NZ710829B2 - Human antibodies to respiratory syncytial virus f protein and methods of use thereof - Google Patents
Human antibodies to respiratory syncytial virus f protein and methods of use thereof Download PDFInfo
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- NZ710829B2 NZ710829B2 NZ710829A NZ71082914A NZ710829B2 NZ 710829 B2 NZ710829 B2 NZ 710829B2 NZ 710829 A NZ710829 A NZ 710829A NZ 71082914 A NZ71082914 A NZ 71082914A NZ 710829 B2 NZ710829 B2 NZ 710829B2
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Abstract
The present invention provides fully human antibodies that bind to respiratory syncytial virus F protein, compositions comprising the antibodies and methods of use. The antibodies of the invention are useful for preventing fusion of the virus with the cell membrane and preventing cell to cell spread of the virus, thereby providing a means of preventing the infection, or treating a patient suffering from the infection and ameliorating one or more symptoms or complications associated with the viral infection. The antibodies may also be useful for diagnosis of an infection by RSV. of the virus, thereby providing a means of preventing the infection, or treating a patient suffering from the infection and ameliorating one or more symptoms or complications associated with the viral infection. The antibodies may also be useful for diagnosis of an infection by RSV.
Description
HUMAN ANTIBODIES TO RESPIRATORY SYNCYTIAL VIRUS F N AND METHODS
OF USE THEREOF
FIELD OF THE INVENTION
The present invention is related to human antibodies and antigen-binding fragments of
human antibodies that specifically bind to Respiratory Syncytial Virus F protein (RSV-F),
compositions comprising these antibodies and methods of using these antibodies.
STATEMENT OF RELATED ART
Respiratory syncytial virus (RSV) is a negative sense, single stranded RNA virus that is
the leading cause of serious respiratory tract infections in s and children, with the primary
infection occurring 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).
(Feigen et s., 1987, In: Textbook of Pediatric Infectious Diseases, W B Saunders,
Philadelphia at pages 1653-1675; New Vaccine Development, Establishing Priorities, Vol. 1,
1985, National Academy Press, Washington D.C. at pages 397-409; Ruuskanen et al., 1993,
Curr. Probl. Pediatr. 23:50-79; Hall et al., 1979, New Engl. J. Med. 300:393-396). Certain
populations of children are at risk for developing an RSV infection and these include preterm
infants (Hall et al., 1979, New Engl. J. Med. 300:393-396), en with congenital
mations of the airway, children with bronchopulmonary dysplasia (Groothuis et al., 1988,
Pediatrics 82:199-203), children with congenital heart disease (MacDonald et al., New Engl. J.
Med. 307:397-400), and children with congenital or acquired immunodeficiency (Ogra et al.,
1988, Pediatr. Infect. Dis. J. 249; and Pohl et al., 1992, J. Infect. Dis. 165:166-169), and
cystic fibrosis (Abman et al., 1988, J. Pediatr. 113:826-830).
RSV can infect the adult population as well. In this population, RSV causes primarily an
upper respiratory tract e, although elderly patients may be at greater risk for a serious
infection and pneumonia , A. S., eds., 1989, Viral Infections of Humans. iology
and Control, 3rd ed., Plenum Medical Book, New York at pages 525-544), as well as adults who
are immunosuppressed, ularly bone marrow transplant patients (Hertz et al., 1989,
Medicine 68:269-281). Other at risk patients include those suffering from congestive heart
failure and those suffering from chronic obstructive pulmonary disease (ie. COPD). There have
also been reports of epidemics among nursing home ts and institutionalized young adults
(Falsey, A. R., 1991, Infect. Control Hosp. Epidemiol. 12:602-608; and Garvie et al., 1980, Br.
Med. J. 53-1254).
While ent s for established RSV disease are d, more severe forms of
the disease of the lower respiratory tract often require considerable supportive care, including
16536800_1 (GHMatters) P40719NZ00
stration of humidified oxygen and respiratory assistance (Fields et al., eds, 1990, Fields
Virology, 2nd ed., Vol. 1, Raven Press, New York at pages 1045-1072).
Ribavirin, which is the only drug approved for ent of infection, has been shown to
be effective in the treatment of pneumonia and bronchiolitis associated with RSV infection, and
has been shown to modify the course of severe RSV disease in immunocompetent children
(Smith et al., 1991, New Engl. J. Med. 325:24-29). The use of ribavirin is limited due to
concerns surrounding its potential risk to pregnant women who may be exposed to the
lized drug while it is being administered in a hospital environment.
Similarly, while a vaccine may be useful, no commercially available vaccine has been
ped to date. l vaccine candidates have been abandoned and others are under
development (Murphy et al., 1994, Virus Res. 32:13-36). The development of a vaccine has
proven to be problematic. In particular, immunization would be required in the ate
neonatal period since the peak incidence of lower respiratory tract disease occurs at 2-5 months
of age. However, it is known that the neonatal immune response is immature at that time. Plus,
the infant at that point in time still has high titers of maternally ed RSV antibody, which
might reduce vaccine immunogenicity (Murphy et al., 1988, J. Virol. 62:3907-3910; and Murphy
et al., 1991, Vaccine 9:185-189).
Two glycoproteins, F and G, on the surface of RSV have been shown to be targets of
neutralizing antibodies (Fields et al., 1990, supra; and Murphy et al., 1994, supra). These two
proteins are also primarily responsible for viral recognition and entry into target cells; G protein
binds to a ic cellular receptor and the F protein promotes fusion of the virus with the cell.
The F protein is also expressed on the surface of infected cells and is responsible for
subsequent fusion with other cells leading to syncytia ion and cell to cell virus spread.
Currently, the only approved approach to prophylaxis of RSV disease is e
immunization. For example, the humanized antibody, palivizumab (SYNAGIS®), which is
specific for an epitope on the F protein, is approved for intramuscular administration to pediatric
patients for tion 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® is a composite of human
(95%) and murine (5%) antibody sequences. (Johnson et al., (1997), J. Infect. Diseases
176:1215-1224 and U.S. Pat. No. 5,824,307).
Although SYNAGIS® has been successfully used for the prevention of RSV ion in
pediatric ts, multiple intramuscular doses of 15 mg/kg of SYNAGIS® are required to
achieve a lactic effect. The necessity for the administration of multiple intramuscular
doses of antibody requires repeated visits to the doctor’s office, which is not only inconvenient
for the patient but can also result in missed doses.
Efforts were made to improve on the therapeutic profile of an anti-RSV-F dy, and
this lead to the identification and pment of motavizumab, also referred to as NUMAX™.
16536800_1 (GHMatters) P40719NZ00
However, clinical testing revealed that certain of the patients being administered motavizumab
were having severe hypersensitivity reactions. Further development of this humanized anti-
RSV-F dy was then discontinued.
Other antibodies to RSV-F protein have been described and can be found in
467; 307, US 7786273; US 7670600; US 7083784; US6818216; US7700735;
US7553489; 172; US7229619; US7425618; US7740851; US7658921; US7704505;
US7635568; US6855493; US6565849; US7582297; 162; US7700720; US6413771;
524; US6537809; US5762905; 786; US7364742; 329; US7488477;
US7867497; US5534411; US6835372; US7482024; US7691603; US8562996; US8568726;
US20100015596; WO2009088159A1. To date, none other than SYNAGIS® has been
approved by a regulatory agency for use in preventing an RSV ion.
Thus, a need still exists for antibodies that specifically bind to an RSV antigen, such as
RSV-F, which are highly potent and which produce no adverse effects that would preclude
al for clinical use.
BRIEF SUMMARY OF THE INVENTION
The invention provides isolated fully human monoclonal antibodies (mAbs) and antigen-
binding fragments thereof that bind specifically to Respiratory Syncytial Virus F protein (RSV-F).
Given the role that the F protein plays in fusion of the virus with the cell and in cell to cell
transmission of the virus, the antibodies described herein provide a method of inhibiting that
process and as such, may be used for preventing infection of a patient exposed to, or at risk for
acquiring an infection with RSV, or for treating and/or ameliorating one or more symptoms
associated with RSV infection in a patient d to, or at risk for acquiring an ion with
RSV, or suffering from infection with RSV. The antibodies described herein may also be used
to prevent or to treat an RSV infection in a patient who may ence a more severe form of
the RSV ion due to an underlying or pre-existing medical condition. A patient who may
benefit from treatment with an antibody of the invention may be a pre-term infant, a full-term
infant born during RSV season ximately late fall (November) through early spring (April))
that is at risk because of other pre-existing or underlying medical conditions including congenital
heart disease or chronic lung disease, a child greater than one year of age with or without an
underlying medical condition, an institutionalized or hospitalized patient, or an elderly adult (> 65
years of age) with or without an underlying l condition, such as tive heart failure
(CHF), or c obstructive pulmonary disease (COPD). A patient who may benefit from such
y may suffer from a medical condition resulting from a compromised pulmonary,
cardiovascular, neuromuscular, or immune system. For example, the patient may suffer from
an abnormality of the airway, or an airway malfunction, a chronic lung disease, a chronic or
congenital heart disease, a neuromuscular disease that mises the handling of
respiratory secretions, or the patient may be immunosuppressed due to severe combined
immunodeficiency disease or severe acquired immunodeficiency disease, or from any other
16536800_1 (GHMatters) P40719NZ00
ying infectious disease or cancerous ion that results in immunosuppression, or the
patient may be immunosuppressed due to treatment with an immunosuppressive drug (e.g. any
drug used for treating a transplant t) or radiation y. A patient who may benefit from
the antibodies of the invention may be a patient that s from chronic obstructive pulmonary
e (COPD), cystic fibrosis (CF), bronchopulmonary dysplasia, congestive heart failure
(CHF), or congenital heart disease.
