators include, but are not
limited to, anti-T cell receptor antibodies (e.g., anti-CD4
antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1.RTM. (IDEC and
SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)),
anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3
(Johnson & Johnson), or Rituxan (IDEC)), anti-CD5 antibodies
(e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7
antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies,
anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)),
anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies
(e.g., MEDI-507 (MedImmune, Inc., International Publication Nos. WO
02/098370 and WO 02/069904), anti-CD11a antibodies (e.g., Xanelim
(Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC))),
CTLA4-immunoglobulin, and LFA-3TIP (Biogen, International
Publication No. WO 93/08656 and U.S. Pat. No. 6,162,432).
[0202] Examples of cytokine receptor modulators include, but are
not limited to, soluble cytokine receptors (e.g., the extracellular
domain of a TNF-.alpha. receptor or a fragment thereof, the
extracellular domain of an IL-1.beta. receptor or a fragment
thereof, and the extracellular domain of an IL-6 receptor or a
fragment thereof), cytokines or fragments thereof (e.g.,
interleukin IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,
IL-11, IL-12, IL-13, IL-15, IL-23, TNF-.alpha., TNF-.beta.,
interferon (IFN)-.alpha., IFN-.beta., IFN-.gamma., and GM-CSF),
anti-cytokine receptor antibodies (e.g., anti-IFN receptor
antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein
Design Labs)), anti-IL-3 receptor antibodies, anti-IL-4 receptor
antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptor
antibodies, anti-IL-12 receptor antibodies, anti-IL-13 receptor
antibodies, anti-IL-15 receptor antibodies, and anti-IL-23 receptor
antibodies), anti-cytokine antibodies (e.g., anti-IFN antibodies,
anti-TNF-.alpha. antibodies, anti-IL-1.beta. antibodies, anti-IL-3
antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g.,
ABX-IL-8 (Abgenix)), anti-IL-12 antibodies, anti-IL-13 antibodies,
anti-IL-15 antibodies, and anti-IL-23 antibodies).
[0203] In a specific embodiment, a cytokine receptor modulator is
IL-3, IL4, IL-10, or a fragment thereof. In another embodiment, a
cytokine receptor modulator is an anti-IL-.beta. antibody,
anti-IL-6 antibody, anti-IL-12 receptor antibody, or
anti-TNF-.alpha. antibody. In another embodiment, a cytokine
receptor modulator is the extracellular domain of a TNF-.alpha.
receptor or a fragment thereof. In certain embodiments, a cytokine
receptor modulator is not a TNF-.alpha. antagonist.
[0204] In one embodiment, a cytokine receptor modulator is a mast
cell modulator. In an alternative embodiment, a cytokine receptor
modulator is not a mast cell modulator. Examples of mast cell
modulators include, but are not limited to stem cell factor (c-kit
receptor ligand) inhibitor (e.g., mAb 7H6, mAb 8H7a, pAb 1337,
FK506, CsA, dexamthasone, and fluconcinonide), c-kit receptor
inhibitor (e.g., STI 571, (formerly known as CGP 57148B)), mast
cell protease inhibitor (e.g., GW-45, GW-58, wortmannin, LY 294002,
calphostin C, cytochalasin D, genistein, KT5926, staurosproine, and
lactoferrin), relaxin ("RLX"), IgE antagonist (e.g., antibodies
rhuMAb-E25 omalizumab, HMK-12 and 6HD5, and mAB Hu-901), IL-3
antagonist, IL-4 antagonists, IL-10 antagonists, and TGF-beta.
[0205] An immunomodulatory agent may be selected to interfere with
the interactions between the T helper subsets (TH1 or TH2) and B
cells to inhibit neutralizing antibody formation. Antibodies that
interfere with or block the interactions necessary for the
activation of B cells by TH (T helper) cells, and thus block the
production of neutralizing antibodies, are useful as
immunomodulatory agents in the methods of the invention. For
example, B cell activation by T cells requires certain interactions
to occur (Durie et al., Immunol. Today, 15(9):406-410 (1994)), such
as the binding of CD40 ligand on the T helper cell to the CD40
antigen on the B cell, and the binding of the CD28 and/or CTLA4
ligands on the T cell to the B7 antigen on the B cell. Without both
interactions, the B cell cannot be activated to induce production
of the neutralizing antibody.
[0206] The CD40 ligand (CD40L)-CD40 interaction is a desirable
point to block the immune response because of its broad activity in
both T helper cell activation and function as well as the absence
of redundancy in its signaling pathway. Thus, in a specific
embodiment of the invention, the interaction of CD40L with CD40 is
transiently blocked at the time of administration of one or more of
the immunomodulatory agents. This can be accomplished by treating
with an agent which blocks the CD40 ligand on the TH cell and
interferes with the normal binding of CD40 ligand on the T helper
cell with the CD40 antigen on the B cell. An antibody to CD40
ligand (anti-CD40L) (available from Bristol-Myers Squibb Co; see,
e.g., European patent application 555,880, published Aug. 18, 1993)
or a soluble CD40 molecule can be selected and used as an
immunomodulatory agent in accordance with the methods of the
invention.
[0207] An immunomodulatory agent may be selected to inhibit the
interaction between TH1 cells and cytotoxic T lymphocytes ("CTLs")
to reduce the occurrence of CTL-mediated killing. An
immunomodulatory agent may be selected to alter (e.g., inhibit or
suppress) the proliferation, differentiation, activity and/or
function of the CD4.sup.+ and/or CD8.sup.+ T cells. For example,
antibodies specific for T cells can be used as immunomodulatory
agents to deplete, or alter the proliferation, differentiation,
activity and/or function of CD4.sup.+ and/or CD8.sup.+ T cells.
[0208] In one embodiment of the invention, an immunomodulatory
agent that reduces or depletes T cells, preferably memory T cells,
is administered to a subject with a RSV infection or a respiratory
condition associated with, potentiated by or potentiating a RSV
infection in accordance with the methods of the invention. See,
e.g., U.S. Pat. No. 4,658,019. In another embodiment of the
invention, an immunomodulatory agent that inactivates CD8.sup.+ T
cells is administered to a subject with a RSV infection or a
respiratory condition associated with, potentiated by or
potentiating a RSV infection in accordance with the methods of the
invention. In a specific embodiment, anti-CD8 antibodies are used
to reduce or deplete CD8.sup.+ T cells.
[0209] In another embodiment, an immunomodulatory agent which
reduces or inhibits one or more biological activities (e.g., the
differentiation, proliferation, and/or effector functions) of TH0,
TH1, and/or TH2 subsets of CD4.sup.+ T helper cells is administered
to a subject with a RSV infection or a respiratory condition
associated with, potentiated by or potentiating a RSV infection in
accordance with the methods of the invention. One example of such
an immunomodulatory agent is IL-4. IL-4 enhances antigen-specific
activity of TH2 cells at the expense of the TH1 cell function (see,
e.g., Yokota et al, 1986 Proc. Natl. Acad. Sci., USA, 83:5894-5898;
and U.S. Pat. No. 5,017,691). Other examples of immunomodulatory
agents that affect the biological activity (e.g., proliferation,
differentiation, and/or effector functions) of T-helper cells (in
particular, TH1 and/or TH2 cells) include, but are not limited to,
IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-23, and
interferon (IFN)-.gamma..
[0210] In another embodiment, an immunomodulatory agent
administered to a subject with a RSV infection or a respiratory
condition associated with, potentiated by or potentiating a RSV
infection in accordance with the methods of the invention is a
cytokine that prevents antigen presentation. In a specific
embodiment, an immunomodulatory agent used in the methods of the
invention is IL-10. IL-10 also reduces or inhibits macrophage
action which involves bacterial elimination.
[0211] An immunomodulatory agent may be selected to reduce or
inhibit the activation, degranulation, proliferation, and/or
infiltration of mast cells. In certain embodiments, the
immunomodulatory agent interferes with the interactions between
mast cells and mast cell activating agents, including, but not
limited to stem cell factors (c-kit ligands), IgE, IL-4,
environmental irritants, and infectious agents. In a specific
embodiment, the immunomodulatory agent reduces or inhibits the
response of mast cells to environmental irritants such as, but not
limited to, pollen, dust mites, tobacco smoke, and/or pet dander.
In another specific embodiment, the immunomodulatory agent reduces
or inhibits the response of mast cells to infectious agents, such
as viruses, bacteria, and fingi. Examples of mast cell modulators
that reduce or inhibit the activation, degranulation,
proliferation, and/or infiltration of mast cells include, but are
not limited to, stem cell factor (c-kit receptor ligand) inhibitors
(e.g., mAb 7H6, mAb 8H7a, and pAb 1337 (see Mendiaz et al., 1996,
Eur J Biochem 293(3):842-849), FK506 and CsA (Ito et al., 1999 Arch
Dermatol Res 291(5):275-283), dexamthasone and fluconcinonide (see
Finooto et al. J Clin Invest 1997 99(7): 1721-1728)), c-kit
receptor inhibitors (e.g., STI 571 (formerly known as CGP 57148B)
(see Heinrich et al., 2000 Blood 96(3):925-932)), mast cell
protease inhibitors (e.g., GW-45 and GW-58 (see Temkin et al., 2002
J Immunol 169(5):2662-2669), wortmannin, LY 294002, calphostin C,
and cytochalasin D (see Vosseller et al., 1997, Mol Biol Cell
1997:909-922), genistein, KT5926, and staurosproine (see Nagai et
al. 1995, Biochem Biophys Res Commun 208(2):576-581), and
lactoferrin (see He et al., 2003 Biochem Pharmacol 65(6):
1007-1015)), relaxin ("RLX") (see Bani et al., 2002 Int
Immunopharmacol 2(8):1195-1294),), IgE antagonists (e.g.,
antibodies rhuMAb-E25 omalizumab (see Finn et al., 2003 J Allergy
Clin Immuno 111(2):278-284; Corren et al., 2003 J Allergy Clin
immuno 111(1):87-90; Busse and Neaville, 2001 Curr Opin Allergy
Clin Immuno 1(1): 105-108; and Tang and Powell, 2001, Eur J Pediatr
160(12): 696-704), HMK-12 and 6HD5 (see Miyajima et al., 2202 Int
Arch Allergy Immuno 128(1):24-32), and mAB Hu-901 (see van Neerven
et al., 2001 Int Arch Allergy Immuno 124(1-3):400), IL-3
antagonist, IL-4 antagonists, IL-10 antagonists, and TGF-beta (see
Metcalfe et al., 1995, Exp Dermatol 4(4 Pt 2):227-230).
[0212] In a preferred embodiment, proteins, polypeptides or
peptides (including antibodies) that are utilized as
immunomodulatory agents are derived from the same species as the
recipient of the proteins, polypeptides or peptides so as to reduce
the likelihood of an immune response to those proteins,
polypeptides or peptides. In another preferred embodiment, when the
subject is a human, the proteins, polypeptides, or peptides that
are utilized as immunomodulatory agents are human or humanized.
[0213] In accordance with the invention, one or more
immunomodulatory agents are administered to a subject with a RSV
infection or a respiratory condition associated with, potentiated
by or potentiating a RSV infection prior to, subsequent to, or
concomitantly with a liquid formulation of an antibody or a
fragment thereof that immunospecifically bind to a RSV antigen.
Preferably, one or more immunomodulatory agents are administered in
combination with a liquid formulation of an antibody or a fragment
thereof that immunospecifically bind to a RSV antigen to a subject
with a RSV infection or a respiratory condition associated with,
potentiated by or potentiating a RSV infection to reduce or inhibit
one or more aspects of the immune response as deemed necessary by
one of skilled in the art. Any technique well-known to one skilled
in the art can be used to measure one or more aspects of the immune
response in a particular subject, and thereby determine when it is
necessary to administer an immunomodulatory agent to said subject.
In a preferred embodiment, a mean absolute lymphocyte count of
approximately 500 cells/mm.sup.3, preferably 600 cells/mm.sup.3,
650 cells/mm.sup.3, 700 cells/mm.sup.3, 750 cells/mm.sup.3, 800
cells/mm.sup.3, 900 cells/mm.sup.3, 1000 cells/mm.sup.3, 1100
cells/mm.sup.3, or 1200 cells/mm.sup.3 is maintained in a subject.
In another preferred embodiment, a subject with a respiratory
condition associated with, potentiated by or potentiating a RSV
infection is not administered an immunomodulatory agent if their
absolute lymphocyte count is 500 cells/mm.sup.3 or less, 550
cells/mm.sup.3 or less, 600 cells/mm.sup.3 or less, 650
cells/mm.sup.3 or less, 700 cells/mm.sup.3 or less, 750
cells/mm.sup.3 or less, or 800 cells/mm.sup.3 or less.
[0214] In a specific embodiment, one or more immunomodulatory
agents are administered in combination with a liquid formulation of
an antibody or a fragment thereof that immunospecifically bind to a
RSV antigen to a subject with a RSV infection or a respiratory
condition associated with, potentiated by or potentiating a RSV
infection so as to transiently reduce or inhibit one or more
aspects of the immune response. Such a transient inhibition or
reduction of one or more aspects of the immune system can last for
hours, days, weeks, or months. Preferably, the transient inhibition
or reduction in one or more aspects of the immune response lasts
for a few hours (e.g., 2 hours, 4 hours, 6 hours, 8 hours, 12
hours, 14 hours, 16 hours, 18 hours, 24 hours, 36 hours, or 48
hours), a few days (e.g., 3 days, 4 days, 5 days, 6 days, 7 days,
or 14 days), or a few weeks (e.g., 3 weeks, 4 weeks, 5 weeks or 6
weeks). The transient reduction or inhibition of one or more
aspects of the immune response enhances the prophylactic and/or
therapeutic effect(s) of a liquid formulation of SYNAGIS.RTM. or an
antigenic fragment thereof.