Because the antibodies of the invention are more ive at neutralization of RSV
compared to known antibodies, lower doses of the antibodies or antibody fragments could be
used to achieve a greater level of protection against infection with RSV, and more effective
treatment and/or amelioration of ms ated with an RSV infection. Accordingly, the
use of lower doses of antibodies or fragments thereof which immunospecifically bind to RSV-F
antigen may result in fewer or less severe adverse events. se, the use of more effective
neutralizing antibodies may result in a diminished need for frequent administration of the
antibodies or antibody fragments than previously envisioned as necessary for the prevention of
infection, or for virus neutralization, or for treatment or amelioration of one or more symptoms
associated with an RSV infection. Symptoms of RSV infection may include a bluish skin color
due to lack of oxygen (hypoxia), breathing difficulty (rapid breathing or shortness of breath),
cough, croupy cough (“seal bark” cough), fever, nasal flaring, nasal congestion (stuffy nose),
apnea, decreased appetite, dehydration, poor feeding, altered mental status, or wheezing.
Such antibodies may be useful when administered prophylactically (prior to exposure to
the virus and infection with the virus) to lessen the severity, or duration of a primary infection
with RSV, or ameliorate at least one symptom associated with the infection. The antibodies may
be used alone or in conjunction with a second agent useful for treating an RSV ion. In
certain embodiments, the antibodies may be given therapeutically (after exposure to and
infection with the virus) either alone, or in conjunction with a second agent to lessen the severity
or duration of the y infection, or to ameliorate at least one m associated with the
infection. In certain embodiments, the antibodies may be used prophylactically as stand-alone
y to protect ts who are at risk for acquiring an infection with RSV, such as those
described above. Any of these patient populations may benefit from treatment with the
antibodies of the invention, when given alone or in conjunction with a second agent, including
for example, an anti-viral therapy, such as ribavirin, or other anti-viral vaccines.
The antibodies of the invention can be full-length (for example, an IgG1 or IgG4
antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab’)2 or scFv
fragment), and may be modified to affect functionality, e.g., to ate al effector
functions (Reddy et al., (2000), J. Immunol. 25-1933).
Accordingly, in a first aspect, the invention provides an isolated antibody or an antigen-
binding fragment thereof that ically binds to Respiratory Syncytial Virus F protein (RSV-F).
[0017a] In one embodiment, the invention provides an isolated antibody or antigen-binding
16536800_1 (GHMatters) P40719NZ00
fragment thereof that specifically binds to Respiratory Syncytial Virus F protein (RSV-F),
wherein the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs
(HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR)
comprising the amino acid sequence of SEQ ID NO: 274; and three light chain CDRs (LCDR1,
LCDR2 and LCDR3) ned within a light chain variable region (LCVR) comprising the amino
acid sequence of SEQ ID NO: 282, wherein the CDRs are identified by the Kabat definition, the
Chothia tion, or the AbM definition.
In one embodiment, the invention provides an isolated antibody or an antigen-binding
fragment thereof that specifically binds to Respiratory Syncytial Virus F protein (RSV-F),
wherein the antibody has one or more of the ing characteristics:
(a) is a fully human monoclonal dy;
(b) interacts with an amino acid sequence comprising amino acid residues ranging
from about position 161 to about position 188 of SEQ ID NO: 354;
(c) interacts with either the serine at position 173 of SEQ ID NO: 354, or the
threonine at position 174 of SEQ ID NO: 354, or both the serine at position 173 of
SEQ ID NO: 354 and the threonine at position 174 of SEQ ID NO: 354;
(d) is capable of lizing respiratory syncytial virus subtype A and subtype B
strains in vitro;
(e) trates the ability to icantly reduce the nasal and/or lung viral load in
vivo in an animal model of RSV infection; or
(f) inhibits fusion of the virus to the cell.
In one ment, the invention provides an isolated antibody or an n-binding
fragment thereof that specifically binds to Respiratory ial Virus F protein (RSV-F),
wherein the antibody interacts with an amino acid sequence comprising amino acid residues
g from about position 161 to about position 188 of SEQ ID NO: 354.
In one embodiment, the antibody is a fully human monoclonal antibody or an antigen-
binding fragment thereof that specifically binds to Respiratory Syncytial Virus F protein (RSV-F),
wherein the antibody or an antigen-binding fragment thereof interacts with an amino acid
sequence comprising amino acid residues ranging from about position 161 to about position 188
of SEQ ID NO: 354, and wherein the antibody neutralizes respiratory syncytial virus subtype A
and/or subtype B strains in vitro and in vivo.
In one embodiment, the invention es an isolated antibody or an antigen-binding
fragment thereof that specifically binds to Respiratory Syncytial Virus F protein (RSV-F),
wherein the antibody or the n-binding fragment thereof demonstrates the ability to
icantly reduce the lung viral load in a mouse model of RSV infection when administered at
a dose ranging from about 0.05 mg/kg to about 0.15 mg/kg.
In one embodiment, the ion provides an isolated antibody or an antigen-binding
fragment thereof that specifically binds to Respiratory Syncytial Virus F protein (RSV-F),
00_1 (GHMatters) P40719NZ00
wherein the antibody or the antigen-binding fragment thereof demonstrates a 1-2 logs r
reduction of nasal and/or lung viral titers as compared to palivizumab in a cotton rat model of
RSV infection when administered at a dose ranging from about 0.62 mg/kg to about 5.0 mg/kg.
In one embodiment, the invention provides an isolated dy or an antigen-binding
fragment thereof that specifically binds to Respiratory Syncytial Virus F protein ),
wherein the antibody or the antigen-binding fragment f demonstrates an ED99 of about
0.15 mg/kg or less when administered in a mouse model of RSV subtype A infection.
In one embodiment, the invention provides an isolated antibody or an antigen-binding
fragment thereof that specifically binds to Respiratory Syncytial Virus F n (RSV-F),
n the antibody or the antigen-binding fragment thereof demonstrates an ED99 of about
0.62 mg/kg or less when administered in a cotton rat model of RSV e A infection.
In one embodiment, the invention provides an isolated antibody or an antigen-binding
fragment thereof that specifically binds to Respiratory Syncytial Virus F protein (RSV-F),
wherein the antibody or the antigen-binding fragment thereof demonstrates an ED99 of about 2.5
mg/kg or less when administered in a cotton rat model of RSV subtype B infection.
In one embodiment, the isolated antibody or an antigen-binding fragment thereof that
specifically binds to Respiratory Syncytial Virus F protein (RSV-F), demonstrates an ED99 that is
about 2 to 3 fold lower than the ED99 for palivizumab or motavizumab.
In one embodiment, the isolated antibody or an antigen-binding fragment thereof that
specifically binds to Respiratory Syncytial Virus F protein (RSV-F), demonstrates a half maximal
inhibitory concentration (IC50 ) of about 2 pM to about 600 pM in a microneutralization assay
ic for RSV subtype A strains of RSV.
In one embodiment, the ed antibody or an antigen-binding fragment thereof that
specifically binds to Respiratory Syncytial Virus F protein (RSV-F), trates a half maximal
inhibitory concentration (IC50 ) of about 6 pM to about 100 pM in a microneutralization assay
specific for RSV subtype B strains of RSV.
In one ment, the isolated dy or an antigen-binding nt f that
specifically binds to RSV-F protein demonstrates a neutralization potency against one or more
subtype A laboratory strains of RSV that is about a 15 to 17 fold improvement over palivizumab,
or demonstrates a neutralization potency against one or more subtype A clinical strains of RSV
that is about 10 to 22 fold improvement over zumab.
In one embodiment, the isolated antibody or an antigen-binding fragment thereof that
ically binds to Respiratory Syncytial Virus F protein ), demonstrates a
neutralization potency against one or more subtype B laboratory strains of RSV that is about a 2
to 5 fold improvement over palivizumab.
In one embodiment, the isolated antibody or an antigen-binding fragment thereof that
specifically binds to Respiratory Syncytial Virus F protein (RSV-F), demonstrates a
16536800_1 (GHMatters) P40719NZ00
neutralization potency t one or more subtype A tory strains or subtype A clinical
s of RSV that is about a 0.5 to 2 fold improvement over AM-22.
In one embodiment, the isolated antibody or an antigen-binding fragment f that
specifically binds to Respiratory Syncytial Virus F protein (RSV-F), demonstrates a
neutralization potency against one or more subtype B laboratory strains of RSV that is about a
2.5 to 17 fold improvement over AM-22.
In one embodiment, the isolated human antibody or an antigen-binding fragment thereof
that specifically binds to atory Syncytial Virus F protein (RSV-F), exhibits a KD ranging
from 1 X 10-7 M to 6 X 10-10 M, as measured by e plasmon resonance.
In one embodiment, the isolated human antibody or an n-binding fragment thereof
that specifically binds to Respiratory Syncytial Virus F protein (RSV-F), exhibits a KD ranging
from 1 X 10-7 M to 9 X 10-9 M, as measured by surface plasmon nce.
In one ment, the isolated human antibody or antigen-binding nt thereof
that specifically binds to atory Syncytial Virus F protein (RSV-F), comprises the three
heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a HCVR amino acid
sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114,
130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322 and 338; and the three light
chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a LCVR amino acid sequence
selected from the group consisting of SEQ ID NOs : 10, 26, 42, 58, 74, 90, 106, 122, 138, 154,
170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330 and 346.
Methods and techniques for identifying CDRs within HCVR and LCVR amino acid
sequences are well known in the art and can be used to identify CDRs within the specified
HCVR and/or LCVR amino acid ces disclosed herein. ary conventions that can
be used to identify the boundaries of CDRs e, e.g. , the Kabat definition, the Chothia
definition, and the AbM definition. In general terms, the Kabat definition is based on sequence
variability, the Chothia definition is based on the location of the structural loop regions, and the
AbM definition is a compromise between the Kabat and Chothia approaches. See , e.g. , Kabat,
"Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md.
(1991); Al-Lazikani et al., (1997), J. Mol. Biol. 273:927-948; and Martin et al., (1989), Proc. Natl.
Acad. Sci. USA 86 :9268-9272. Public databases are also available for identifying CDR
sequences within an antibody.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof
that specifically binds to Respiratory Syncytial Virus F protein ), comprises a heavy chain
variable region (HCVR) having an amino acid sequence selected from the group consisting of
SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274,
290, 306, 322 and 338.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof
that specifically binds to Respiratory Syncytial Virus F protein ), comprises a light chain
16536800_1 (GHMatters) P40719NZ00
variable region (LCVR) having an amino acid sequence ed from the group consisting of
SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266,
282, 298, 314, 330 and 346.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof
that specifically binds to Respiratory Syncytial Virus F protein (RSV-F), comprises a heavy chain
variable region (HCVR) having an amino acid sequence selected from the group consisting of
SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274,
290, 306, 322 and 338; and a light chain variable region (LCVR) having an amino acid
sequence selected from the group consisting of SEQ ID NOs : 10, 26, 42, 58, 74, 90, 106, 122,
138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, 314, 330 and 346.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof
that specifically binds to Respiratory Syncytial Virus F protein (RSV-F), comprises the heavy
chain amino acid sequence of SEQ ID NO: 363 and the light chain amino acid sequence of SEQ
ID NO: 364.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof
that specifically binds to Respiratory ial Virus F protein ), ses a
HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs:
SEQ ID NO: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 130/138, 146/154,
162/170, 178/186, 194/202, 210/218, 226/234, 0, 6, 274/282, 290/298, 306/314,
322/330 and 338/346.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof
that ically binds to Respiratory Syncytial Virus F protein (RSV-F), comprises a
HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs:
274/282 and 338/346.
In one embodiment, the isolated human dy or antigen-binding fragment thereof
that specifically binds to Respiratory Syncytial Virus F protein (RSV-F), comprises:
(a) a HCDR3 domain having an amino acid sequence selected from the group
ting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232,
248, 264, 280, 296, 312, 328, and 344; and
(b) a LCDR3 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240,
256, 272, 288, 304, 320, 336 and 352.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof
that specifically binds to Respiratory Syncytial Virus F protein (RSV-F), r comprises:
(c) a HCDR1 domain having an amino acid sequence selected from the group
ting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228,
244, 260, 276, 292, 308, 324 and 340;
16536800_1 (GHMatters) P40719NZ00
(d) a HCDR2 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230,
246, 262, 278, 294, 310, 326 and 342;
(e) a LCDR1 domain having an amino acid ce selected from the group
consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236,
252, 268, 284, 300, 316, 332 and 348; and
(f) a LCDR2 domain having an amino acid sequence ed from the group
consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238,
254, 270, 286, 302, 318, 334 and 350.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof
that specifically binds to Respiratory Syncytial Virus F n (RSV-F) ses:
(a) a HCDR1 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212,
228, 244, 260, 276, 292, 308, 324 and 340;
(b) a HCDR2 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214,
230, 246, 262, 278, 294, 310, 326 and 342;
(c) a HCDR3 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216,
232, 248, 264, 280, 296, 312, 328, and 344;
(d) a LCDR1 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204,
220, 236, 252, 268, 284, 300, 316, 332 and 348;
(e) a LCDR2 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206,
222, 238, 254, 270, 286, 302, 318, 334 and 350; and
(f) a LCDR3 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208,
224, 240, 256, 272, 288, 304, 320, 336 and 352.
In one embodiment, the isolated human antibody or antigen-binding nt thereof
that specifically binds to Respiratory Syncytial Virus F protein (RSV-F) comprises:
(a) a HCDR1 domain having an amino acid ce selected from the group
consisting of SEQ ID NOs: 276 and 340;
(b) a HCDR2 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 278 and 342;
(c) a HCDR3 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 280 and 344;
16536800_1 (GHMatters) P40719NZ00
(d) a LCDR1 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 284 and 348;
(e) a LCDR2 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 286 and 350; and
(f) a LCDR3 domain having an amino acid ce selected from the group
consisting of SEQ ID NOs: 288 and 352.
In one embodiment, the isolated human antibody or antigen binding fragment thereof
that specifically binds to RSV-F comprises the HCDR1, HCDR2 and HCDR3 amino acid
ces of SEQ ID NOs: 276, 278 and 280, respectively and LCDR1, LCDR2 and LCDR3
amino acid sequences of SEQ ID NOs: 284, 286 and 288, respectively.
In one ment, the isolated human antibody or antigen binding fragment f
that specifically binds to RSV-F comprises the HCDR1, HCDR2 and HCDR3 amino acid
sequences of SEQ ID NOs: 340, 342 and 344, respectively and LCDR1, LCDR2 and LCDR3
amino acid sequences of SEQ ID NOs: 348, 350 and 352, respectively.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof
that specifically binds to Respiratory Syncytial Virus F protein (RSV-F) competes for specific
binding to RSV-F with an antibody or antigen-binding fragment comprising heavy and light chain
sequence pairs selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58,
66/74, 82/90, 98/106, 114/122, 130/138, 4, 162/170, 178/186, 194/202, 210/218,
226/234, 242/250, 258/266, 274/282, 290/298, 306/314, 322/330 and 338/346.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof,
which comprises heavy and light chain sequence pairs selected from the group consisting of
SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, , 114/122, 130/138, 146/154,
162/170, 178/186, 194/202, 8, 226/234, 242/250, 258/266, 274/282, 290/298, 306/314,
322/330 and 338/346, and which specifically binds to atory Syncytial Virus F protein
(RSV-F), does not compete for specific binding to RSV-F with palivizumab, motavizumab, or
AM-22.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof
that specifically binds to Respiratory ial Virus F protein (RSV-F) binds the same epitope
on RSV-F that is recognized by an antibody comprising heavy and light chain ce pairs
selected from the group consisting of SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90,
98/106, 114/122, 130/138, 146/154, 162/170, 178/186, 194/202, 8, 226/234, 242/250,
258/266, 274/282, 290/298, 4, 322/330 and 338/346.
In one embodiment, the isolated human antibody or antigen-binding fragment thereof,
which comprises heavy and light chain sequence pairs selected from the group consisting of
SEQ ID NOs: 2/10, 18/26, 34/42, 50/58, 66/74, 82/90, 98/106, 114/122, 8, 146/154,
162/170, 178/186, 194/202, 8, 226/234, 242/250, 258/266, 274/282, 290/298, 4,
322/330 and 338/346, and which specifically binds to Respiratory ial Virus F protein
16536800_1 ters) P40719NZ00
(RSV-F), does not bind the same epitope on RSV-F as palivizumab or motavizumab.
In one embodiment, the ion provides a fully human monoclonal antibody or
antigen-binding fragment thereof that specifically binds to RSV-F, wherein the antibody or
fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR
having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34,
50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322 and 338,
or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at
least 99% ce identity; (ii) comprises a LCVR having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186,
202, 218, 234, 250, 266, 282, 298, 314, 330 and 346, or a substantially similar sequence
thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence ty; (iii)
ses a HCDR3 domain having an amino acid sequence selected from the group ting
of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264,
280, 296, 312, 328, and 344, or a substantially similar sequence thereof having at least 90%, at
least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having an
amino acid ce selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96,
112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336 and 352, or a
substantially similar sequence f having at least 90%, at least 95%, at least 98% or at least
99% sequence identity; (iv) comprises a HCDR1 domain having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148,
164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324 and 340, or a substantially similar
sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence
identity; (v) a HCDR2 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230,
246, 262, 278, 294, 310, 326 and 342, or a substantially similar sequence thereof having at
least 90%, at least 95%, at least 98% or at least 99% sequence identity; (vi) a LCDR1 domain
having an amino acid sequence ed from the group consisting of SEQ ID NOs: 12, 28, 44,
60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332 and 348,
or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at
least 99% ce identity; (vii) and a LCDR2 domain having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158,
174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334 and 350, or a substantially similar
sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence
identity; (viii) exhibits a KD ranging from about 1 X 10-7 M to about 6 X 10-10 M as measured by
surface plasmon resonance; (ix) is e of neutralizing respiratory syncytial virus subtype A
and/or subtype B strains in vitro; (x) demonstrates the ability to significantly re duce the viral
load in a mouse model of RSV infection when administered at a dose ranging from about 0.05
mg/kg to about 0.15 mg/kg; (xi) demonstrates a 1 to 2 logs greater reduction of nasal and/or
00_1 (GHMatters) P40719NZ00
lung viral titers in a cotton rat model of RSV infection at a dose ranging from about 0.62 mg/kg
to about 5.0 mg/kg when compared to palivizumab; (xii) demonstrates an effective dose 99
(ED 99 ) ranging from about 0.15 mg/kg to about 2.5 mg/kg when administered in an animal
model of RSV ion (e.g. a mouse model or a cotton rat model); or (xiii) demonstrates a half
maximal tory concentration (IC50 ) of about 2 pM to about 15 pM in a microneutralization
assay specific for RSV subtype A strains of RSV and a half maximal inhibitory concentration
(IC 50 ) of about 6 pM to about 100 pM in a microneutralization assay.