[0215] Nucleic acid molecules encoding proteins, polypeptides, or
peptides with immunomodulatory activity or proteins, polypeptides,
or peptides with immunomodulatory activity can be administered to a
subject with a RSV infection or a respiratory condition associated
with, potentiated by or potentiating a RSV infection in accordance
with the methods of the invention. Further, nucleic acid molecules
encoding derivatives, analogues, or fragments of proteins,
polypeptides, or peptides with immunomodulatory activity, or
derivatives, analogues, or fragments of proteins, polypeptides, or
peptides with immunomodulatory activity can be administered to a
subject with a RSV infection or a respiratory condition associated
with, potentiated by or potentiating a RSV infection in accordance
with the methods of the invention. Preferably, such derivatives,
analogues, and fragments retain the immunomodulatory activity of
the full-length, wild-type protein, polypeptide, or peptide.
[0216] Preferably, agents that are commercially available and known
to function as immunomodulatory agents are used in the methods of
the invention. The immunomodulatory activity of an agent can be
determined in vitro and/or in vivo by any technique well-known to
one skilled in the art, including, e.g., by CTL assays,
proliferation assays, and immunoassays (e.g. ELISAs) for the
expression of particular proteins such as co-stimulatory molecules
and cytokines.
[0217] 5.1.1.1. Interleukin-9 Antagonists
[0218] In a specific embodiment, a liquid formulation of the
invention is used in combination with a interleukin-9 (IL-9)
antagonists to prevent or treat a RSV infection or a respiratory
condition that associated with, potentiated by or potentiates a RSV
infection. The terms "IL-9 antagonist" or "IL-9 antagonists" as
used herein, refer to any agent that blocks, inhibits, reduces, or
neutralizes the function, activity and/or expression of an IL-9
protein, polypeptide or peptide. An IL-9 antagonist may inhibit a
pathologic cellular or humoral phenotype associated with or
resulting from IL-9 expression and/or activity (e.g., decreased
secretion of mucin, the differentiation of IL-9 expressing cells
into a mucin-secreting cell, the secretion of inflammatory agents,
the proliferation, migration, and increase in volume of cells
(e.g., immune and smooth muscle cells), the secretion of
extracellular matrix molecules or matrix metalloproteinases and/or
the binding of IL-9 to the IL-9 receptor ("IL-9R")). IL-9
antagonists are disclosed in U.S. Application Nos. 60/462,307 filed
Apr. 11, 2003, and 60/462,259 filed Apr. 11, 2003, both of which
are incorporated herein by reference.
[0219] IL-9 antagonists include, but are not limited to,
proteinaceous agents (e.g., proteins, polypeptides, peptides,
fusion proteins, antibodies, and antibody fragments), nucleic acid
molecules (e.g., IL-9 antisense nucleic acid molecules, triple
helices, RNAi, or nucleic acid molecules encoding proteinaceous
agents), organic molecules, inorganic molecules, small organic
molecules, drugs, and small inorganic molecules that block,
inhibit, reduce or neutralize a function, an activity and/or the
expression of an IL-9 polypeptide, the function, an activity,
and/or expression of the IL-9R or a subunit thereof, and/or the
binding of an IL-9 polypeptide to the IL-9R or a subunit thereof.
In various embodiments, an IL-9 antagonist reduces the function,
activity, and/or expression of another molecule other than an IL-9
polypeptide or the IL-9R or a subunit thereof. In other
embodiments, an IL-9 antagonist reduces the function, activity,
and/or expression of an IL-9 polypeptide, the function, activity,
and/or expression of the IL-9R or a subunit thereof, and/or the
binding of an IL-9 polypeptide to the IL-9R or a subunit thereof.
In particular embodiments, an IL-9 antagonist reduces the function,
activity and/or expression of an IL-9 polypeptide, the function,
activity, and/or expression of the IL-9R or a subunit thereof,
and/or the binding of an IL-9 polypeptide to the IL-9R or a subunit
thereof by at least 10%, at least 15%, at least 20%, at least 25%,
at least 30%, at least 35%, at least 40%, at least 45%, at least
50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%
or at least 99% relative to a control such as PBS.
[0220] In a preferred embodiment, a liquid formulation of the
invention is administered in combination with a IL-9 antagonist to
a subject in need thereof to prevent, treat, manage or ameliorating
wheezing associated with a RSV infection. In certain cases,
wheezing precedes the onset or development of a RSV infection. In a
specific embodiment, the invention provides methods of preventing,
treating, ameliorating, or managing wheezing associated with a RSV
infection, said methods comprising administering to a subject in
need thereof an effective amount of one or more IL-9 antagonists in
combination with a liquid formulation of the invention. In other
embodiments, the invention provides methods of preventing the onset
and/or development of asthma (which may be associated with,
potentiated by or potentiates a RSV infection) in subjects with
wheezing, said method comprising administering to said subject an
effective amount of one or more IL-9 antagonists in combination
with an effective amount of a liquid formulation of the
invention.
[0221] 5.5.2 Anti-Inflammatory Agents
[0222] Any anti-inflammatory agent, including agents useful in
therapies for inflammatory disorders, well-known to one of skilled
in the art can be used in the compositions and methods of the
invention. Non-limiting examples of anti-inflammatory agents
include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal
anti-inflammatory drugs, anticholinergics (e.g., atropine sulfate,
atropine methylnitrate, and ipratropium bromide (ATROVENT.TM.)),
beta2-agonists (e.g., albuterol (VENTOLIN.TM. and PROVENTIL.TM.),
bitolterol (TORNALATE.TM.), levalbuterol (XOPONEX.TM.),
metaproterenol (ALUPENT.TM.), pirbuterol (MAXAIR.TM.), terbutlaine
(BRETHAIRE.TM. and BRETHINE.TM.), albuterol (PROVENTIL.TM.,
REPETABS.TM., and VOLMAX.TM.), formoterol (FORADIL AEROLIZER.TM.),
and salmeterol (SEREVENT.TM. and SEREVENT DISKUS.TM.)), and
methylxanthines (e.g., theophylline (UNIPHYL.TM., THEO-DUR.TM.,
SLO-BID.TM., AND TEHO-42.TM.)). Examples of NSAIDs include, but are
not limited to, aspirin, ibuprofen, celecoxib (CELEBREX.TM.),
diclofenac (VOLTAREN.TM.), etodolac (LODINE.TM.), fenoprofen
(NALFON.TM.), indomethacin (INDOCIN.TM.), ketoralac (TORADOL.TM.),
oxaprozin (DAYPRO.TM.), nabumetone (RELAFEN.TM.), sulindac
(CLINORIL.TM.), tolmetin (TOLECTYN.TM.), rofecoxib (VIOXX.TM.),
naproxen (ALEVE.TM., NAPROSYN.TM.), ketoprofen (ACTRON.TM.) and
nabumetone (RELAFEN.TM.). Such NSAIDs function by inhibiting a
cyclooxgenase enzyme (e.g., COX-1 and/or COX-2). Examples of
steroidal anti-inflammatory drugs include, but are not limited to,
glucocorticoids, dexamethasone (DECADRON.TM.), corticosteroids
(e.g., methylprednisolone (MEDROL.TM.)), cortisone, hydrocortisone,
prednisone (PREDNISONE.TM. and DELTASONE.TM.), prednisolone
(PRELONE.TM. and PEDIAPRED.TM.), triamcinolone, azulfidine, and
inhibitors of eicosanoids (e.g., prostaglandins, thromboxanes, and
leukotrienes (see Table 2, infra, for non-limiting examples of
leukotriene and typical dosages of such agents)).
[0223] In certain embodiments, the anti-inflammatory agent is an
agent useful in the prevention, management, treatment, and/or
amelioration of asthma or one or more symptoms thereof.
Non-limiting examples of such agents include adrenergic stimulants
(e.g., catecholamines (e.g., epinephrine, isoproterenol, and
isoetharine), resorcinols (e.g., metaproterenol, terbutaline, and
fenoterol), and saligenins (e.g. salbutamol)), adrenocorticoids,
blucocorticoids, corticosteroids (e.g., beclomethadonse,
budesonide, flunisolide, fluticasone, triamcinolone,
methylprednisolone, prednisolone, and prednisone), other steroids,
beta2-agonists (e.g., albuterol, bitolterol, fenoterol,
isoetharine, metaproterenol, pirbuterol, salbutamol, terbutaline,
formoterol, salmeterol, and albutamol terbutaline),
anti-cholinergics (e.g., ipratropium bromide and oxitropium
bromide), IL-4 antagonists (including antibodies), IL-5 antagonists
(including antibodies), IL-13 antagonists (including antibodies),
PDE4-inhibitor, NF-Kappa-.beta. inhibitor, VLA-4 inhibitor, CpG,
anti-CD23, selectin antagonists (TBC 1269), mast cell protease
inhibitors (e.g., tryptase kinase inhibitors (e.g., GW-45, GW-58,
and genisteine), phosphatidylinositide-3' (PI3)-kinase inhibitors
(e.g., calphostin C), and other kinase inhibitors (e.g.,
staurosporine) (see Temkin et al., 2002 J Immunol 169(5):2662-2669;
Vosseller et al., 1997 Mol. Biol. Cell 8(5):909-922; and Nagai et
al., 1995 Biochem Biophys Res Commun 208(2):576-581)), a C3
receptor antagonists (including antibodies), immunosuppressant
agents (e.g., methotrexate and gold salts), mast cell modulator
(e.g., cromolyn sodium (INTAL.TM.) and nedocromil sodium
(TILADE.TM.)), and mucolytic agents (e.g., acetylcysteine)). In a
specific embodiment, the anti-inflammatory agent is a leukotriene
inhibitor (e.g., montelukast (SINGULAIR.TM.), zafirlukast
(ACCOLATE.TM.), pranlukast (ONON.TM.), or zileuton (ZYFLO.TM.).
[0224] In certain embodiments, the anti-inflammatory agent is an
agent useful in preventing, treating, managing, or ameliorating
allergies or one or more symptoms thereof. Non-limiting examples of
such agents include antimmediator drugs (e.g., antihistamine, see
Table 4 for non-limiting examples of antihistamine and typical
dosages of such agents), corticosteroids, decongestants,
sympathomimetic drugs (e.g., .alpha.-adrenergic and
.beta.-adrenergic drugs), TNX901 (Leung et al., 2003, N Engl J Med
348(11):986-993), IgE antagonists (e.g., antibodies rhuMAb-E25
omalizumab (see Finn et al., 2003 J Allergy Clin Immuno
111(2):278-284; Corren et al., 2003 J Allergy Clin Immuno
111(1):87-90; Busse and Neaville, 2001 Curr Opin Allergy Clin
Immuno 1(1): 105-108; and Tang and Powell, 2001, Eur J Pediatr
160(12): 696-704), HMK-12 and 6HD5 (see Miyajima et al., 2202 Int
Arch Allergy Immuno 128(1):24-32), and mAB Hu-901 (see van Neerven
et al., 2001 Int Arch Allergy Immuno 124(1-3):400), theophylline
and its derivatives, glucocorticoids, and immunotherapies (e.g.,
repeated long-term injection of allergen, short course
desensitization, and venom immunotherapy).
[0225] Anti-inflammatory therapies and their dosages, routes of
administration, and recommended usage are known in the art and have
been described in such literature as the Physician's Desk Reference
(57th ed., 2003) and The Merk Manual (17th ed., 1999).
[0226] 5.5.3 Anti-Viral Agents
[0227] Any anti-viral agent well-known to one of skilled in the art
for the treatment, prevention, management, or amelioration of a RSV
infection or a respiratory condition associated with, potentiated
by or potentiating a RSV infection can be used in the compositions
and methods of the invention. Non-limiting examples of anti-viral
agents include proteins, polypeptides, peptides, fusion proteins
antibodies, nucleic acid molecules, organic molecules, inorganic
molecules, and small molecules that inhibit and/or reduce the
attachment of a virus to its receptor, the internalization of a
virus into a cell, the replication of a virus, or release of virus
from a cell. In particular, anti-viral agents include, but are not
limited to, nucleoside analogues (e.g., zidovudine, acyclovir,
gangcyclovir, vidarabine, idoxuridine, trifluridine, and
ribavirin), foscarnet, amantadine, rimantadine, saquinavir,
indinavir, ritonavir, alpha-interferons and other interferons, and
AZT.