In one embodiment, the invention provides a fully human monoclonal antibody or
antigen-binding fragment f that specifically binds to RSV-F, wherein the antibody or
fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR
having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34,
50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322 and 338,
or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at
least 99% sequence ty; (ii) comprises a LCVR having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186,
202, 218, 234, 250, 266, 282, 298, 314, 330 and 346, or a substantially similar sequence
thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence ty; (iii)
comprises a HCDR3 domain having an amino acid sequence selected from the group consisting
of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264,
280, 296, 312, 328, and 344, or a ntially similar sequence thereof having at least 90%, at
least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having an
amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96,
112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336 and 352, or a
substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least
99% sequence identity; (iv) comprises a HCDR1 domain having an amino acid sequence
ed from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148,
164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324 and 340, or a substantially similar
sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% ce
identity; (v) a HCDR2 domain having an amino acid ce selected from the group
consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230,
246, 262, 278, 294, 310, 326 and 342, or a substantially similar ce thereof having at
least 90%, at least 95%, at least 98% or at least 99% sequence identity; (vi) a LCDR1 domain
having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44,
60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332 and 348,
or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at
least 99% sequence identity; (vii) and a LCDR2 domain having an amino acid sequence
ed from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158,
174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334 and 350, or a substantially similar
16536800_1 (GHMatters) P40719NZ00
ce thereof having at least 90%, at least 95%, at least 98% or at least 99% ce
identity; (viii) exhibits a KD ranging from about 1 X 10-7 M to about 6 X 10-10 M; (ix) is capable of
neutralizing respiratory ial virus subtype A and/or subtype B strains in vitro; (x)
demonstrates the ability to icantly reduce the viral load in an animal model of RSV infection
(e.g. a mouse model) when administered at a dose ranging from about 0.05 mg/kg to about 0.15
mg/kg; (xi) demonstrates a 1 to 2 logs greater reduction of nasal and/or lung viral titers in an
animal model of RSV infection (e.g. a cotton rat model) at a dose ranging from about 0.62
mg/kg to about 5.0 mg/kg when compared to palivizumab; (xii) demonstrates an effective dose
99 (ED99 ) ranging from about 0.05 mg/kg to about 2.5 mg/kg when administered in an animal
model of RSV infection (e.g. a mouse model or a cotton rat model); (xiii) demonstrates an ED99
that is about 2 to 3 fold lower than the ED99 for palivizumab or motavizumab; (xiv) demonstrates
a neutralization potency against one or more subtype A tory strains of RSV that is about
to 17 fold improvement over palivizumab, or demonstrates a neutralization potency against
one or more subtype A clinical strains of RSV that is about a 10-22 fold improvement over
palivizumab; (xv) demonstrates a neutralization potency against one or more subtype B
laboratory strains of RSV that is about a 2 to 5 fold improvement over palivizumab; (xvi)
demonstrates a neutralization potency against one or more subtype A laboratory strains or
subtype A clinical s of RSV that is about 0.5 to 2 fold improvement over AM-22; (xvii)
demonstrates a neutralization potency against one or more subtype B laboratory s of RSV
that is about a 2.5 to 17 fold improvement over AM-22.
In one embodiment, the invention es a fully human onal antibody or
antigen-binding fragment thereof that specifically binds to RSV-F, wherein the antibody or
fragment thereof exhibits one or more of the following characteristics: (i) comprises a HCVR
having an amino acid sequence selected from the group ting of SEQ ID NOs: 2, 18, 34,
50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, 306, 322 and 338,
or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at
least 99% sequence identity; (ii) comprises a LCVR having an amino acid sequence selected
from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186,
202, 218, 234, 250, 266, 282, 298, 314, 330 and 346, or a ntially similar sequence
thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (iii)
comprises a HCDR3 domain having an amino acid sequence selected from the group consisting
of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264,
280, 296, 312, 328, and 344, or a substantially similar ce thereof having at least 90%, at
least 95%, at least 98% or at least 99% sequence identity; and a LCDR3 domain having an
amino acid sequence selected from the group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96,
112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, 320, 336 and 352, or a
ntially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least
99% sequence identity; (iv) ses a HCDR1 domain having an amino acid sequence
16536800_1 (GHMatters) P40719NZ00
selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148,
164, 180, 196, 212, 228, 244, 260, 276, 292, 308, 324 and 340, or a ntially similar
sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence
identity; (v) a HCDR2 domain having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230,
246, 262, 278, 294, 310, 326 and 342, or a substantially similar sequence thereof having at
least 90%, at least 95%, at least 98% or at least 99% sequence identity; (vi) a LCDR1 domain
having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44,
60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, 316, 332 and 348,
or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at
least 99% sequence identity; (vii) and a LCDR2 domain having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158,
174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334 and 350, or a substantially similar
sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% ce
identity; (viii) exhibits a KD ranging from about 1 X 10-7 M to about 6 X 10-10 M; (ix) is capable of
neutralizing respiratory syncytial virus subtype A and/or subtype B strains in vitro; (x)
trates the ability to significantly reduce the viral load in an mammal having an RSV
infection; (xi) interacts with an amino acid sequence comprising amino acid residues ranging
from about on 161 to about position 188 of SEQ ID NO: 354; (xii) interacts with either the
serine at position 173 of SEQ ID O: 354, or the threonine at position 174 of SEQ ID NO: 354, or
both the serine at position 173 of SEQ ID O: 354, and the threonine at position 174 of SEQ ID
NO: 354; (xiii) inhibits fusion of RSV to the host cell; (xiv) does not compete with
palivizumab or AM-22 for binding to RSV-F.
In one embodiment, the invention provides an isolated human monoclonal antibody that
specifically binds Respiratory ial Virus F protein (RSV-F), or an antigen-binding fragment
thereof, wherein the antibody or antigen-binding fragment thereof interacts with an amino acid
sequence comprising amino acid residues ranging from about position 161 to about position 188
of SEQ ID NO: 354.
In one ment, the invention provides an isolated human monoclonal antibody that
specifically binds RSV-F, or an antigen-binding fragment thereof, wherein the dy or
antigen-binding fragment thereof interacts with at least one amino acid sequence selected from
the group consisting of SEQ ID NO: 355 and 356.
In one embodiment, the invention provides an isolated human monoclonal dy that
ically binds RSV-F, or an antigen-binding fragment thereof, wherein the antibody or
antigen-binding nt thereof interacts with at least one amino acid residue within residues
161 h 188 of SEQ ID NO: 354.
In one embodiment, the invention provides an isolated human monoclonal dy that
specifically binds RSV-F, or an n-binding fragment thereof, wherein the antibody or
16536800_1 (GHMatters) P40719NZ00
antigen-binding fragment thereof interacts with at least one amino acid residue within SEQ ID
NO: 355 or SEQ ID NO:356.
In one embodiment, the invention es an isolated human monoclonal antibody that
specifically binds RSV-F, or an antigen-binding fragment thereof, wherein the antibody or
antigen-binding nt thereof cts with either the serine at on 173 of SEQ ID NO:
354, or the threonine at position 174 of SEQ ID NO: 354, or both the serine at position 173 of
SEQ ID NO: 354 and the threonine at position 174 of SEQ ID NO: 354.
In one embodiment, the invention provides an isolated human monoclonal antibody or
antigen-binding fragment thereof that specifically binds to Respiratory ial Virus F protein
(RSV-F), wherein the antibody or antigen-binding fragment thereof interacts with an amino acid
sequence sing amino acid residues ranging from about position 161 to about position 188
of SEQ ID NO: 354, and wherein the antibody or antigen-binding fragment thereof comprises
three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within the heavy chain
variable region (HCVR) amino acid sequence of SEQ ID NO: 274; and three light chain CDRs
(LCDR1, LCDR2 and LCDR3) contained within the light chain variable region (LCVR) amino
acid sequence of SEQ ID NO: 282.
In one embodiment, the invention provides an isolated human monoclonal antibody or
antigen-binding fragment thereof that ically binds to Respiratory Syncytial Virus F protein
(RSV-F), wherein the antibody or n-binding fragment f comprises:
(a) a HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 276;
(b) a HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 278;
(c) a HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 280;
(d) a LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 284;
(e) a LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 286; and
(f) a LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 288.
In one embodiment, the invention provides an isolated human monoclonal antibody, or
an antigen-binding fragment thereof, that binds specifically to RSV-F, wherein the antibody
comprises the three HCDRs contained within the heavy chain le region (HCVR) amino
acid sequence of SEQ ID NO: 274; and the three light chain CDRs (LCDR1, LCDR2 and
LCDR3) contained within the light chain variable region (LCVR) amino acid sequence of SEQ ID
NO: 282 and n the antibody or antigen-binding fragment thereof interacts with at least
one amino acid sequence selected from the group consisting of SEQ ID NO: 355 and 356.
In one ment, the invention provides an isolated human monoclonal antibody, or
an antigen-binding fragment thereof, that binds specifically to RSV-F, wherein the antibody
comprises the three HCDRs contained within the heavy chain variable region (HCVR) amino
acid sequence of SEQ ID NO: 274; and the three light chain CDRs (LCDR1, LCDR2 and
LCDR3) contained within the light chain variable region (LCVR) amino acid sequence of SEQ ID
NO: 282 and wherein the antibody or antigen-binding fragment thereof interacts with at least
16536800_1 (GHMatters) P40719NZ00
one amino acid residue within residues 161 through 188 of SEQ ID NO: 354.
In one embodiment, the invention provides an isolated human monoclonal dy, or
an n-binding fragment thereof, that binds specifically to RSV-F, wherein the dy
comprises the three HCDRs ned within the heavy chain le region (HCVR) amino
acid sequence of SEQ ID NO: 274; and the three light chain CDRs (LCDR1, LCDR2 and
LCDR3) contained within the light chain variable region (LCVR) amino acid sequence of SEQ ID
NO: 282 and wherein the dy or n-binding fragment thereof interacts with at least
one amino acid residue within SEQ ID NO: 355 or SEQ ID NO:356.
In one embodiment, the invention provides an isolated human monoclonal antibody, or
an antigen-binding fragment thereof, that binds specifically to RSV-F, n the dy
comprises the three HCDRs contained within the heavy chain variable region (HCVR) amino
acid sequence of SEQ ID NO: 274; and the three light chain CDRs (LCDR1, LCDR2 and
LCDR3) contained within the light chain variable region (LCVR) amino acid sequence of SEQ ID
NO: 282, wherein the antibody or antigen-binding fragment thereof interacts with either the
serine at position 173 of SEQ ID NO: 354, or the threonine at position 174 of SEQ ID NO: 354,
or both the serine at on 173 of SEQ ID NO: 354 and the threonine at position 174 of SEQ
ID NO: 354.
In one embodiment, the invention provides an isolated human dy, or an antigen-
binding nt thereof that does not cross-compete for binding to RSV-F with palivizumab, or
motavizumab.
In one embodiment, the invention provides an isolated human antibody, or an n-
binding fragment thereof that does not cross-compete for binding to RSV-F with AM-22.
In one embodiment, the invention provides an isolated human antibody, or an antigen-
binding fragment thereof that does not bind the same epitope on RSV-F as palivizumab.
In one embodiment, the invention provides an isolated human antibody, or an antigen-
binding fragment thereof that does not bind the same epitope on RSV-F as motavizumab.
In one embodiment, the invention provides an isolated human monoclonal antibody, or
an antigen-binding fragment thereof that does not bind to an epitope on RSV-F ranging from
about amino acid residue 255 to about amino acid residue 276 of SEQ ID NO: 354.
In one embodiment, the isolated human monoclonal antibody, or an n-binding
fragment thereof does not bind to the same epitope on RSV-F as palivizumab, wherein the
epitope ranges from about amino acid residue 255 to about amino acid residue 276 of SEQ ID
NO: 354.
In a second aspect, the invention es nucleic acid molecules encoding antibodies
or fragments thereof that specifically bind to RSV-F. Recombinant expression vectors carrying
the nucleic acids of the invention, and host cells into which such vectors have been introduced,
are also assed by the invention, as are methods of producing the antibodies by culturing
the host cells under conditions permitting production of the antibodies, and recovering the
16536800_1 (GHMatters) P40719NZ00
antibodies produced.
In one embodiment, the ion provides an antibody or fragment thereof comprising a
HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:
1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, 305, 321,
and 337 or a substantially cal sequence having at least 90%, at least 95%, at least 98%, or
at least 99% homology thereof.
In one embodiment, the HCVR is encoded by a nucleic acid sequence ed from the
group consisting of SEQ ID NO: 273 and 337.
In one embodiment, the antibody or fragment thereof further comprises a LCVR encoded
by a nucleic acid ce selected from the group ting of SEQ ID NO: 9, 25, 41, 57, 73,
89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, 313, 329, and 345, or a
substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99%
homology thereof.
In one embodiment, the LCVR is encoded by a nucleic acid sequence selected from the
group consisting of SEQ ID NO: 281 and 345.
In one embodiment, the invention also provides an antibody or antigen-binding nt
of an antibody comprising a HCDR3 domain encoded by a nucleotide sequence selected from
the group consisting of SEQ ID NO: 7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199,
215, 231, 247, 263, 279, 295, 311, 327, and 343 or a substantially similar sequence f
having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; and a LCDR3
domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:
, 31, 47, 63, 79, 95, 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, 319,
335, and 351, or a substantially similar sequence thereof having at least 90%, at least 95%, at
least 98% or at least 99% sequence identity.
In one embodiment, the ion provides an antibody or fragment thereof r
comprising a HCDR1 domain encoded by a nucleotide sequence selected from the group
consisting of SEQ ID NO: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243,
259, 275, 291, 307, 323, and 339, or a substantially similar sequence thereof having at least
90%, at least 95%, at least 98% or at least 99% ce identity; a HCDR2 domain encoded
by a nucleotide ce selected from the group consisting of SEQ ID NO: 5, 21, 37, 53, 69,
85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, 309, 325, and 341, or a
substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least
99% sequence identity; a LCDR1 domain encoded by a nucleotide sequence selected from the
group consisting of SEQ ID NO: 11, 27, 43, 59, 75, 91, 107, 123, 139, 155, 171, 187, 203, 219,
235, 251, 267, 283, 299, 315, 331, and 347, or a substantially similar sequence thereof having
at least 90%, at least 95%, at least 98% or at least 99% sequence ty; and a LCDR2
domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NO:
13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, 317,
16536800_1 (GHMatters) P40719NZ00
333, and 349, or a ntially similar sequence thereof having at least 90%, at least 95%, at
least 98% or at least 99% sequence identity.
In a third aspect, the invention features a human antibody or antigen-binding fragment
specific for RSV-F sing a HCVR encoded by nucleotide sequence segments derived from
VH, DH and JH germline sequences, and a LCVR encoded by nucleotide ce segments
derived from VK and JK germline sequences.
The invention encompasses antibodies having a modified glycosylation pattern. In some
applications, modification to remove rable glycosylation sites may be useful, or e.g.,
l of a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function
(see Shield et al. (2002) JBC 277:26733). In other applications, modification of galactosylation
can be made in order to modify complement dependent cytotoxicity (CDC).
In a fourth aspect, the invention provides a pharmaceutical composition comprising at
least one isolated fully human monoclonal antibody or antigen-binding fragment thereof that
binds to RSV-F and a pharmaceutically able carrier or diluent. In one embodiment, the
invention provides a pharmaceutical composition comprising two fully human monoclonal
antibodies or antigen-binding fragments thereof, which either bind to the same epitope or bind to
two ent epitopes on RSV-F and a pharmaceutically acceptable carrier or diluent. It is to be
understood that any combination of antibodies as described herein may be used in a
pharmaceutical composition to e the desired results in the patient population in need of
such therapy. For example, two antibodies that recognize and/or bind RSV-F may be used in a
composition. Alternatively, two antibodies, one that izes and/or binds RSV-F and a
second antibody that binds to another antigen on RSV (e.g . RSV-G) may be used in a
composition. In one embodiment, two antibodies, one that recognizes and/or binds RSV-F and
a second antibody that binds to a metapneumovirus antigen may be used in a composition.
Alternatively, two or more antibodies may be used in a composition, one that recognizes and/or
binds to RSV-F, one that binds to a metapneumovirus antigen and one that binds to an
influenza virus n or to any other virus that causes respiratory diseases.
In one embodiment, the ceutical composition comprises an antibody that binds
RSV-F and has a HCVR/LCVR amino acid sequence pair selected from the group ting of
SEQ ID NOs: 274/282 and 338/346.
In one embodiment, the pharmaceutical composition comprises an antibody that binds
RSV-F and has a CVR amino acid sequence pair consisting of SEQ ID NOs: 274/282.
In one embodiment, the pharmaceutical composition comprises an antibody that binds
RSV-F and has a HCVR/LCVR amino acid sequence pair consisting of SEQ ID NOs: 6.
In one embodiment, the pharmaceutical composition comprises at least one antibody
that binds RSV-F, wherein the antibody ses the three heavy chain complementarity
determining s (HCDR1, HCDR2 and HCDR3) contained within any one of the heavy chain
variable region (HCVR) amino acid sequences selected from the group ting of SEQ ID
16536800_1 (GHMatters) P40719NZ00
NOs: 274 and 338; and the three light chain complementarity determining regions (LCDR1,
LCDR2 and LCDR3) contained within any one of the light chain variable region (LCVR) amino
acid sequences selected from the group consisting of SEQ ID NOs: 282 and 346.
In one embodiment, the antibodies of the invention, or itions containing one or
more antibodies of the ion may be used to neutralize RSV from any subtype A or subtype
B strain of RSV.
In one embodiment, the invention features a ition, which is a combination of an
dy or antigen-binding fragment of an antibody of the invention, and a second therapeutic
agent.
The second therapeutic agent may be a small molecule drug, a protein/polypeptide, an
antibody, a nucleic acid molecule, such as an anti-sense molecule, or a siRNA. The second
therapeutic agent may be synthetic or naturally derived.