[0228] In a specific embodiment, the anti-viral agent is an
antibody that is immunospecific for a non-RSV viral antigen. As
used herein, the term "viral antigen" includes, but is not limited
to, any viral peptide, polypeptide and protein (e.g., influenza
virus neuraminidase, influenza virus hemagglutinin, and herpes
simplex virus glycoprotein (e.g., gB, gC, gD, and gE)) that is
capable of eliciting an immune response. Antibodies useful in this
invention for prevention, management, treatment, and/or
amelioration of a non-RSV viral infectious disease that may
potentiate or potentiated by a RSV infection include, but are not
limited to, antibodies against antigens of pathogenic viruses,
including as examples and not by limitation: adenovirdiae (e.g.,
mastadenovirus and aviadenovirus), herpesviridae (e.g., herpes
simplex virus 1, herpes simplex virus 2, herpes simplex virus 5,
and herpes simplex virus 6), leviviridae (e.g., levivirus,
enterobacteria phase MS2, allolevirus), poxyiridae (e.g.,
chordopoxyirinae, parapoxvirus, avipoxvirus, capripoxvirus,
leporiipoxvirus, suipoxvirus, molluscipoxvirus, and
entomopoxyirinae), papovaviridae (e.g., polyomavirus and
papillomavirus), paramyxoviridae (e.g., paramyxovirus,
parainfluenza virus 1, mobillivirus (e.g., measles virus),
rubulavirus (e.g., mumps virus), pneumonovirinae (e.g.,
pneumovirus, human respiratory syncytial virus), and
metapneumovirus (e.g., avian pneumovirus and human
metapneumovirus)), picornaviridae (e.g., enterovirus, rhinovirus,
hepatovirus (e.g., human hepatits A virus), cardiovirus, and
apthovirus), reoviridae (e.g., orthoreovirus, orbivirus, rotavirus,
cypovirus, fijivirus, phytoreovirus, and oryzavirus), retroviridae
(e.g., mammalian type B retroviruses, mammalian type C
retroviruses, avian type C retroviruses, type D retrovirus group,
BLV-HTLV retroviruses, lentivirus (e.g. human immunodeficiency
virus 1 and human immunodeficiency virus 2), spumavirus),
flaviviridae (e.g., hepatitis C virus), hepadnaviridae (e.g.,
hepatitis B virus), togaviridae (e.g., alphavirus (e.g., sindbis
virus) and rubivirus (e.g., rubella virus)), rhabdoviridae (e.g.,
vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus, and
necleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocytic
choriomeningitis virus, Ippy virus, and lassa virus), and
coronaviridae (e.g., coronavirus and torovirus).
[0229] A specific example of antibodies available useful for the
prevention, management, treatment, and/or amelioration of a viral
infectious disease include, but are not limited to, PRO542
(Progenics), which is a CD4 fusion antibody useful for the
treatment of HIV infection.
[0230] In a specific embodiment, the anti-viral agent used in the
compositions and methods of the invention inhibits or reduces a
pulmonary or respiratory virus infection, inhibits or reduces the
replication of a virus that causes a pulmonary or respiratory
infection, or inhibits or reduces the spread of a virus that causes
a pulmonary or respiratory infection to other cells or subjects. In
another specific embodiment, the anti-viral agent used in the
compositions and methods of the invention inhibits or reduces
infection, inhibits or reduces the replication, or inhibits or
reduces the spread to other cells or subjects by RSV, or another
virus of which an infection can be potentiated by or potentiating a
RSV infection, e.g., hMPV, or PIV. Examples of such agents include,
but are not limited to, nucleoside analogues, such as zidovudine,
acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and
ribavirin, as well as foscarnet, amantadine, rimantadine,
saquinavir, indinavir, ritonavir, and the alpha-interferons. See
U.S. Provisional Patent Application 60/398,475 filed Jul. 25, 2002,
entitled "Methods of Treating and Preventing RSV, HMPV, and PIV
Using Anti-RSV, Anti-HMPV, and Anti-PIV Antibodies" and U.S. patent
application Ser. No. 10/371,122 filed Feb. 21, 2003, which are
incorporated herein by reference in its entirety.
[0231] Anti-viral therapies and their dosages, routes of
administration and recommended usage are known in the art and have
been described in such literature as the Physician's Desk Reference
(57th ed., 2003). Additional information on respiratory viral
infections is available in Cecil Textbook of Medicine (18th ed.,
1988).
[0232] 5.5.4 Anti-Bacterial Agents
[0233] Anti-bacterial agents and therapies well-known to one of
skilled in the art for the prevention, treatment, management, or
amelioration of a respiratory condition associated with,
potentiated by or potentiating a RSV infection (e.g., a bacterial
respiratory infection) can be used in the compositions and methods
of the invention. Non-limiting examples of anti-bacterial agents
include proteins, polypeptides, peptides, fusion proteins,
antibodies, nucleic acid molecules, organic molecules, inorganic
molecules, and small molecules that inhibit and/or reduce a
bacterial infection, inhibit and/or reduce the replication of
bacteria, or inhibit and/or reduce the spread of bacteria to other
cells or subjects. Specific examples of anti-bacterial agents
include, but are not limited to, antibiotics such as penicillin,
cephalosporin, imipenem, axtreonam, vancomycin, cycloserine,
bacitracin, chloramphenicol, erythromycin, clindamycin,
tetracycline, streptomycin, tobramycin, gentamicin, amikacin,
kanamycin, neomycin, spectinomycin, trimethoprim, norfloxacin,
rifampin, polymyxin, amphotericin B, nystatin, ketocanazole,
isoniazid, metronidazole, and pentamidine.
[0234] In certain embodiments, the anti-bacterial agent is an agent
that inhibits or reduces a pulmonary or respiratory bacterial
infection, inhibits or reduces the replication of a bacteria that
causes a pulmonary or respiratory infection, or inhibits or reduces
the spread of a bacteria that causes a pulmonary or respiratory
infection to other cells or subjects. In cases in which the
pulmonary or respiratory bacterial infection is a mycoplasma
infection (e.g., pharyngitis, tracheobronchitis, and pneumonia),
the anti-bacterial agent is preferably a tetracycline,
erythromycin, or spectinomycin. In cases in which the pulmonary or
respiratory bacterial infection is tuberculosis, the anti-bacterial
agent is preferably rifampcin, isonaizid, pyranzinamide,
ethambutol, and streptomycin. In cases in which the pulmonary or
respiratory bacterial infection is pneumonia caused by an aerobic
gram negative bacilli (GNB), the anti-bacterial agent is preferably
penicillin, first, second, or third generation cephalosporin (e.g.,
cefaclor, cefadroxil, cephalexin, or cephazolin), erythomycin,
clindamycin, an aminoglycoside (e.g., gentamicin, tobramycin, or
amikacine), or a monolactam (e.g., aztreonam). In cases in which
the respiratory infection is recurrent aspiration pneumonia, the
anti-bacterial agent is preferably penicillin, an aminoglycoside,
or a second or third generation cephalosporin.
[0235] Anti-bacterial therapies and their dosages, routes of
administration and recommended usage are known in the art and have
been described in such literature as the Physician's Desk Reference
(57th ed., 2003), Cecil Textbook of Medicine (18th ed., 1988), and
The Merk Manual of Diagnosis and Therapy (17th ed. 1999).
[0236] 5.5.5 Anti-Fungal Agents
[0237] Anti-fungal agents and therapies well known to one of
skilled in the art for prevention, management, treatment, and/or
amelioration of a respiratory condition associated with,
potentiated by or potentiating a RSV infection (e.g., a fungal
respiratory infection) can be used in the compositions and methods
of the invention. Non-limiting examples of anti-fungal agents
include proteins, polypeptides, peptides, fusion proteins,
antibodies, nucleic acid molecules, organic molecules, inorganic
molecules, and small molecules that inhibit and/or reduce fungal
infection, inhibit and/or reduce the replication of fungi, or
inhibit and/or reduce the spread of fungi to other subjects.
Specific examples of anti-fungal agents include, but are not
limited to, azole drugs (e.g., miconazole, ketoconazole
(NIZORAL.RTM.), caspofungin acetate (CANCIDAS.RTM.), imidazole,
triazoles (e.g., fluconazole (DIFLUCAN.RTM.)), and itraconazole
(SPORANOX.RTM.)), polyene (e.g., nystatin, amphotericin B
(FUNGIZONE.RTM.), amphotericin B lipid complex
("ABLC")(ABELCET.RTM.), amphotericin B colloidal dispersion
("ABCD")(AMPHOTEC.RTM.), liposomal amphotericin B (AMBISONE.RTM.)),
potassium iodide (KI), pyrimidine (e.g., flucytosine
(ANCOBON.RTM.)), and voriconazole (VFEND.RTM.).
[0238] In certain embodiments, the anti-fungal agent is an agent
that inhibits or reduces a respiratory fungal infection, inhibits
or reduces the replication of a fungus that causes a pulmonary or
respiratory infection, or inhibits or reduces the spread of a
fungus that causes a pulmonary or respiratory infection to other
subjects. In cases in which the pulmonary or respiratory fungal
infection is Blastomyces dermatitidis, the anti-fungal agent is
preferably itraconazole, amphotericin B, fluconazole, or
ketoconazole. In cases in which the pulmonary or respiratory fungal
infection is pulmonary aspergilloma, the anti-fungal agent is
preferably amphotericin B, liposomal amphotericin B, itraconazole,
or fluconazole. In cases in which the pulmonary or respiratory
fungal infection is histoplasmosis, the anti-fungal agent is
preferably amphotericin B, itraconazole, fluconazole, or
ketoconazole. In cases in which the pulmonary or respiratory fungal
infection is coccidioidomycosis, the anti-fungal agent is
preferably fluconazole or amphotericin B. In cases in which the
pulmonary or respiratory fungal infection is cryptococcosis, the
anti-fungal agent is preferably amphotericin B, fluconazole, or
combination of the two agents. In cases in which the pulmonary or
respiratory fungal infection is chromomycosis, the anti-fungal
agent is preferably itraconazole, fluconazole, or flucytosine. In
cases in which the pulmonary or respiratory fungal infection is
mucomycosis, the anti-fungal agent is preferably amphotericin B or
liposomal amphotericin B. In cases in which the pulmonary or
respiratory fungal infection is pseudoallescheriasis, the
anti-fungal agent is preferably itraconazole ore miconazole.
[0239] Anti-fungal therapies and their dosages, routes of
administration, and recommended usage are known in the art and have
been described in such literature as Dodds et al., 2000
Pharmacotherapy 20(11) 1335-1355, the Physician's Desk Reference
(57th ed., 2003) and the Merk Manual of Diagnosis and Therapy (17th
ed., 1999).
[0240] 5.6 Methods of Administering the Antibody Formulations
[0241] The invention provides methods of treatment, prophylaxis,
and amelioration of a RSV infection, one or more symptoms thereof,
or a respiratory condition associated with, potentiated by or
potentiating a RSV infection by administrating to a subject of an
effective amount of liquid formulations of the invention. The
subject is preferably a mammal such as non-primate (e.g., cows,
pigs, horses, cats, dogs, rats etc.) and a primate (e.g. monkey
such as a cynomolgous monkey and a human). In a preferred
embodiment, the subject is a human. In another preferred
embodiment, the subject is a human infant or a human infant born
prematurely.
[0242] Various delivery systems are known and can be used to
administer a liquid formulation of the present invention. Methods
of administering antibody liquid formulations of the present
invention include, but are not limited to, parenteral
administration (e.g., intradermal, intramuscular, intraperitoneal,
intravenous and subcutaneous), epidural administration, topical
administration, pulmonary administration, and mucosal
administration (e.g., intranasal and oral routes). In a specific
embodiment, liquid formulations of the present invention are
administered intramuscularly, intravenously, or subcutaneously and,
preferably, intramuscularly. The formulations may be administered
by any convenient route, for example by infusion or bolus
injection, by absorption through epithelial or mucocutaneous
linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and
may be administered together with other biologically active agents.
Administration can be systemic or local. In addition, pulmonary
administration can be employed, e.g., by use of an inhaler or
nebulizer.
[0243] The invention also provides that a liquid formulation of the
present invention is packaged in a hermetically sealed container
such as an ampoule or sachette indicating the quantity of antibody
or antibody fragment. Preferably, the liquid formulations of the
present invention are in a hermetically sealed container indicating
the quantity and concentration of the antibody or antibody
fragment. Preferably, the liquid formulation of the present
invention is supplied in a hermetically sealed container at least
15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70
mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 150 mg/ml, 200 mg/ml, 250
mg/ml, or 300 mg/ml and, most preferably, 105 mg/ml, in a quantity
of 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15
ml, or 20 ml and, most preferably, 1.2 ml.
[0244] The amount of the liquid formulations of the present
invention which will be effective in the treatment, prevention,
management or amelioration of a RSV infection, one or more symptoms
thereof, or a respiratory condition associated with, potentiated by
or potentiating a RSV infection can be determined by standard
clinical techniques. For example, the dosage of a liquid
formulation which will be effective in the treatment, prevention,
management or amelioration of a RSV infection, one or more symptoms
thereof, or a respiratory condition associated with, potentiated by
or potentiating a RSV infection can be determined by administering
the formulation to a cotton rat, measuring the RSV titer after
challenging the cotton rat with 10.sup.5 pfu of RSV and comparing
the RSV titer to that obtain for a cotton rat not administered the
formulation. Accordingly, a dosage that results in a 2 log decrease
or a 99% reduction in RSV titer in the cotton rat challenged with
10.sup.5 pfu of RSV relative to the cotton rat challenged with
10.sup.5 pfu of RSV but not administered the formulation is the
dosage of the formulation that can be administered to a human for
the treatment, prevention, management or amelioration of a RSV
infection, one or more symptoms thereof, or a respiratory condition
associated with, potentiated by or potentiating a RSV infection.