The second eutic agent may be any agent that is advantageously ed with
the antibody or fragment thereof of the invention, for example, an antiviral agent (e.g. ribavirin),
a vaccine specific for RSV, or a vaccine specific for influenza virus, or a vaccine specific for
metapneumovirus (MPV), an siRNA specific for an RSV antigen, an siRNA specific for an
influenza virus antigen, an siRNA specific for a eumovirus (MPV) antigen, a second
antibody specific for an RSV antigen, or a metapneumovirus (MPV) antigen, or an influenza
antigen, an anti-IL4R antibody, an anti-RSV-G dy or a NSAID. In certain embodiments,
the second eutic agent may be an agent that helps to counteract or reduce any possible
side effect(s) associated with the antibody or antigen-binding fragment of an antibody of the
invention, if such side effect(s) should occur.
It will also be appreciated that the antibodies and pharmaceutically acceptable
compositions of the present invention can be employed in combination therapies, that is, the
antibodies and pharmaceutically acceptable compositions can be administered concurrently
with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures.
The particular combination of therapies (therapeutics or procedures) to employ in a combination
regimen will take into account compatibility of the desired therapeutics and/or procedures and
the d therapeutic effect to be ed. It will also be appreciated that the therapies
employed may achieve a desired effect for the same disorder (for e, an dy may be
administered concurrently with another agent used to treat the same disorder), or they may
achieve different effects (e.g., control of any e effects). As used herein, additional
therapeutic agents that are normally administered to treat or prevent a ular disease, or
condition, are appropriate for the disease, or ion, being treated.
When multiple therapeutics are co-administered, dosages may be adjusted
accordingly, as is recognized in the pertinent art.
A fifth aspect of the invention es a method for preventing infection with respiratory
syncytial virus in a patient in need f, or for treating a patient suffering from an infection
16536800_1 (GHMatters) P40719NZ00
with RSV, or for ameliorating at least one symptom or complication associated with the RSV
infection, the method sing administering one or more antibodies or antigen-binding
fragments thereof as described herein, or a pharmaceutical composition comprising one or
more antibodies of the invention or nts thereof, as described herein, to a patient in need
thereof, such that the RSV infection is prevented, or at least one symptom or complication
ated with the infection is ameliorated, alleviated or reduced in severity and/or on.
In a related embodiment, the invention provides a ceutical composition
comprising one or more antibodies of the invention, alone or in ation with a second
therapeutic agent, for use in preventing a respiratory syncytial virus (RSV) infection in a patient
in need thereof, or for treating a patient suffering from an RSV infection, or for ameliorating at
least one symptom or complication associated with the infection, wherein the infection is either
prevented, or at least one symptom or complication associated with the infection is ted,
ameliorated, or lessened in severity and/or duration.
In one embodiment, the invention provides a pharmaceutical composition comprising
one or more antibodies of the invention, alone or in combination with a second therapeutic
agent in the manufacture of a medicament for preventing a respiratory ial virus (RSV)
ion in a patient in need thereof, or for treating a patient suffering from an RSV infection, or
for ameliorating at least one symptom or complication associated with the infection, wherein the
infection is either prevented, or at least one symptom or complication associated with the
ion is ted, ameliorated, or lessened in ty and/or duration.
In one embodiment, a t in need of treatment with an antibody of the ion, or
an n-binding fragment f is a patient who may experience a more severe form of the
RSV infection due to an underlying or pre-existing l ion. In one embodiment, the
method provides for preventing the development of infection with RSV in a patient at risk
thereof, the method comprising administering to the patient an ive amount of an antibody
or an antigen-binding fragment thereof that binds to the F protein of RSV, or a pharmaceutical
ition comprising an effective amount of an antibody or an antigen-binding fragment
thereof that binds to the F protein of RSV such that the infection is either prevented,
ameliorated, or lessened in severity and/or duration, or at least one symptom or complication
associated with the infection is prevented, or ameliorated, or lessened in severity or duration. In
one embodiment, the administering of the isolated human RSV-F antibody or an antigen-binding
fragment thereof results in prevention of recurrent wheezing in the patient. In one embodiment,
the administering of the isolated human RSV-F antibody or an antigen-binding fragment thereof
results in prevention of RSV-associated asthma in a child. In one embodiment, the
administering of the isolated human RSV-F antibody or an antigen-binding fragment thereof
results in prevention of an RSV infection caused by a subtype A or a subtype B respiratory
syncytial virus.
In one embodiment, the at least one symptom or complication associated with the RSV
16536800_1 (GHMatters) P40719NZ00
infection that may be treated with an antibody of the invention, or an antigen-binding fragment
thereof, may be selected from the group consisting of hypoxia, a bluish skin color due to lack of
, breathing difficulty (e.g .,rapid breathing or shortness of breath), cough, croupy cough
(“seal bark” cough), fever, nasal flaring, stuffy nose, wheezing, pneumonia, apnea, dehydration,
poor feeding, altered mental status, decreased appetite, or iolitis.
In one ment, the t at risk of developing an RSV infection, who may benefit
from treatment with the antibodies of the invention, or with a ition comprising one or
more antibodies of the invention, may be ed from the group consisting of a pre-term infant,
a full term infant who is compromised due to some other underlying medical condition and/or is
exposed during the peak season for RSV, a child greater than or equal to one year of age with
or without an underlying medical condition (e.g. ital heart disease, chronic lung disease,
cystic fibrosis, immunodeficiency, a neuromuscular er), an institutionalized or hospitalized
patient, an elderly patient (≥ 65 years of age) with or without an underlying medical condition
such as congestive heart failure or chronic obstructive pulmonary disease), a patient who is
immunocompromised due to underlying illness or due to administration of immunosuppressive
eutics, a patient who has some underlying medical condition that may pre-dispose them
to acquiring an RSV infection, for example, c obstructive pulmonary disease (COPD),
congestive heart failure, cystic fibrosis, bronchopulmonary dysplasia, airway malfunction,
chronic lung disease, a cancer patient, or a transplant patient who is on immunosuppressive
therapy.
In one embodiment, a patient who is a candidate for therapy with an antibody of the
invention may suffer from a condition resulting from a compromised pulmonary, cardiovascular,
neuromuscular, or immune system. The condition may be selected from the group consisting of
an ality of the airway, a chronic lung disease, a chronic heart disease, a neuromuscular
disease that compromises the handling of respiratory secretions and immunosuppression. The
chronic lung disease may be chronic obstructive pulmonary disease , cystic fibrosis, or
bronchopulmonary dysplasia. The c heart disease may be congestive heart failure (CHF),
or congenital heart disease. The neuromuscular disease or condition may be a
neurodegenerative disease, or an ity to handle and/or eliminate respiratory secretions due
to an injury or accident to the nervous system, e.g. a stroke, or a spinal cord injury. The
immunosuppression may be the result of severe combined immunodeficiency or severe
acquired immunodeficiency, or may be a result of any other ious disease or cancerous
condition that leads to immunosuppression, or is a result of treatment with immunosuppressant
drug therapy or radiation therapy.
In one embodiment, the antibody is administered lactically (administered prior to
development of the ion) to a patient at risk for developing an RSV infection, or at risk for
developing at least one symptom or complication associated with the RSV ion. The
16536800_1 (GHMatters) P40719NZ00
patients who are candidates for treatment with the antibodies of the invention may be
administered the compositions comprising one or more antibodies by any route of delivery
le for administration, ing but not limited to intravenous injection, intramuscular
ion, or subcutaneous injection.
In one embodiment, the antibody is administered therapeutically (administered after the
development of the infection) to a patient to ameliorate or reduce the severity and/or duration of
at least one m or cation associated with the RSV infection.
In one ment, the antibodies of the ion may be stered to the patient
in combination with one or more therapeutic agents useful for treating a RSV infection. The one
or more therapeutic agents may be selected from the group consisting of an antiviral agent; a
vaccine specific for RSV, a vaccine specific for influenza virus, or a vaccine ic for
metapneumovirus (MPV); an siRNA specific for an RSV antigen or a metapneumovirus (MPV)
antigen; a second antibody specific for an RSV antigen or a metapneumovirus (MPV) antigen;
an anti-IL4R antibody, an antibody specific for an influenza virus antigen, an anti-RSV-G
antibody and a NSAID.
A sixth aspect of the ion provides an immunogenic composition, or a vaccine, that
when administered to an individual, preferably a human, induces an immune se in such
individual to a Respiratory Syncytial Virus (RSV) antigen.
In one embodiment, the immunogenic composition, or vaccine, comprises an RSV
n, for example, an RSV-F protein, polypeptide, or an immunogenic fragment thereof, or an
epitope contained within and/or obtained from an n of the RSV-F polypeptide or a
fragment thereof, and/or ses DNA and/or RNA which encodes and expresses an e
from an antigen of the RSV-F polypeptide, or other polypeptides of the invention.
In one embodiment of the invention, the immunogenic composition, or vaccine, may
comprise the RSV-F protein as shown in SEQ ID NO: 354. In one embodiment of the invention,
the immunogenic composition, or vaccine, may comprise a RSV-F polypeptide fragment
comprising residues 161 through 188 of SEQ ID NO: 354. In one embodiment of the invention,
the immunogenic composition, or vaccine, may comprise one or more amino acid residues
contained within SEQ ID NO: 355 and/or SEQ ID NO: 356. In one embodiment of the invention,
the immunogenic composition, or vaccine, may comprise SEQ ID NO: 355 and/or SEQ ID NO:
356.