The dosage of a liquid formulation which will be effective in the
treatment, prevention, management or amelioration of a RSV
infection, one or more symptoms thereof, or a respiratory condition
associated with, potentiated by or potentiating a RSV infection can
be determined by administering the formulation to an animal model
(e.g., a cotton rat or monkey) and measuring the serum titer of
antibodies or fragments thereof that immunospecifically bind to a
RSV antigen. Accordingly, a dosage of the formulation that results
in a serum titer of at least 1 .mu.g/ml, preferably 2 .mu.g/ml, 5
.mu.g/ml, 10 .mu.g/ml, 20 .mu.g/ml, 25 .mu.g/ml, at least 35
.mu.g/ml, at least 40 .mu.g/ml, at least 50 .mu.g/ml, at least 75
.mu.g/ml, at least 100 .mu.g/ml, at least 125 .mu.g/ml, at least
150 .mu.g/ml, at least 200 .mu.g/ml, at least 250 .mu.g/ml, at
least 300 .mu.g/ml, at least 350 .mu.g/ml, at least 400 .mu.g/ml,
or at least 450 .mu.g/ml can be administered to a human for the
treatment, prevention, management or amelioration of a RSV
infection, one or more symptoms thereof, or a respiratory condition
associated with, potentiated by or potentiating a RSV infection. In
addition, in vitro assays may optionally be employed to help
identify optimal dosage ranges.
[0245] The precise dose to be employed in the formulation will also
depend on the route of administration, and the seriousness of the
RSV infection, and should be decided according to the judgment of
the practitioner and each patients circumstances. Effective doses
may be extrapolated from dose-response curves derived from in vitro
or animal model (e.g., the cotton rat or Cynomolgous monkey) test
systems.
[0246] For antibodies, proteins, polypeptides, peptides and fusion
proteins, the dosage administered to a patient may be about 1 mg/kg
to 30 mg/kg of the patients body weight. Preferably, the dosage
administered to a patient is between 10 mg/kg and 20 mg/kg of the
patients body weight, more preferably 15 mg/kg of the patients body
weight. Generally, human antibodies have a longer half-life within
the human body than antibodies from other species due to the immune
response to the foreign polypeptides. Thus, lower dosages of human
antibodies and less frequent administration is often possible.
Further, the dosage, volume and frequency of administration of
liquid formulations of the present invention may be reduced by
increasing the concentration of an antibody or a fragment thereof
in the formulations, increasing affinity and/or avidity of the
antibody or a fragment thereof, and/or increasing the half-life of
the antibody or a fragment thereof.
[0247] Exemplary doses of a small molecule include milligram or
microgram amounts of the small molecule per kilogram of subject or
sample weight (e.g., about 1 microgram per kilogram to about 500
milligrams per kilogram, about 100 micrograms per kilogram to about
5 milligrams per kilogram, or about 1 microgram per kilogram to
about 50 micrograms per kilogram).
[0248] In one embodiment, the liquid formulations of the present
invention are administered to a mammal, preferably a human, to
prevent, treat, manage or ameliorate a RSV infection or one or more
symptoms thereof. In another embodiment, the liquid formulations of
the invention are administered to a human with cystic fibrosis,
bronchopulmonary dysplasia, congenital heart disease, congenital
immunodeficiency or acquired immunodeficiency, or to a human who
has had a bone marrow transplant to prevent, treat, mange or
ameliorate a RSV infection or one or more symptoms thereof. In
another embodiment, the liquid formulations of the invention are
administered to a human infant, preferably a human infant born
prematurely or a human infant at risk of hospitalization for a RSV
infection to prevent, treat, manage or ameliorate a RSV infection
or one or more symptoms thereof. In another embodiment, the liquid
formulations of the invention are administered to an elderly person
to prevent, treat, manage or ameliorate a RSV infection or one or
more symptoms thereof.
[0249] In a specific embodiment, a subject, preferably a human, is
administered a stable liquid formulation of the present invention
for the treatment, prevention or amelioration of one or more
symptoms associated with a RSV infection in an amount effective for
decreasing RSV titers. In accordance with this embodiment, an
effective amount of the liquid formulations of the present
invention reduces the RSV titers in the lung as measured, for
example, by the concentration of RSV in sputum samples or a lavage
from the lungs from a subject. In another embodiment, a subject,
preferably a human, is administered an antibody liquid formulation
of the present invention for the treatment, prevention or
amelioration of symptoms associated with a RSV infection in an
amount effective for inducing an immune response in the
subject.
[0250] In another embodiment, a subject, preferably a human, is
administered a first dose of a liquid formulation of the present
invention comprising 30 mg/kg or less, 15 mg/kg or less, 10 mg/kg
or less, 5 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less, or 0.5
mg/kg or less of an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen and, preferably has equal
to or higher affinity, equal to or higher avidity, and/or equal to
or longer half-life than previously known antibodies (e.g.,
SYNAGIS.RTM.) for the prevention of a RSV infection in an amount
effective to induce a serum titer of at least 1 .mu.g/ml,
preferably at least 2 .mu.g/ml, at least 5 .mu.g/ml, at least 10
.mu.g/ml, at least 15 .mu.g/ml, at least 20 .mu.g/ml, at least 25
.mu.g/ml, at least 30 .mu.g/ml, at least 35 .mu.g/ml, at least 40
.mu.g/ml 20 days (preferably 25, 30, 35, 40 days) after the
administration of the first dose and prior to the administration of
a subsequent dose. In a specific embodiment, a liquid formulation
of the present invention comprises an antibody having a VH domain
of SEQ ID NO:9 and a VL domain of SEQ ID NO:13; a VH domain of SEQ
ID NO:17 and a VL domain of SEQ ID NO:21; a VH domain of SEQ ID
NO:24 and a VL domain of SEQ ID NO:26; a VH domain of SEQ ID NO:28
and a VL domain of SEQ ID NO:30; a VH domain of SEQ ID NO:33 and a
VL domain of SEQ ID NO:34; a VH domain of SEQ ID NO:36 and a VL
domain of SEQ ID NO:38; a VH domain of SEQ ID NO:40 and a VL domain
of SEQ ID NO:42; a VH domain of SEQ ID NO:44 and a VL domain of SEQ
ID NO:46; a VH domain of SEQ ID NO:48 and a VL domain of SEQ ID
NO:49; a VH domain of SEQ ID NO:51 and a VL domain of SEQ ID NO:52;
a VH domain of SEQ ID NO:7 and a VL domain of SEQ ID NO:54; a VH
domain of SEQ ID NO:55 and a VL domain of SEQ ID NO:56; a VH domain
of SEQ ID NO:55 and a VL domain of SEQ ID NO:58; a VH domain of SEQ
ID NO:78 and a VL domain of SEQ ID NO:56; a VH domain of SEQ ID
NO:9 and a VL domain of SEQ ID NO:60; a VH domain of SEQ ID NO:78
and a VL domain of SEQ ID NO:62; a VH domain of SEQ ID NO:78 and a
VL domain of SEQ ID NO:64; a VH domain of SEQ ID NO:78 and a VL
domain of SEQ ID NO:65; a VH domain of SEQ ID NO:67 and a VL domain
of SEQ ID NO:68 (see Table 1 and 2, supra) a VH domain of SEQ ID
NO:70 and a VL domain of SEQ ID NO:71; a VH domain of SEQ ID NO:48
and a VL domain of SEQ ID NO:74; a VH domain of SEQ ID NO:48 and a
VL domain of SEQ ID NO:11; or a VH domain of SEQ ID NO:48 and a VL
domain of SEQ ID NO:76 (see Table 1 and 2, supra).
[0251] In another embodiment, a subject, preferably a human, is
administered a first dose of a liquid formulations of the present
invention comprising 30 mg/kg or less, 15 mg/kg or less, 10 mg/kg
or less, 5 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less or 0.5
mg/kg or less of an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen and, preferably, has a
higher affinity and/or higher avidity than previously known
antibodies (e.g., SYNAGIS.RTM.) for the treatment or amelioration
of a RSV infection or a symptom thereof in an amount effective to
induce a serum titer of at least 1 .mu.g/ml, preferably at least 2
.mu.g/ml, at least 5 .mu.g/ml, at least 10 .mu.g/ml, at least 15
.mu.g/ml, at least 20 .mu.g/ml, or at least 25 .mu.g/ml 20 days
(preferably 25, 30, 35, 40 days) after the administration of the
first dose and prior to the administration of subsequent dose.
Preferably, the serum titer of said antibodies or antibody
fragments is less than 30 .mu.g/ml 30 days after the administration
of the first dose and prior to the administration of a subsequent
dose. Preferably, said antibodies have a VH domain of SEQ ID NO:9
and a VL domain of SEQ ID NO: 13; a VH domain of SEQ ID NO:17 and a
VL domain of SEQ ID NO:21; a VH domain of SEQ ID NO:24 and a VL
domain of SEQ ID NO:26; a VH domain of SEQ ID NO:28 and a VL domain
of SEQ ID NO:30; a VH domain of SEQ ID NO:33 and a VL domain of SEQ
ID NO:34; a VH domain of SEQ ID NO:36 and a VL domain of SEQ ID
NO:38; a VH domain of SEQ ID NO:40 and a VL domain of SEQ ID NO:42;
a VH domain of SEQ ID NO:44 and a VL domain of SEQ ID NO:46; a VH
domain of SEQ ID NO:48 and a VL domain of SEQ ID NO:49; a VH domain
of SEQ ID NO:51 and a VL domain of SEQ ID NO:52; a VH domain of SEQ
ID NO:7 and a VL domain of SEQ ID NO:54; a VH domain of SEQ ID
NO:55 and a VL domain of SEQ ID NO:56; a VH domain of SEQ ID NO:55
and a VL domain of SEQ ID NO:58; a VH domain of SEQ ID NO:78 and a
VL domain of SEQ ID NO:56; a VH domain of SEQ ID NO:9 and a VL
domain of SEQ ID NO:60; a VH domain of SEQ ID NO:78 and a VL domain
of SEQ ID NO:62; a VH domain of SEQ ID NO:78 and a VL domain of SEQ
ID NO:64; a VH domain of SEQ ID NO:78 and a VL domain of SEQ ID
NO:65; a VH domain of SEQ ID NO:67 and a VL domain of SEQ ID NO:68
(see Table 1 and 2, supra) a VH domain of SEQ ID NO:70 and a VL
domain of SEQ ID NO:71; a VH domain of SEQ ID NO:48 and a VL domain
of SEQ ID NO:74; a VH domain of SEQ ID NO:48 and a VL domain of SEQ
ID NO: 11; or a VH domain of SEQ ID NO:48 and a VL domain of SEQ ID
NO:76 (see Table 1 and 2, supra)
[0252] In another embodiment, a subject, preferably a human, is
administered a first dose of a liquid formulation of the present
invention comprising 30 mg/kg or less, 15 mg/kg or less, 5 mg/kg or
less, 3 mg/kg or less, 1 mg/kg or less or 0.5 mg/kg or less of an
antibody or a fragment thereof which has increased in vivo
half-life and which immunospecifically binds to a RSV antigen with
higher affinity and/or higher avidity than previously known
antibodies (e.g., SYNAGIS.RTM.) for the prevention of a RSV
infection in an amount effective to induce a serum titer of at
least 1 .mu.g/ml, preferably at least 2 .mu.g/ml, at least 5
.mu.g/ml, at least 10 .mu.g/ml, at least 15 .mu.g/ml, at least 20
.mu.g/ml, or at least 25 .mu.g/ml 25 days (preferably 30, 35, or 40
days) after the administration of the first dose and prior to the
administration of a subsequent dose. Preferably, the serum titer of
said antibodies or antibody fragments is less than 30 .mu.g/ml 30
days after the administration of the first dose and prior to the
administration of a subsequent dose. Preferably, the said
antibodies have a VH domain of SEQ ID NO:9 and a VL domain of SEQ
ID NO:13; a VH domain of SEQ ID NO:17 and a VL domain of SEQ ID
NO:21; a VH domain of SEQ ID NO:24 and a VL domain of SEQ ID NO:26;
a VH domain of SEQ ID NO:28 and a VL domain of SEQ ID NO:30; a VH
domain of SEQ ID NO:33 and a VL domain of SEQ ID NO:34; a VH domain
of SEQ ID NO:36 and a VL domain of SEQ ID NO:38; a VH domain of SEQ
ID NO:40 and a VL domain of SEQ ID NO:42; a VH domain of SEQ ID
NO:44 and a VL domain of SEQ ID NO:46; a VH domain of SEQ ID NO:48
and a VL domain of SEQ ID NO:49; a VH domain of SEQ ID NO:51 and a
VL domain of SEQ ID NO:52; a VH domain of SEQ ID NO:7 and a VL
domain of SEQ ID NO:54; a VH domain of SEQ ID NO:55 and a VL domain
of SEQ ID NO:56; a VH domain of SEQ ID NO:55 and a VL domain of SEQ
ID NO:58; a VH domain of SEQ ID NO:78 and a VL domain of SEQ ID
NO:56; a VH domain of SEQ ID NO:9 and a VL domain of SEQ ID NO:60;
a VH domain of SEQ ID NO:78 and a VL domain of SEQ ID NO:62; a VH
domain of SEQ ID NO:78 and a VL domain of SEQ ID NO:64; a VH domain
of SEQ ID NO:78 and a VL domain of SEQ ID NO:65; a VH domain of SEQ
ID NO:67 and a VL domain of SEQ ID NO:68 (see Table 1 and 2, supra)
a VII domain of SEQ ID NO:70 and a VL domain of SEQ ID NO:71; a VH
domain of SEQ ID NO:48 and a VL domain of SEQ ID NO:74; a VH domain
of SEQ ID NO:48 and a VL domain of SEQ ID NO: 11; or a VH domain of
SEQ ID NO:48 and a VL domain of SEQ ID NO:76 (see Table 1 and 2,
supra).