In a related aspect, the invention provides a method for inducing an immune response
in an individual, particularly a mammal, preferably , by administering to an individual an
immunogenic composition, or a vaccine, comprising a RSV-F protein, or an immunogenic
fragment f, or a RSV-F antigen or an immunogenic fragment thereof comprising one or
more epitopes ned within the RSV-F antigen or fragment thereof, adequate to produce an
16536800_1 (GHMatters) P40719NZ00
antibody and/or a T cell immune response to protect the individual from infection, particularly
infection with atory Syncytial Virus (RSV).
In one embodiment, methods are provided for using the immunogenic compositions, or
vaccines of the invention for inducing an immune response that results in inhibiting, or slowing
the progression of cell to cell viral . Methods are also provided for ameliorating at least
one symptom associated with RSV infection by administering an immunogenic composition, or a
vaccine, comprising at least one RSV-F n, or one or more epitopes contained within the
RSV-F antigen, which when administered will induce an immune response in the individual.
For example, in one embodiment the invention provides a method of inducing an
immune response in an individual comprising delivering to the individual an immunogenic
composition, or vaccine comprising, an RSV-F antigen (e.g . the amino acid sequence shown in
SEQ ID NO: 354), or an antigenic fragment thereof, ( e.g. a polypeptide comprising residues 161
through 188 of SEQ ID NO: 354), or a nucleic acid vector comprising a nucleotide sequence to
direct expression of such viral polypeptide, or a fragment or a variant thereof, in vivo in order to
induce an immune se.
In one ment of the invention, the polypeptide to be used in an immunogenic
composition or in a vaccine for inducing an immune response in an dual comprises
residues 161 through 188 of SEQ ID NO: 354. In one embodiment of the invention, the
polypeptide to be used in an genic composition or in a vaccine for ng an immune
response in an dual comprises one or more amino acid residues ned within SEQ ID
NO: 355 and/or SEQ ID NO: 356. In one embodiment of the invention, the polypeptide to be
used in an immunogenic ition or in a vaccine for inducing an immune response in an
individual comprises SEQ ID NO: 355 and/or SEQ ID NO: 356. In one embodiment of the
invention, the immunogenic composition, or vaccine, may elicit an antibody response or a T cell
se specific for the RSV-F antigen of RSV, n the antibodies generated interact with
either the serine at position 173 of SEQ ID NO: 354, or the ine at position 354, or both the
serine at position 173 of SEQ ID NO: 354 and the threonine at position 174 of SEQ ID NO: 354.
In certain embodiments of the invention, the immunogenic composition, or vaccine may
comprise an immunogenic polypeptide and/or polynucleotide of the invention, or a combination
thereof, together with a suitable carrier/excipient, such as a pharmaceutically acceptable
carrier/excipient. The immunogenic composition, or vaccine of the invention may also include
adjuvants for enhancing the immunogenicity of the formulation.
In certain embodiments, it is advantageous for the RSV-F antigens or fragments thereof
to be formulated into immunogenic compositions, or vaccines that comprise immunogenic,
preferably logically effective, amounts of onal antigens to elicit immunity to other
pathogens, preferably s and/or bacteria. Such additional antigens may include an
influenza virus antigen, an antigen from metapneumovirus or from a coronavirus, an antigen
from Haemophilus influenzae, Streptococcus pneumonia, or Bordetella pertussis. Other RSV
00_1 ters) P40719NZ00
antigens may be included in the immunogenic compositions, or vaccines, such as the RSV-G
rotein, or immunogenic fragments thereof, the HN protein, or derivatives thereof.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. A schematic diagram of the RSV-F protein.
Figure 2A and 2B. Demonstrates that H1H3592P3 blocks viral entry by inhibiting fusion
of virus and cell membranes.
DETAILED DESCRIPTION
Before the t methods are bed, it is to be understood that this invention is not
limited to particular s, and experimental conditions described, as such methods and
conditions may vary. It is also to be understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art to which this invention
belongs. As used herein, the term "about," when used in reference to a particular recited
numerical value, means that the value may vary from the recited value by no more than 1%.
For example, as used herein, the expression "about 100" includes 99 and 101 and all values in
between (e.g. , 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can
be used in the practice or g of the t invention, preferred methods and als are
now described.
Definitions
“Respiratory Syncytial Virus-F protein”, also referred to as “RSV-F” is a type I
transmembrane surface protein, which has an N terminal cleaved signal e and a
membrane anchor near the C terminus (Collins, P.L. et al., (1984), PNAS (USA) 81:7683-7687).
The RSV-F protein is synthesized as an inactive 67 KDa precursor denoted as F0 r, L.J.;
et al., Virology (2000), 271 ,122–131. The F0 protein is activated proteolytically in the Golgi
complex by a furin-like protease at two sites, yielding two disulfide linked polypeptides, F2 and
F1, from the N and C terminal, respectively. There is a 27 amino acid peptide released called
”. There are furin cleavage sites (FCS) on either side of the pep27 (Collins, P.L.; Mottet,
G. (1991), J.Gen.Virol. , 72 : 101; Sugrue, R.J , et al. (2001), J..Gen.Virol. , 82 ,1375–
1386). The F2 subunit consists of the Heptad repeat C (HRC), while the F1 ns the fusion
polypeptide (FP), heptad repeat A (HRA), domain I, domain II, heptad repeat B (HRB),
transmembrane (TM) and asmic domain (CP) (See Sun, Z. et al. s (2013), 5:211-
225). The RSV-F protein plays a role in fusion of the virus particle to the cell membrane, and is
16536800_1 (GHMatters) P40719NZ00
expressed on the surface of ed cells, thus playing a role in cell to cell ission of the
virus and syncytia formation. The amino acid sequence of the RSV-F protein is provided in
GenBank as accession number AAX23994 and is also referred to herein as SEQ ID NO: 354.
A genetically engineered construct of the RSV-F protein is shown herein as having the
amino acid sequence of SEQ ID NO: 353.
The term "laboratory strain" as used herein refers to a strain of RSV (subtype A or B)
that has been passaged extensively in in vitro cell culture. A "laboratory " can acquire
adaptive mutations that may affect their biological properties. A "clinical strain" as used herein
refers to an RSV isolate (subtype A or B), which is obtained from an infected individual and
which has been isolated and grown in tissue culture at low passage.
The term “effective dose 99” or “ED99 ” refers to the dosage of an agent that produces a
desired effect of 99% reduction of viral forming plaques relative to the isotype (negative) control.
In the t invention, the ED99 refers to the dosage of the anti-RSV-F antibodies that will
neutralize the virus infection (ie.g. reduce 99% of viral load) in vivo, as described in Example 5.
The term “IC50” refers to the “half maximal inhibitory concentration”, which value
measures the effectiveness of compound (e.g. anti-RSV-F antibody) tion towards a
biological or biochemical utility. This quantitative e indicates the quantity required for a
ular inhibitor to inhibit a given biological process by half.
“Palivizumab”, also referred to as “SYNAGIS®”, is a humanized anti-RSV-F antibody
with heavy and light chain variable domains having the amino acid sequences as set forth in
US7635568 and 5824307 (also shown herein as SEQ ID NO: 361 for the heavy chain of the
antibody and SEQ ID NO: 362 for the light chain of the antibody). This antibody, which
immunospecifically binds to the RSV-F protein, is currently FDA-approved for the passive
immunoprophylaxis of serious RSV disease in isk children and is administered
intramuscularly at recommended monthly doses of 15 mg/kg of body weight throughout the RSV
season (November h April in the rn hemisphere). SYNAGIS® is composed of 95%
human and 5% murine antibody sequences. See also Johnson et al., (1997), J. Infect. Diseases
-1224.
“Motavizumab”, also referred to as “NUMAX™”, is an enhanced potency RSV-F-specific
humanized onal antibody derived by in vitro affinity maturation of the complementaritydetermining
regions of the heavy and light chains of palivizumab. For reference purposes, the
amino acid ce of the NUMAX™ antibody is disclosed in U.S Patent Publication
2003/0091584 and in U.S. Pat. No. 216 and in Wu et al., (2005) J. Mol. Bio. 350(1):126-
144 and in Wu, et al. (2007) J. Mol. Biol. 368:652-665. It is also shown herein as SEQ ID NO:
359 for the heavy chain and as SEQ ID NO: 360 for the light chain of the antibody.
As used herein, the terms "treat," "treatment" and "treating" refer to the reduction or
ration of the progression, severity, and/or duration of an upper and/or lower atory
16536800_1 (GHMatters) P40719NZ00
tract RSV infection, otitis media, or a symptom or respiratory condition related thereto (such as
asthma, wheezing, or a combination thereof) resulting from the administration of one or more
therapies (including, but not limited to, the stration of one or more prophylactic or
eutic agents). In specific embodiments, such terms refer to the reduction or inhibition of
the replication of RSV, the inhibition or reduction in the spread of RSV to other tissues or
subjects (e.g., the spread to the lower respiratory tract), the inhibition or reduction of infection of
a cell with a RSV, or the amelioration of one or more symptoms ated with an upper and/or
lower respiratory tract RSV infection or otitis media.