[0253] In another embodiment, a subject, preferably a human, is
administered a first dose of a liquid formulation of the present
invention comprising 30 mg/kg or less, 15 mg/kg or less, 5 mg/kg or
less, 3 mg/kg or less, 1 mg/kg or less, or 0.5 mg/kg or less of an
antibody or a fragment thereof which has increased in vivo
half-lives and which immunospecifically bind to a RSV antigen with
higher affinity and/or higher avidity than previously known
antibodies (e.g., SYNAGIS.RTM.) for the treatment or amelioration
of a RSV infection or one or more symptoms thereof in an amount
effective to induce a serum titer of at least 1 .mu.g/ml,
preferably at least 2 .mu.g/ml, at least 5 .mu.g/ml, at least 10
.mu.g/ml, at least 15 .mu.g/ml, at least 20 .mu.g/ml, or at least
25 .mu.g/ml 25 days (preferably 30, 35, or 40 days) after the
administration of the first dose and prior to the administration of
a subsequent dose. Preferably, the serum titer of said antibodies
or antibody fragments is less than 30 .mu.g/ml 30 days after the
administration of the first dose and prior to the administration of
a subsequent dose. Preferably, the said antibodies have a VH domain
of SEQ ID NO:9 and a VL domain of SEQ ID NO:13; a VH domain of SEQ
ID NO:17 and a VL domain of SEQ ID NO:21; a VH domain of SEQ ID
NO:24 and a VL domain of SEQ ID NO:26; a VH domain of SEQ ID NO:28
and a VL domain of SEQ ID NO:30; a VH domain of SEQ ID NO:33 and a
VL domain of SEQ ID NO:34; a VH domain of SEQ ID NO:36 and a VL
domain of SEQ ID NO:38; a VH domain of SEQ ID NO:40 and a VL domain
of SEQ ID NO:42; a VH domain of SEQ ID NO:44 and a VL domain of SEQ
ID NO:46; a VH domain of SEQ ID NO:48 and a VL domain of SEQ ID
NO:49; a VH domain of SEQ ID NO:51 and a VL domain of SEQ ID NO:52;
a VH domain of SEQ ID NO:7 and a VL domain of SEQ ID NO:54; a VH
domain of SEQ ID NO:55 and a VL domain of SEQ ID NO:56; a VH domain
of SEQ ID NO:55 and a VL domain of SEQ ID NO:58; a VH domain of SEQ
ID NO:78 and a VL domain of SEQ ID NO:56; a VH domain of SEQ ID
NO:9 and a VL domain of SEQ ID NO:60; a VH domain of SEQ ID NO:78
and a VL domain of SEQ ID NO:62; a VH domain of SEQ ID NO:78 and a
VL domain of SEQ ID NO:64; a VH domain of SEQ ID NO:78 and a VL
domain of SEQ ID NO:65; a VH domain of SEQ ID NO:67 and a VL domain
of SEQ ID NO:68 (see Table 1 and 2, supra) a VH domain of SEQ ID
NO:70 and a VL domain of SEQ ID NO:71; a VH domain of SEQ ID NO:48
and a VL domain of SEQ ID NO:74; a VH domain of SEQ ID NO:48 and a
VL domain of SEQ ID NO: 11; or a VH domain of SEQ ID NO:48 and a VL
domain of SEQ ID NO:76 (see Table 1 and 2, supra).
[0254] In another embodiment, a subject, preferably a human, is
administered a first dose of a liquid formulation of the present
invention comprising approximately 30 mg/kg or less, 15 mg/kg or
less (preferably 10 mg/kg or less, 5 mg/kg or less, 3 mg/kg or
less, 1 mg/kg or less, or 0.5 mg/kg or less) of an antibody or a
fragment thereof which has increased in vivo half-life for the
prevention, treatment or amelioration of a RSV infection or one or
more symptoms thereof in an amount effective to induce a serum
titer of at least 1 .mu.g/ml, preferably at least 30 .mu.g/ml, at
least 35 .mu.g/ml, at least 40 .mu.g/ml, or at least 50 .mu.g/ml 25
days (preferably 30, 35, or 40 days) after the administration of
the first dose and prior to the administration of a subsequent
dose.
[0255] The present invention encompasses liquid formulations for
pulmonary delivery comprising one or more antibodies or fragments
thereof which immunospecifically bind to one or more RSV antigens.
Preferably, such antibodies and antibody fragments have a higher
affinity and/or a higher avidity than previously known antibodies
(e.g., SYNAGIS.RTM.).
[0256] In one embodiment, a subject, preferably a human, is
administered a first dose of a liquid formulation of the present
invention for pulmonary delivery comprising 30 mg/kg or less, 15
mg/kg or less, 5 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less,
0.5 mg/kg or less, or 0.01 mg/kg or less of an antibody or a
fragment thereof that immunospecifically binds to a RSV antigen
and, preferably, has higher affinity and/or higher avidity than
previously known antibodies (e.g., SYNAGIS.RTM.) for the
prevention, treatment or amelioration of a RSV infection or a
symptom thereof in an amount effective to induce a titer of at
least 20 ng per mg of lung protein (preferably at least 40 ng/mg,
at least 60 ng/mg, at least 80 ng/mg, at least 50 ng/mg, at least
75 ng/mg, at least 100 ng/mg, or at least 150 ng/mg) in an
intubation sample or lavage from the lungs of said mammal 20 days
(preferably 25, 30, 35, or 40 days) after the administration of the
first dose and prior to the administration of a subsequent dose.
Preferably, the serum titer of said antibodies or antibody
fragments is less than 100 ng/ml of protein 30 days after the
administration of the first dose and prior to the administration of
a subsequent dose. Preferably, said antibodies have a VH domain of
SEQ ID NO:9 and a VL domain of SEQ ID NO:13; a VH domain of SEQ ID
NO:17 and a VL domain of SEQ ID NO:21; a VH domain of SEQ ID NO:24
and a VL domain of SEQ ID NO:26; a VH domain of SEQ ID NO:28 and a
VL domain of SEQ ID NO:30; a VH domain of SEQ ID NO:33 and a VL
domain of SEQ ID NO:34; a VH domain of SEQ ID NO:36 and a VL domain
of SEQ ID NO:38; a VH domain of SEQ ID NO:40 and a VL domain of SEQ
ID NO:42; a VH domain of SEQ ID NO:44 and a VL domain of SEQ ID
NO:46; a VH domain of SEQ ID NO:48 and a VL domain of SEQ ID NO:49;
a VH domain of SEQ ID NO:51 and a VL domain of SEQ ID NO:52; a VH
domain of SEQ ID NO:7 and a VL domain of SEQ ID NO:54; a VH domain
of SEQ ID NO:55 and a VL domain of SEQ ID NO:56; a VH domain of SEQ
ID NO:55 and a VL domain of SEQ ID NO:58; a VH domain of SEQ ID
NO:78 and a VL domain of SEQ ID NO:56; a VH domain of SEQ ID NO:9
and a VL domain of SEQ ID NO:60; a VH domain of SEQ ID NO:78 and a
VL domain of SEQ ID NO:62; a VH domain of SEQ ID NO:78 and a VL
domain of SEQ ID NO:64; a VH domain of SEQ ID NO:78 and a VL domain
of SEQ ID NO:65; a VH domain of SEQ ID NO:67 and a VL domain of SEQ
ID NO:68 (see Table 1 and 2, supra) a VH domain of SEQ ID NO:70 and
a VL domain of SEQ ID NO:71; a VH domain of SEQ ID NO:48 and a VL
domain of SEQ ID NO:74; a VH domain of SEQ ID NO:48 and a VL domain
of SEQ ID NO:11; or a VH domain of SEQ ID NO:48 and a VL domain of
SEQ ID NO:76 (see Table 1 and 2, supra).
[0257] The present invention encompasses liquid formulations of the
present invention for pulmonary delivery comprising an antibody or
a fragment thereof which has increased in vivo half-life and which
immunospecifically binds to a RSV antigen and, preferably, has a
higher affinity and/or a higher avidity than previously known
antibodies (e.g., SYNAGIS.RTM.).
[0258] In another embodiment, a subject, preferably a human, is
administered a first dose of a liquid formulation of the present
invention 10 mg/kg or less, 5 mg/kg or less, 3 mg/kg or less, 1
mg/kg or less, or 0.5 mg/kg or less of an antibody or a fragment
thereof for the prevention treatment or amelioration of a RSV
infection or one or more symptoms thereof in an amount effective to
induce a serum titer of at least 35 .mu.g/ml, at least 40 .mu.g/ml,
at least 50 .mu.g/ml, at least 80 .mu.g/ml, at least 100 .mu.g/ml,
at least 120 .mu.g/ml, at least 150 .mu.g/ml, at least 200
.mu.g/ml, at least 250 .mu.g/ml, or at least 300 .mu.g/ml 20 days
(preferably 25, 30, 35 or 40 days) after the administration of the
first dose. In another embodiment, a mammal, preferably a human, is
administered a first dose of a liquid formulation of the present
invention comprising approximately 15 mg/kg of an antibody or a
fragment thereof for the prevention, treatment or amelioration of
one or more symptoms associated with a RSV infection in an amount
effective to induce a serum titer of at least 100 .mu.g/ml, at
least 125 :g/ml, at least 150 .mu.g/ml, at least 200 .mu.g/ml, at
least 250 .mu.g/ml, at least 300 .mu.g/ml, at least 350 .mu.g/ml,
at least 400 .mu.g/ml, or at least 450:g/ml 20 days (preferably 25,
30, 35 or 40 days) after the administration of the first dose. The
term "approximately 15 mg/kg" as used herein refers to a range of
between 14 mg/kg and 16 mg/kg.
[0259] In another embodiment, a subject, preferably a human, is
administered a dose of a liquid formulation of the present
invention comprising an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen for the prevention a RSV
infection in an amount effective to induce a prophylactically
effective serum titer of 20 .mu.g/ml or less, 15 .mu.g/ml or less,
10 .mu.g/ml or less, 8 .mu.g/ml or less, 5 .mu.g/ml or less, 3
.mu.g/ml or less, 1 .mu.g/ml or less, or 0.5 .mu.g/ml or less 30
days after the administration of the dose, wherein said
prophylactically effective serum titer is the serum titer that
reduces the incidence of RSV infection in the human or the serum
titer in a cotton rat that results in a RSV titer 5 days after
challenge with 10.sup.5 pfu RSV that is 99% lower than the RSV
titer in the cotton rat 5 days after challenge with 10.sup.5 pfu of
RSV in a cotton rat not administered the dose prior to challenge.
Preferably, the dose of the therapeutic or pharmaceutical
composition comprises 15 mg/kg or less, 10 mg/kg or less, 5 mg/kg
or less, 3 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less of an
antibody or a fragment thereof.
[0260] In yet another embodiment, a subject, preferably a human, is
administered a dose of a liquid formulation of the present
invention comprising an antibody or a fragment thereof that
immunospecifically binds to a RSV antigen for the treatment or
amelioration of one or more symptoms associated with a RSV
infection in an amount effective to induce a therapeutically
effective serum titer of 25 .mu.g/ml or less, 20 .mu.g/ml or less,
15 .mu.g/ml or less, 10 .mu.g/ml or less, 8 .mu.g/ml or less, 5
.mu.g/ml or less, 3 .mu.g/ml or less, 1 .mu.g/ml or less, or 0.5
.mu.g/ml or less 30 days after the administration of the dose,
wherein said therapeutically effective serum titer is the serum
titer that reduces the severity or length of RSV infection or is
the serum titer in a cotton rat that results in a RSV titer in the
rat 5 days after challenge with 10.sup.5 pfu RSV that is 99% lower
than the RSV titer 5 days after challenge with 10.sup.5 pfu of RSV
in a cotton rat not administered the dose prior to challenge.
Preferably, the dose of the liquid formulation of the present
invention comprises 15 mg/kg or less, 12 mg/kg or less, 10 mg/kg or
less, 5 mg/kg or less, 3 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg
or less of an antibody or a fragment thereof.
[0261] In a specific embodiment, formulations of the present
invention are administered once a month just prior to or during the
RSV season. In another embodiment, the formulations are
administered every two months just prior to or during the RSV
season. In yet another embodiment, the stable formulations of the
present invention are administered once just prior to or during the
RSV season. The term "RSV season" refers to the season when RSV
infection is most likely to occur. Typically, the RSV season in the
northern hemisphere commences in November and lasts through
April.
[0262] In one embodiment, approximately 5 mg/kg or less (preferably
1.5 mg/kg or less) of an antibody or fragment thereof, in the
liquid formulations of the present invention, which
immunospecifically binds to a RSV antigen with a higher avidity
and/or higher affinity than previously known antibodies such as,
e.g., SYNAGIS.RTM., is administered five times, 3 times, or 1 to 2
times during a RSV season to a mammal, preferably a human. In
another embodiment, approximately 1.5 mg/kg of an antibody or a
fragment thereof, in the formulations of the present invention,
which immunospecifically binds to a RSV antigen with a higher
avidity and/or a higher affinity than known antibodies such as,
e.g., SYNAGIS.RTM., is administered monthly five times during a RSV
season to a mammal, preferably a human, intramuscularly. In another
embodiment, 3 mg/kg of an antibody or a fragment thereof in the
present formulation which immunospecifically binds to a RSV antigen
with a higher avidity and/or a higher affinity than known
antibodies such as, e.g., SYNAGIS.RTM. is administered monthly
three times during a RSV season to a mammal, preferably a human,
intramuscularly. In yet another embodiment, 5 mg/kg of an antibody
or a fragment thereof in the formulation which immunospecifically
binds to a RSV antigen with a higher avidity and/or a higher
affinity than known antibodies such as, e.g., SYNAGIS.RTM. is
administered monthly one to two times during a RSV season to a
mammal, preferably a human, intramuscularly.
[0263] In a specific embodiment, 15 mg/kg of anti-RSV antibodies or
an antigen-binding fragment thereof in the liquid formulation of
the present invention is administered to a mammal, preferably a
human, intramuscularly five times during a RSV season, wherein said
antibodies or an antibody fragment has an increased in vivo
half-life. In another embodiment, approximately 5 mg/kg or less
(preferably 1.5 mg/kg or less) of an antibody or fragment thereof
in the liquid formulation of the present invention which
immunospecifically binds to a RSV antigen with a higher avidity
and/or higher affinity than previously known antibodies such as,
e.g., SYNAGIS.RTM., is administered five times, 3 times, or 1 to 2
times during a RSV season to a mammal, preferably a human. In
another embodiment, 3 mg/kg of antibody or a fragment thereof in
the liquid formulation of the present invention which
immunospecifically binds to a RSV antigen with a higher avidity
and/or a higher affinity known antibodies such as, e.g.,
SYNAGIS.RTM. and which has an increased in vivo half-life is
administered monthly three times during a RSV season to a mammal,
preferably a human, intramuscularly. In another embodiment, 5 mg/kg
of antibody or a fragment thereof in the liquid formulation of the
present invention which immunospecifically binds to a RSV antigen
with a higher avidity and/or a higher affinity than known
antibodies such as, e.g., SYNAGIS.RTM. and which has an increased
in vivo half-life is administered to a mammal, preferably a human,
intramuscularly twice times during a RSV season.
[0264] 5.7 Biological Assays
[0265] 5.7.1 Immunospecificity of the Antibodies of the
Invention
[0266] Antibodies of the present invention or fragments thereof may
be characterized in a variety of ways well-known to one of skill in
the art. In particular, antibodies of the invention or fragments
thereof may be assayed for the ability to immunospecifically bind
to an epitope of a respiratory syncytial virus. Such an assay may
be performed in solution (e.g., Houghten, 1992, Bio/Techniques
13:412-421), on beads (Lam, 1991, Nature 354:82-84), on chips
(Fodor, 1993, Nature 364:555-556), on bacteria (U.S. Pat. No.
5,223,409), on spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and
5,223,409), on plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci.
USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science
249:386-390; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA
87:6378-6382; and Felici, 1991, J. Mol. Biol. 222:301-310) (each of
these references is incorporated herein in its entirety by
reference). An antibody of the invention or a fragment thereof in a
liquid formulation of the present invention can be assayed for its
specificity and affinity.
[0267] An antibody or a fragment thereof of the present invention
may be assayed for immunospecific binding to a RSV antigen and
cross-reactivity with other antigens by any method known in the
art. Immunoassays which can be used to analyze immunospecific
binding and cross-reactivity include, but are not limited to,
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and
well-known in the art (see, e.g., Ausubel et al., eds., 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley &
Sons, Inc., New York, which is incorporated by reference herein in
its entirety).
[0268] 5.7.2 In Vitro and In Vivo Assays
[0269] An antibody or a fragment thereof, the liquid formulations
of the invention, or a combination therapy of the present invention
can be tested in vitro and/or in vivo in various assays or suitable
animal model systems for its activity.
[0270] A liquid formulation of the present invention for treating,
managing, preventing, or ameliorating a RSV infection or one or
more symptoms thereof can be tested for its ability to inhibit
viral replication or reduce viral load in in vitro assays. For
example, viral replication can be assayed by a plaque assay such as
described, e.g., by Johnson et al., 1997, Journal of Infectious
Diseases 176:1215-1224 176:1215-1224. A liquid formulation of the
invention administered according to the methods of the invention
can also be assayed for their ability to inhibit or down-regulate
the expression of viral polypeptides. Techniques known to those of
skill in the art, including, but not limited to, western blot
analysis, northern blot analysis, and RT-PCR can be used to measure
the expression of viral polypeptides and/or viral titers.
[0271] A liquid formulation of the invention can be tested in
suitable animal model systems prior to use in humans. Such animal
model systems include, but are not limited to, rats, mice, chicken,
cows, monkeys, pigs, dogs, rabbits, etc. Any animal system
well-known in the art may be used. Several aspects of the procedure
may vary; said aspects include, but are not limited to, the
temporal regime of administering the therapies (e.g., prophylactic
and/or therapeutic agents) whether such therapies are administered
separately or as an admixture, and the frequency of administration
of the therapies.
[0272] Animal models can be used to assess the efficacy of the
methods of the invention for treating, managing, preventing, or
ameliorating a RSV infection or one or more symptom thereof. Animal
models for RSV infection include, but are not limited to, those as
described by, e.g., Piedimonte et al., Am J Physiol 1999,
277:L831-L840; McArthur-Vaughan et al., J. Med. Primatol. 2002,
31(2):61-73; and Byrd et al., Clin. Infect. Dis. 1997,
25(6):1363-8. In a specific embodiment, cotton rats are
administered a liquid formulation comprising an antibody or a
fragment thereof that immunospecifically binds to a RSV antigen
according to the methods of the invention, challenged with 10.sup.5
pfu of RSV, and four or more days later, the rats are sacrificed
and RSV titer and anti-RSV antibody serum titer is determined.
Accordingly, a dosage that results in a 2 log decrease or a 99%
reduction in RSV titer in the cotton rat challenged with 10.sup.5
pfu of RSV relative to the cotton rat challenged with 10.sup.5 pfu
of RSV but not administered the formulation is the dosage of the
formulation that can be administered to a human for the treatment,
prevention or amelioration of a RSV infection or one or more
symptoms thereof. Further, this embodiment, the tissues (e.g., the
lung tissues) from the sacrificed rats can be examined for
histological changes.
[0273] The administration of a liquid formulation of the invention
according to the methods of the present invention can be tested for
its ability to decrease the time course of a RSV infection by at
least 25%, preferably at least 50%, at least 60%, at least 75%, at
least 85%, at least 95%, or at least 99% relative to a negative
control. A liquid formulation of the invention can also be tested
for its ability to increase the survival period of humans suffering
from a RSV infection by at least 25%, preferably at least 50%, at
least 60%, at least 75%, at least 85%, at least 95%, or at least
99% relative to a negative control. Further, a liquid formulation
of the invention can be tested for its ability reduce the
hospitalization period of a human suffering from RSV infection by
at least 60%, preferably at least 75%, at least 85%, at least 95%,
or at least 99% relative to a negative control. Techniques known to
those of skill in the art can be used to analyze the function of a
liquid formulation of the invention in vivo.
[0274] Further, any in vitro or in vivo assays known to those
skilled in the art can be used to evaluate the prophylactic and/or
therapeutic utility of a liquid formulation of the invention
disclosed herein for a RSV infection or one or more symptoms
thereof.
[0275] 5.7.3 Toxicity Assays
[0276] The toxicity and/or efficacy of the prophylactic and/or
therapeutic protocols of the instant invention can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for determining the LD50 (the dose lethal to 50% of
the population) and the ED50 (the dose therapeutically effective in
50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD50/ED50. Therapies that exhibit large
therapeutic indices are preferred. While therapies that exhibit
toxic side effects may be used, care should be taken to design a
delivery system that targets such agents to the site of affected
tissue in order to minimize potential damage to uninfected cells
and, thereby, reduce side effects.
[0277] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage of the
prophylactic and/or therapeutic agents for use in humans. The
dosage of such agents lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage may vary within this range depending upon the dosage
form employed and the route of administration utilized. For any
therapy used in the method of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC50 (i.e., the
concentration of the test compound that achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
[0278] 5.8 Kits
[0279] The invention provides a pharmaceutical pack or kit
comprising one or more containers filled with a liquid formulation
of the invention for the prevention, treatment, management or
amelioration of a RSV infection, one or more symptoms thereof, or a
respiratory condition associated with, potentiated by or
potentiating a RSV infection. The invention also provides a
pharmaceutical pack or kit comprising one or more containers filled
with a liquid formulation of the invention for the detection,
diagnosis or monitoring of a RSV infection. In a specific
embodiment, the liquid formulations of the invention comprise an
antibody or a fragment thereof that immunospecifically binds to a
RSV antigen recombinantly fused or chemically conjugated to another
moiety, including but not limited to, a heterologous protein, a
heterologous polypeptide, a heterologous peptide, a large molecule,
a small molecule, a marker sequence, a diagnostic or detectable
agent, a therapeutic moiety, a drug moiety, a radioactive metal
ion, a second antibody, and a solid support.
[0280] The present invention provides kits that can be used in the
above methods. In one embodiment, a kit comprises a liquid
formulation of the invention, in one or more containers. In another
embodiment, a kit comprises a liquid formulation of the invention,
in one or more containers, and one or more other prophylactic or
therapeutic agents useful for the prevention, management or
treatment of a RSV infection, one or more symptoms thereof, or a
respiratory condition associated with, potentiated by or
potentiating a RSV infection in one or more other containers.
Preferably, the kit further comprises instructions for preventing,
treating, managing or ameliorating a RSV infection (e.g., using the
liquid formulations of the invention alone or in combination with
another prophylactic or therapeutic agent), as well as side effects
and dosage information for method of administration. Optionally
associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration.
[0281] 5.9 Use of Liquid Formulations in the Diagnosis of RSV
Infection
[0282] The liquid formulations of the invention comprising labeled
antibodies or fragments, derivatives and analogues thereof that
immunospecifically bind to a RSV antigen can be used for diagnostic
purposes to detect, diagnose, or monitor a RSV infection. Such
diagnostic techniques are known in the art, including but not
limited to, those disclosed in International Publication No. WO
01/58483, U.S. Pat. No. 6,248,326, Pecheur et al., The FASEB J.
16(10):1266-8 (2002), Almed et al., The Journal of Histochemistry
& Cytochemistry 50:1371-1379 (2002), all of which are
incorporated herein by reference. In a preferred embodiment,
antibodies which immunospecifically bind to a RSV antigen are used
for diagnostic purposes to detect, diagnosis, or monitor a RSV
infection. The detection or diagnosis of a infection can be
conducted utilizing an effective amount (i.e., an amount effective
to be able to detect the expression of a RSV antigen) of a liquid
formulation of the invention in an in vitro assay using techniques
well-known to one of skilled in the art, including but not limited
to, assaying a sample taken from a subject, wherein such sample can
be, but not limited to, secretions from a subject's respiratory
tract (e.g., sputum and saliva) and blood.
[0283] The liquid formulations of the present invention can be used
in any in vitro immunoassay known in the art, such as ELISA, to
detect, diagnose or monitor a RSV infection. In specific
embodiments, the invention provides methods of detecting,
diagnosing or monitoring a RSV infection, said methods comprising:
a) combining an effective amount of a liquid formulation of the
invention comprising a labeled antibody or antibody fragment that
immunospecifically binds to a RSV antigen with a sample from a
subject; b) waiting for a time interval to permit the labeled
antibody or antibody fragment to preferentially bind to a RSV
antigen if present in the sample; c) removing the unbound
antibodies from the sample, and d) detecting the labeled antibody
or antibody fragment in the sample. In specific embodiments, an
antibody or fragment of the liquid formulations of the invention is
not labeled, and a second labeled antibody or antibody fragment
that recognizes the antibody or fragment of the liquid formulation
of the invention can be used.
[0284] In some embodiments, monitoring of an infection is carried
out by repeating the method for diagnosing the infection, for
example, one month after initial diagnosis, six month after initial
diagnosis, and one year after initial diagnosis.
[0285] The liquid formulations of the invention can also be
administered to a subject to detect, diagnose or monitor a RSV
infection.
6. EXAMPLES
Stability Study
[0286] An antibody formulation of the present invention comprising,
in an aqueous carrier, 25 mM of histidine, 1.6 mM of glycine, and
an anti-RSV antibody is prepared according to the following
protocol:
[0287] For a 1 kg solution of buffer: In 800 g water, 3.875 g
histidine (free base) and 0.12 g glycine are dissolved. The pH is
adjusted with 6 N HCl to 6.0.+-.0.2. Water is added to bring the
total mass up to 1.0 kg (qs).
[0288] For the difiltration: After the chromatography steps in the
purification process, the antibody is concentrated to 150.+-.15
g/L. The concentrated product is difiltered into formulation
buffer. The formulated product is diluted to a target manufacturing
concentration of 103.+-.3 g/L.
[0289] For a stability study, two formulations are prepared: one
contains 105 mg/ml of the antibody and the other contained 160
mg/ml of the antibody. The stability of each formulation is
measured using HPSEC in terms of degrees of aggregate formation and
fragmentation during the storage at 2-8.degree. C. for up to 15
months and at 38-42.degree. C. for up to 1 year. For the HPSEC
analysis, typically, Tosohaas G3000WXL column with a mobile phase
containing 0.1 M sodium phosphate and 0.1 M sodium sulfate, pH 6.8,
is used at a flow rate of 0.8 ml/min. A sample containing 250 mg of
protein in an appropriate volume is injected into the column and
protein peaks are detected by 280 nm UV and/or fluorescence (280 nm
excitation and 340 nm emission).
EQUIVALENTS
[0290] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0291] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference.
[0292] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
Sequence CWU 1
1
20917PRTHomo sapiens 1Thr Ser Gly Met Ser Val Gly1 5216PRTHomo
sapiens 2Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu
Lys Ser1 5 10 15310PRTHomo sapiens 3Ser Met Ile Thr Asn Trp Tyr Phe
Asp Val1 5 10410PRTHomo sapiens 4Lys Cys Gln Leu Ser Val Gly Tyr
Met His1 5 1057PRTHomo sapiens 5Asp Thr Ser Lys Leu Ala Ser1
569PRTHomo sapiens 6Phe Gln Gly Ser Gly Tyr Pro Phe Thr1
57120PRTHomo sapiens 7Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu
Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly
Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln
Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile Trp Trp Asp
Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile
Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Lys Val Thr
Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser
Met Ile Thr Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110Gly Thr
Thr Val Thr Val Ser Ser 115 1208106PRTHomo sapiensmisc_featureVL
Domain 8Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val
Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Cys Gln Leu Ser Val Gly
Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg
Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln
Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 1059120PRTHomo sapiensmisc_featureVH Domain 9Gln Val
Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr
Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25
30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro
Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn
Gln Val65 70 75 80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr
Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Ser Met Ile Thr Asn Phe Tyr Phe
Asp Val Trp Gly Ala 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
120107PRTHomo sapiens 10Thr Ala Gly Met Ser Val Gly1 511106PRTHomo
sapiens 11Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Arg Val
Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe
Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 1051210PRTHomo sapiens 12Ser Met Ile Thr Asn Phe
Tyr Phe Asp Val1 5 1013106PRTHomo sapiens 13Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Phe
Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Phe Ser Gly Tyr Pro Phe Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 1051410PRTHomo
sapiens 14Ser Ala Ser Ser Ser Val Gly Tyr Met His1 5 10157PRTHomo
sapiens 15Asp Thr Phe Lys Leu Ala Ser1 5169PRTHomo sapiens 16Phe
Gln Phe Ser Gly Tyr Pro Phe Thr1 517120PRTHomo sapiens 17Gln Val
Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr
Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Pro 20 25
30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn Pro
Ser 50 55 60Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn
Gln Val65 70 75 80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr
Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Asp Met Ile Phe Asn Phe Tyr Phe
Asp Val Trp Gly Ala 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
120187PRTHomo sapiens 18Thr Pro Gly Met Ser Val Gly1 51916PRTHomo
sapiens 19Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn Pro Ser Leu
Lys Asp1 5 10 152010PRTHomo sapiens 20Asp Met Ile Phe Asn Phe Tyr
Phe Asp Val1 5 1021106PRTHomo sapiensmisc_featureVL Domain 21Asp
Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Ser Leu Ser Ser Arg Val Gly Tyr Met
20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
Tyr 35 40 45Asp Thr Phe Tyr Leu Ser Ser Gly Val Pro Ser Arg Phe Ser
Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser
Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 1052210PRTHomo sapiens 22Ser Leu Ser Ser Arg Val Gly Tyr Met
His1 5 10237PRTHomo sapiens 23Asp Thr Phe Tyr Leu Ser Ser1
524120PRTHomo sapiensmisc_featureVH Domain 24Gln Val Thr Leu Arg
Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu
Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Pro 20 25 30Gly Met Ser
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu
Ala Asp Ile Trp Trp Asp Gly Lys Lys His Tyr Asn Pro Ser 50 55 60Leu
Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75
80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Asp Met Ile Phe Asn Phe Tyr Phe Asp Val Trp Gly
Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115 1202516PRTHomo
sapiens 25Asp Ile Trp Trp Asp Gly Lys Lys His Tyr Asn Pro Ser Leu
Lys Asp1 5 10 1526106PRTHomo sapiens 26Asp Ile Gln Met Thr Gln Ser
Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys Ser Leu Ser Ser Arg Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Arg Gly
Leu Pro Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp
Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90
95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105277PRTHomo sapiens
27Asp Thr Arg Gly Leu Pro Ser1 528120PRTHomo sapiens 28Gln Val Thr
Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu
Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Pro 20 25 30Gly
Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40
45Trp Leu Ala Asp Ile Trp Trp Asp Gly Lys Lys His Tyr Asn Pro Ser
50 55 60Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
Val65 70 75 80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala
Thr Tyr Tyr 85 90 95Cys Ala Arg Asp Met Ile Phe Asn Trp Tyr Phe Asp
Val Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
1202910PRTHomo sapiens 29Asp Met Ile Phe Asn Trp Tyr Phe Asp Val1 5
1030106PRTHomo sapiens 30Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Pro
Ser Ser Arg Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Met Arg Leu Ala Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr
Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 1053110PRTHomo sapiens 31Ser Pro
Ser Ser Arg Val Gly Tyr Met His1 5 10327PRTHomo sapiens 32Asp Thr
Met Arg Leu Ala Ser1 533120PRTHomo sapiens 33Gln Val Thr Leu Arg
Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu
Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Pro 20 25 30Gly Met Ser
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu
Ala Asp Ile Trp Trp Asp Gly Lys Lys His Tyr Asn Pro Ser 50 55 60Leu
Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75
80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Asp Met Ile Phe Asn Trp Tyr Phe Asp Val Trp Gly
Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115 12034106PRTHomo
sapiens 34Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Leu Ser Ser Arg Val
Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr 35 40 45Asp Thr Phe Lys Leu Ser Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe
Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys 100 105357PRTHomo sapiens 35Asp Thr Phe Lys Leu Ser
Ser1 536120PRTHomo sapiens 36Gln Val Thr Leu Arg Glu Ser Gly Pro
Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe
Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30Gly Met Ser Val Gly Trp Ile
Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile Trp
Trp Asp Gly Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Asp Arg Leu
Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Lys
Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala
Arg Asp Met Ile Phe Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 1203716PRTHomo sapiens 37Asp
Ile Trp Trp Asp Gly Lys Lys Asp Tyr Asn Pro Ser Leu Lys Asp1 5 10
1538106PRTHomo sapiens 38Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala
Ser Ser Arg Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Phe Lys Leu Ser Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr
Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 1053910PRTHomo sapiens 39Ser Ala
Ser Ser Arg Val Gly Tyr Met His1 5 1040120PRTHomo sapiens 40Gln Val
Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr
Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25
30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu
35 40 45Trp Leu Ala Asp Ile Trp Trp Asp Gly Lys Lys Ser Tyr Asn Pro
Ser 50 55 60Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn
Gln Val65 70 75 80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr
Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Asp Met Ile Phe Asn Phe Tyr Phe
Asp Val Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
1204116PRTHomo sapiens 41Asp Ile Trp Trp Asp Gly Lys Lys Ser Tyr
Asn Pro Ser Leu Lys Asp1 5 10 1542106PRTHomo sapiens 42Asp Ile Gln
Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Ser Leu Ser Ser Arg Val Gly Tyr Met 20 25 30His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40
45Asp Thr Met Tyr Gln Ser Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr
Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
105437PRTHomo sapiens 43Asp Thr Met Tyr Gln Ser Ser1 544120PRTHomo
sapiens 44Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro
Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu
Ser Thr Ala 20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly
Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile Trp Trp Asp Gly Lys Lys
Ser Tyr Asn Pro Ser 50 55 60Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp
Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Lys Val Thr Asn Met Asp
Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Asp Met Ile Phe
Asn Phe Tyr Phe Asp Val Trp Gly Gln 100 105 110Gly Thr Thr Val Thr
Val Ser Ser 115 1204516PRTHomo sapiens 45Asp Ile Trp Trp Asp Asp
Lys Lys Ser Tyr Asn Pro Ser Leu Lys Asp1 5 10 1546106PRTHomo
sapiens 46Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Leu Pro Ser
Ser Arg Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Met Tyr Gln Ala Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr
Tyr Cys Phe Gln Phe Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys 100 1054710PRTHomo sapiens 47Leu Pro Ser
Ser Arg Val Gly Tyr Met His1 5 1048120PRTHomo sapiens 48Gln Val Thr
Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu
Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30Gly
Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40
45Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn Pro Ser
50 55 60Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
Val65 70 75 80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala
Thr Tyr Tyr 85 90 95Cys Ala Arg Asp Met Ile Phe Asn Phe Tyr Phe Asp
Val Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
12049106PRTHomo sapiens 49Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala
Ser Ser Arg Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Phe Phe Leu Asp Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr
Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105507PRTHomo sapiens 50Asp Thr Phe
Phe Leu Asp Ser1 551120PRTHomo sapiens 51Gln Val Thr Leu Arg Glu
Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr
Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30Gly Met Ser Val
Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala
Asp Ile Trp Trp Asp Asp Lys Lys Ser Tyr Asn Pro Ser 50 55 60Leu Lys
Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75
80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr
85 90 95Cys Ala Arg Asp Met Ile Phe Asn Trp Tyr Phe Asp Val Trp Gly
Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115 12052106PRTHomo
sapiens 52Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Arg Val
Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr 35 40 45Asp Thr Arg Tyr Gln Ser Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe
Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105537PRTHomo sapiens 53Asp Thr Arg Tyr Gln Ser
Ser1 554106PRTHomo sapiensmisc_featureVL Domain 54Asp Ile Gln Met
Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp
Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55
60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65
70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe
Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
10555120PRTHomo sapiens 55Gln Val Thr Leu Arg Glu Ser Gly Pro Ala
Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr Leu Thr Cys Thr Phe Ser
Gly Phe Ser Leu Ser Thr Ala 20 25 30Gly Met Ser Val Gly Trp Ile Arg
Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp Leu Ala Asp Ile Trp Trp
Asp Asp Lys Lys Asp Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65 70 75 80Val Leu Lys Val
Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg
Asp Met Ile Phe Asn Trp Tyr Phe Asp Val Trp Gly Ala 100 105 110Gly
Thr Thr Val Thr Val Ser Ser 115 12056106PRTHomo
sapiensmisc_featureVL Domain 56Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser
Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Phe Lys Leu Ala
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala
Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 10557106PRTHomo
sapiensmisc_featureVL Domain 57Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser
Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Tyr Lys Gln Thr
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala
Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105587PRTHomo sapiens 58Asp Thr
Tyr Lys Gln Thr Ser1 559106PRTHomo sapiensmisc_featureVL Domain
59Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr
Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr 35 40 45Asp Thr Arg Tyr Leu Ser Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly
Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 10560106PRTHomo sapiens 60Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Phe Lys Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe
Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Phe Tyr Pro Phe Thr 85 90 95Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105619PRTHomo sapiens 61Phe
Gln Gly Ser Phe Tyr Pro Phe Thr1 562106PRTHomo
sapiensmisc_featureVL Domain 62Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser
Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Phe Lys Leu Thr
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala
Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105637PRTHomo sapiens 63Asp Thr
Phe Lys Leu Thr Ser1 564106PRTHomo sapiensmisc_featureVL Domain
64Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr
Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr 35 40 45Asp Thr Phe Arg Leu Ala Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly
Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 10565106PRTHomo sapiens 65Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Phe Arg Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe
Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105667PRTHomo sapiens 66Asp
Thr Phe Arg Leu Ala Ser1 567120PRTHomo sapiens 67Gln Val Thr Leu
Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10 15Thr Leu Thr
Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala 20 25 30Gly Met
Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45Trp
Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn Pro Ser 50 55
60Leu Lys Asp Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val65
70 75 80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp Thr Ala Thr Tyr
Tyr 85 90 95Cys Ala Arg Asp Met Ile Phe Asn Trp Tyr Phe Asp Val Trp
Gly Ala 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
12068106PRTHomo sapiens 68Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala
Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Tyr Arg His Ala Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr
Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105697PRTHomo sapiens 69Asp Thr Tyr
Arg His Ser Ser1 570106PRTHomo sapiens 70Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30His Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Tyr
Lys Gln Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 10571106PRTHomo
sapiens 71Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Leu Ser Ser Ser Val
Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr 35 40 45Asp Thr Phe Phe His Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Phe
Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 1057210PRTHomo sapiens 72Ser Leu Ser Ser Ser Val
Gly Tyr Met His1 5 10737PRTHomo sapiens 73Asp Thr Phe Phe His Arg
Ser1 574106PRTHomo sapiens 74Asp Ile Gln Met Thr Gln Ser Pro Ser
Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser
Ala Ser Ser Arg Val Gly Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Leu Leu Leu Asp
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75 80Asp Phe Ala
Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr 85 90 95Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys 100 105757PRTHomo sapiens 75Asp Thr
Leu Leu Leu Asp Ser1 576106PRTHomo sapiens 76Asp Ile Gln Met Thr
Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Ser Ala Ser Ser Arg Val Gly Tyr Met 20 25 30His Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr
Ser Phe Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly
Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp65 70 75
80Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr
85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105777PRTHomo
sapiens 77Asp Thr Ser Phe Leu Asp Ser1 578120PRTHomo sapiens 78Gln
Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10
15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ala
20 25 30Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu
Glu 35 40 45Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn
Pro Ser 50 55 60Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys
Asn Gln Val65 70 75 80Val Leu Lys Val Thr Asn Met Asp Pro Ala Asp
Thr Ala Thr Tyr Tyr 85 90 95Cys Ala Arg Asp Met Ile Thr Asn Phe Tyr
Phe Asp Val Trp Gly Ala 100 105 110Gly Thr Thr Val Thr Val Ser Ser
115 1207910PRTHomo sapiens 79Asp Met Ile Thr Asn Phe Tyr Phe Asp
Val1 5
108010PRTHomo sapiens 80Lys Cys Gln Ser Ser Val Gly Tyr Met His1 5
10817PRTHomo sapiens 81Asp Thr Ser Tyr Leu Ala Ser1 58216PRTHomo
sapiens 82Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn Pro Ser Leu
Lys Ser1 5 10 158310PRTHomo sapiens 83Asp Met Ile Thr Asn Trp Tyr
Phe Asp Val1 5 108410PRTHomo sapiens 84Lys Cys Gln Ser Arg Val Gly
Tyr Met His1 5 10857PRTHomo sapiens 85Asp Thr Ser Tyr Leu Ser Ser1
58616PRTHomo sapiens 86Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn
Pro Ser Leu Lys Asp1 5 10 158710PRTHomo sapiens 87Lys Cys Gln Leu
Arg Val Gly Tyr Met His1 5 10887PRTHomo sapiens 88Asp Thr Lys Lys
Leu Ser Ser1 58910PRTHomo sapiens 89Lys Leu Gln Leu Ser Val Gly Tyr
Met His1 5 10907PRTHomo sapiens 90Asp Thr Phe Tyr Leu Ser Ser1
59116PRTHomo sapiens 91Asp Ile Trp Trp Asp Asp Lys Lys His Tyr Asn
Pro Ser Leu Lys Ser1 5 10 159210PRTHomo sapiens 92Lys Leu Gln Ser
Ser Val Gly Tyr Met His1 5 109316PRTHomo sapiens 93Asp Ile Trp Trp
Asp Asp Lys Lys His Tyr Asn Pro Ser Leu Lys Ser1 5 10 159410PRTHomo
sapiens 94Ser Met Ile Phe Asn Trp Tyr Phe Asp Val1 5 109510PRTHomo
sapiens 95Lys Leu Gln Ser Arg Val Gly Tyr Met His1 5 10967PRTHomo
sapiens 96Asp Thr Phe Lys Leu Ser Ser1 59710PRTHomo sapiens 97Ser
Met Ile Phe Asn Phe Tyr Phe Asp Val1 5 109810PRTHomo sapiens 98Lys
Leu Gln Leu Arg Val Gly Tyr Met His1 5 10997PRTHomo sapiens 99Asp
Thr Phe Tyr Leu Ala Ser1 510016PRTHomo sapiens 100Asp Ile Trp Trp
Asp Gly Lys Lys Asp Tyr Asn Pro Ser Leu Lys Ser1 5 10
1510110PRTHomo sapiens 101Lys Leu Ser Leu Ser Val Gly Tyr Met His1
5 101027PRTHomo sapiens 102Asp Thr Ser Lys Leu Pro Ser1
510316PRTHomo sapiens 103Asp Ile Trp Trp Asp Gly Lys Lys Asp Tyr
Asn Pro Ser Leu Lys Asp1 5 10 1510410PRTHomo sapiens 104Lys Leu Ser
Ser Ser Val Gly Tyr Met His1 5 101057PRTHomo sapiens 105Asp Thr Ser
Gly Leu Ala Ser1 510616PRTHomo sapiens 106Asp Ile Trp Trp Asp Gly
Lys Lys His Tyr Asn Pro Ser Leu Lys Ser1 5 10 1510710PRTHomo
sapiens 107Lys Leu Ser Ser Arg Val Gly Tyr Met His1 5 101087PRTHomo
sapiens 108Asp Thr Ser Gly Leu Pro Ser1 510916PRTHomo sapiens
109Asp Ile Trp Trp Asp Asp Lys Lys Ser Tyr Asn Pro Ser Leu Lys Ser1
5 10 1511010PRTHomo sapiens 110Lys Leu Ser Leu Arg Val Gly Tyr Met
His1 5 1011116PRTHomo sapiens 111Asp Ile Trp Trp Asp Asp Lys Lys
Ser Tyr Asn Pro Ser Leu Lys Ser1 5 10 1511210PRTHomo sapiens 112Lys
Cys Ser Leu Ser Val Gly Tyr Met His1 5 101137PRTHomo sapiens 113Asp
Thr Arg Lys Leu Ala Ser1 511416PRTHomo sapiens 114Asp Ile Trp Trp
Asp Gly Lys Lys Ser Tyr Asn Pro Ser Leu Lys Ser1 5 10
1511510PRTHomo sapiens 115Lys Cys Ser Ser Ser Val Gly Tyr Met His1
5 101167PRTHomo sapiens 116Asp Thr Arg Gly Leu Ala Ser1
511710PRTHomo sapiens 117Lys Cys Ser Leu Arg Val Gly Tyr Met His1 5
101187PRTHomo sapiens 118Asp Thr Arg Lys Leu Pro Ser1 511910PRTHomo
sapiens 119Lys Cys Ser Leu Arg Val Gly Tyr Met His1 5
1012010PRTHomo sapiens 120Ser Leu Ser Leu Ser Val Gly Tyr Met His1
5 101217PRTHomo sapiens 121Asp Thr Met Lys Leu Ala Ser1
512210PRTHomo sapiens 122Ser Leu Ser Ser Ser Val Gly Tyr Met His1 5
101237PRTHomo sapiens 123Asp Thr Ser Arg Leu Ala Ser1 51247PRTHomo
sapiens 124Asp Thr Ser Leu Leu Ala Ser1 512510PRTHomo sapiens
125Ser Leu Ser Leu Arg Val Gly Tyr Met His1 5 101267PRTHomo sapiens
126Asp Thr Ser Leu Leu Asp Ser1 512710PRTHomo sapiens 127Ser Cys
Gln Leu Ser Val Gly Tyr Met His1 5 101287PRTHomo sapiens 128Asp Thr
Ser Lys Leu Asp Ser1 512910PRTHomo sapiens 129Ser Cys Gln Ser Ser
Val Gly Tyr Met His1 5 1013010PRTHomo sapiens 130Ser Cys Gln Ser
Arg Val Gly Tyr Met His1 5 101317PRTHomo sapiens 131Asp Thr Leu Lys
Leu Asp Ser1 513210PRTHomo sapiens 132Ser Cys Gln Leu Arg Val Gly
Tyr Met His1 5 101337PRTHomo sapiens 133Asp Thr Leu Leu Leu Ala
Ser1 513410PRTHomo sapiens 134Ser Leu Gln Leu Ser Val Gly Tyr Met
His1 5 101357PRTHomo sapiens 135Asp Thr Leu Lys Leu Ala Ser1
513610PRTHomo sapiens 136Ser Leu Gln Ser Ser Val Gly Tyr Met His1 5
101377PRTHomo sapiens 137Asp Thr Ser Lys Leu Ser Ser1 513810PRTHomo
sapiens 138Ser Leu Gln Ser Arg Val Gly Tyr Met His1 5 101397PRTHomo
sapiens 139Asp Thr Ser Lys Gln Ala Ser1 514010PRTHomo sapiens
140Ser Leu Gln Leu Arg Val Gly Tyr Met His1 5 101417PRTHomo sapiens
141Asp Thr Ser Lys Gln Ser Ser1 514210PRTHomo sapiens 142Ser Cys
Ser Leu Ser Val Gly Tyr Met His1 5 101437PRTHomo sapiens 143Asp Thr
Ser Tyr Leu Ala Ser1 514410PRTHomo sapiens 144Ser Cys Ser Ser Ser
Val Gly Tyr Met His1 5 101457PRTHomo sapiens 145Asp Thr Ser Tyr Leu
Ser Ser1 514610PRTHomo sapiens 146Ser Cys Ser Ser Arg Val Gly Tyr
Met His1 5 101477PRTHomo sapiens 147Asp Thr Ser Tyr Gln Ala Ser1
514810PRTHomo sapiens 148Ser Cys Ser Leu Arg Val Gly Tyr Met His1 5
101497PRTHomo sapiens 149Asp Thr Ser Tyr Gln Ser Ser1 515010PRTHomo
sapiens 150Lys Pro Ser Ser Arg Val Gly Tyr Met His1 5 101517PRTHomo
sapiens 151Asp Thr Met Tyr Gln Ala Ser1 515210PRTHomo sapiens
152Lys Pro Ser Leu Arg Val Gly Tyr Met His1 5 1015310PRTHomo
sapiens 153Lys Pro Ser Ser Ser Val Gly Tyr Met His1 5 101547PRTHomo
sapiens 154Asp Thr Met Lys Gln Ala Ser1 515510PRTHomo sapiens
155Lys Pro Ser Leu Ser Val Gly Tyr Met His1 5 101567PRTHomo sapiens
156Asp Thr Met Lys Gln Ser Ser1 515710PRTHomo sapiens 157Lys Pro
Gln Ser Arg Val Gly Tyr Met His1 5 101587PRTHomo sapiens 158Asp Thr
Met Tyr Leu Ala Ser1 515910PRTHomo sapiens 159Lys Pro Gln Leu Arg
Val Gly Tyr Met His1 5 101607PRTHomo sapiens 160Asp Thr Met Tyr Leu
Ser Ser1 516110PRTHomo sapiens 161Lys Pro Gln Ser Ser Val Gly Tyr
Met His1 5 101627PRTHomo sapiens 162Asp Thr Met Lys Leu Ala Ser1
516310PRTHomo sapiens 163Lys Pro Gln Leu Ser Val Gly Tyr Met His1 5
101647PRTHomo sapiens 164Asp Thr Met Lys Leu Ser Ser1 51657PRTHomo
sapiens 165Asp Thr Ser Lys Leu Ser Ser1 516610PRTHomo sapiens
166Ser Pro Ser Leu Arg Val Gly Tyr Met His1 5 101677PRTHomo sapiens
167Asp Thr Ser Lys Leu Ser Ser1 516810PRTHomo sapiens 168Ser Pro
Ser Ser Ser Val Gly Tyr Met His1 5 1016910PRTHomo sapiens 169Ser
Pro Ser Leu Ser Val Gly Tyr Met His1 5 101707PRTHomo sapiens 170Asp
Thr Arg Tyr Gln Ala Ser1 517110PRTHomo sapiens 171Ser Pro Gln Ser
Arg Val Gly Tyr Met His1 5 101727PRTHomo sapiens 172Asp Thr Arg Lys
Gln Ser Ser1 517310PRTHomo sapiens 173Ser Pro Gln Leu Arg Val Gly
Tyr Met His1 5 101747PRTHomo sapiens 174Asp Thr Arg Lys Leu Ala
Ser1 51757PRTHomo sapiens 175Asp Thr Arg Lys Leu Ser Ser1
517610PRTHomo sapiens 176Ser Pro Gln Ser Ser Val Gly Tyr Met His1 5
1017710PRTHomo sapiens 177Ser Pro Gln Leu Ser Val Gly Tyr Met His1
5 101787PRTHomo sapiens 178Asp Thr Arg Tyr Leu Ala Ser1
517910PRTHomo sapiens 179Lys Ala Gln Ser Arg Val Gly Tyr Met His1 5
1018010PRTHomo sapiens 180Lys Ala Gln Leu Arg Val Gly Tyr Met His1
5 1018110PRTHomo sapiens 181Lys Ala Gln Ser Ser Val Gly Tyr Met
His1 5 1018210PRTHomo sapiens 182Lys Ala Gln Leu Ser Val Gly Tyr
Met His1 5 1018310PRTHomo sapiens 183Lys Ala Ser Ser Arg Val Gly
Tyr Met His1 5 1018410PRTHomo sapiens 184Lys Ala Ser Leu Arg Val
Gly Tyr Met His1 5 1018510PRTHomo sapiens 185Lys Ala Ser Ser Ser
Val Gly Tyr Met His1 5 1018610PRTHomo sapiens 186Lys Ala Ser Leu
Ser Val Gly Tyr Met His1 5 1018710PRTHomo sapiens 187Ser Ala Ser
Leu Arg Val Gly Tyr Met His1 5 1018810PRTHomo sapiens 188Ser Ala
Ser Leu Ser Val Gly Tyr Met His1 5 1018910PRTHomo sapiens 189Ser
Ala Gln Ser Arg Val Gly Tyr Met His1 5 1019010PRTHomo sapiens
190Ser Ala Gln Leu Arg Val Gly Tyr Met His1 5 1019110PRTHomo
sapiens 191Ser Ala Gln Ser Ser Val Gly Tyr Met His1 5
1019210PRTHomo sapiens 192Leu Pro Ser Leu Ser Val Gly Tyr Met His1
5 1019310PRTHomo sapiens 193Leu Pro Ser Ser Ser Val Gly Tyr Met
His1 5 1019410PRTHomo sapiens 194Leu Pro Ser Leu Arg Val Gly Tyr
Met His1 5 1019510PRTHomo sapiens 195Leu Cys Ser Ser Arg Val Gly
Tyr Met His1 5 1019610PRTHomo sapiens 196Leu Cys Ser Leu Ser Val
Gly Tyr Met His1 5 1019710PRTHomo sapiens 197Leu Cys Ser Ser Ser
Val Gly Tyr Met His1 5 1019810PRTHomo sapiens 198Leu Cys Ser Leu
Arg Val Gly Tyr Met His1 5 1019910PRTHomo sapiens 199Leu Pro Gln
Ser Arg Val Gly Tyr Met His1 5 1020010PRTHomo sapiens 200Leu Pro
Gln Leu Ser Val Gly Tyr Met His1 5 1020110PRTHomo sapiens 201Leu
Pro Gln Ser Ser Val Gly Tyr Met His1 5 1020210PRTHomo sapiens
202Leu Pro Gln Leu Arg Val Gly Tyr Met His1 5 1020310PRTHomo
sapiens 203Leu Cys Gln Ser Arg Val Gly Tyr Met His1 5
1020410PRTHomo sapiens 204Leu Cys Gln Leu Ser Val Gly Tyr Met His1
5 1020510PRTHomo sapiens 205Leu Cys Gln Ser Ser Val Gly Tyr Met
His1 5 1020610PRTHomo sapiens 206Leu Cys Gln Leu Arg Val Gly Tyr
Met His1 5 1020710PRTHomo sapiens 207Ser Ala Gln Leu Ser Val Gly
Tyr Met His1 5 1020810PRTHomo sapiens 208Asp Met Ile Thr Asn Phe
Tyr Phe Asp Val1 5 102099PRTHomo sapiens 209Phe Gln Phe Ser Gly Tyr
Pro Phe Tyr1 5
* * * * *