As used herein, the terms "prevent," "preventing," and "prevention" refer to the
prevention or inhibition of the development or onset of an upper and/or lower respiratory tract
RSV infection, otitis media or a respiratory condition related thereto in a subject, the prevention
or inhibition of the progression of an upper respiratory tract RSV infection to a lower respiratory
tract RSV infection, otitis media or a respiratory ion related thereto resulting from the
administration of a therapy (e.g., a prophylactic or therapeutic agent), the prevention of a
symptom of an upper and/or lower tract RSV infection, otitis media or a respiratory condition
related thereto, or the administration of a combination of therapies (e.g., a ation of
prophylactic or therapeutic agents).
The term "antibody", as used herein, is intended to refer to immunoglobulin molecules
comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected
by disulfide bonds (i.e. , "full antibody molecules"), as well as multimers thereof (e.g.
IgM) or n-binding fragments thereof. Each heavy chain is comprised of a heavy chain
variable region (“HCVR” or “VH”) and a heavy chain constant region ised of domains CH1,
CH2 and CH3). Each light chain is comprised of a light chain variable region (“LCVR or “VL”) and
a light chain constant region (CL). The V H and VL s can be further subdivided into regions
of hypervariability, termed mentarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR). Each VH and VL is composed
of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments of the invention, the
FRs of the antibody (or antigen binding fragment thereof) may be identical to the human
germline sequences, or may be lly or artificially modified. An amino acid consensus
sequence may be defined based on a side-by-side analysis of two or more CDRs.
tution of one or more CDR residues or omission of one or more CDRs is also
possible. dies have been described in the scientific literature in which one or two CDRs
can be dispensed with for binding. Padlan et al. (1995 FASEB J. 9:133-139) analyzed the
t regions between antibodies and their antigens, based on hed crystal structures,
and concluded that only about one fifth to one third of CDR residues actually contact the
antigen. Padlan also found many dies in which one or two CDRs had no amino acids in
16536800_1 (GHMatters) P40719NZ00
contact with an antigen (see also, Vajdos et al. 2002 J Mol Biol 320:415-428).
CDR residues not contacting antigen can be identified based on previous studies (for
example residues H60-H65 in CDRH2 are often not required), from regions of Kabat CDRs
lying e Chothia CDRs, by lar modeling and/or empirically. If a CDR or e(s)
thereof is omitted, it is usually substituted with an amino acid ing the corresponding
position in another human antibody sequence or a sus of such sequences. Positions for
substitution within CDRs and amino acids to tute can also be selected empirically.
Empirical tutions can be conservative or non-conservative substitutions.
The fully human monoclonal antibodies disclosed herein may comprise one or more
amino acid tutions, insertions and/or deletions in the framework and/or CDR regions of the
heavy and light chain variable domains as compared to the corresponding germline sequences.
Such mutations can be readily ascertained by comparing the amino acid sequences disclosed
herein to germline sequences available from, for example, public antibody ce databases.
The present invention es antibodies, and antigen-binding nts thereof, which are
derived from any of the amino acid sequences disclosed herein, n one or more amino
acids within one or more framework and/or CDR regions are mutated to the corresponding
residue(s) of the germline sequence from which the antibody was d, or to the
corresponding residue(s) of another human germline sequence, or to a conservative amino acid
substitution of the corresponding germline residue(s) (such sequence changes are referred to
herein collectively as ine mutations"). A person of ordinary skill in the art, starting with the
heavy and light chain variable region sequences disclosed herein, can easily produce numerous
antibodies and antigen-binding fragments which comprise one or more individual germline
mutations or combinations thereof. In certain embodiments, all of the framework and/or CDR
residues within the VH and/or VL domains are mutated back to the residues found in the original
germline sequence from which the antibody was derived. In other embodiments, only certain
residues are mutated back to the original germline sequence, e.g. , only the mutated es
found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the
mutated es found within CDR1, CDR2 or CDR3. In other embodiments, one or more of
the ork and/or CDR residue(s) are mutated to the corresponding residue(s) of a different
germline sequence (i.e. , a germline sequence that is different from the germline sequence from
which the antibody was originally derived). Furthermore, the antibodies of the present invention
may contain any combination of two or more germline mutations within the framework and/or
CDR regions, e.g. , wherein certain individual es are mutated to the corresponding residue
of a particular germline sequence while certain other residues that differ from the original
germline sequence are maintained or are mutated to the corresponding residue of a different
germline sequence. Once obtained, antibodies and antigen-binding fragments that contain one
or more germline mutations can be easily tested for one or more desired property such as,
16536800_1 (GHMatters) P40719NZ00
improved binding specificity, increased binding affinity, improved or enhanced nistic or
agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies
and antigen-binding nts obtained in this general manner are encompassed within the
present ion.
The present invention also includes fully monoclonal antibodies comprising variants of
any of the HCVR, LCVR, and/or CDR amino acid sequences sed herein having one or
more conservative substitutions. For example, the present ion includes antibodies having
HCVR, LCVR, and/or CDR amino acid sequences with, e.g. , 10 or fewer, 8 or fewer, 6 or fewer,
4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and/or
CDR amino acid sequences disclosed herein.
The term "human antibody", as used herein, is intended to include antibodies having
variable and constant regions derived from human germline immunoglobulin sequences. The
human mAbs of the invention may include amino acid residues not encoded by human ne
immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
However, the term "human antibody", as used herein, is not intended to include mAbs in which
CDR sequences derived from the germline of another mammalian species (e.g., mouse), have
been grafted onto human FR sequences.
The term "recombinant" lly refers to any protein, polypeptide, or cell expressing a
gene of interest that is produced by genetic engineering methods. The term "recombinant" as
used with respect to a n or polypeptide, means a polypeptide produced by expression of a
recombinant polynucleotide. The proteins used in the immunogenic compositions of the
invention may be isolated from a natural source or produced by c engineering s.
The antibodies of the ion may, in some embodiments, be recombinant human
antibodies. The term "recombinant human antibody", as used , is intended to e all
human antibodies that are prepared, expressed, d or isolated by recombinant means,
such as antibodies expressed using a recombinant expression vector ected into a host cell
(described further below), antibodies isolated from a recombinant, combinatorial human
antibody library (described further below), antibodies isolated from an animal (e.g., a mouse)
that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids
Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means
that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
Such recombinant human antibodies have variable and constant regions derived from human
germline immunoglobulin sequences. In certain embodiments, however, such recombinant
human antibodies are subjected to in vitro mutagenesis (or, when an animal enic for
human Ig sequences is used, in vivo somatic nesis) and thus the amino acid ces
of the VH and VL regions of the recombinant antibodies are sequences that, while derived from
and related to human germline VH and VL sequences, may not naturally exist within the human
16536800_1 (GHMatters) P40719NZ00
antibody germline repertoire in vivo.
The term "specifically binds," or “binds specifically to”, or the like, means that an
antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively
stable under physiologic ions. Specific binding can be characterized by an equilibrium
iation nt of at least about 1x10-6 M or less (e.g ., a smaller KD denotes a tighter
binding). Methods for determining whether two molecules specifically bind are well known in the
art and include, for e, equilibrium dialysis, surface plasmon resonance, and the like. As
described herein, antibodies have been identified by surface plasmon resonance, e.g .,
BIACORE™, which bind specifically to RSV-F. Moreover, multi-specific antibodies that bind to
RSV-F protein and one or more additional antigens or a bi-specific that binds to two different
regions of RSV-F are eless considered antibodies that “specifically bind”, as used .
The term “high affinity” antibody refers to those mAbs having a g affinity to RSV-F,
expressed as KD, of at least 10-9 M; more preferably 10-10 M, more preferably 10-11 M, more
preferably 10-12 M as ed by surface plasmon resonance, e.g., BIACORE™ or solutionaffinity
ELISA.
By the term “slow off rate”, “Koff” or “kd” is meant an antibody that dissociates from RSV-
F, with a rate nt of 1 x 10-3 s-1 or less, preferably 1 x 10-4 s-1 or less, as determined by
surface plasmon resonance, e.g ., BIACORE™.
The terms "antigen-binding portion" of an antibody, en-binding fragment" of an
antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable,
synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an
antigen to form a complex. The terms "antigen-binding portion" of an antibody, or "antibody
fragment”, as used herein, refers to one or more fragments of an antibody that retains the ability
to bind to RSV-F.
The specific embodiments, antibody or antibody fragments of the invention may be
conjugated to a therapeutic moiety (“immunoconjugate”), such as an antibiotic, a second anti-
RSV-7 antibody, a vaccine, or a toxoid, or any other therapeutic moiety useful for treating a RSV
infection.
An ted antibody", as used herein, is intended to refer to an antibody that is
substantially free of other antibodies (Abs) having different antigenic icities (e.g., an
isolated antibody that specifically binds RSV-F, or a fragment thereof, is substantially free of
Abs that specifically bind antigens other than RSV-F.
A “blocking antibody” or a "neutralizing antibody", as used herein (or an "antibody that
neutralizes RSV-F activity"), is intended to refer to an antibody whose binding to RSV-F results
in inhibition of at least one biological activity of RSV-F. For example, an antibody of the
invention may aid in blocking the fusion of RSV to a host cell, or prevent syncytia formation, or
prevent the primary disease caused by RSV. Alternatively, an antibody of the ion may
00_1 (GHMatters) P40719NZ00
demonstrate the y to ameliorate at least one symptom of the RSV infection. This inhibition
of the biological activity of RSV-F can be assessed by measuring one or more indicators of
RSV-F biological activity by one or more of several standard in vitro assays (such as a
neutralization assay, as described herein) or in vivo assays known in the art (for example,
animal models to look at protection from challenge with RSV following administration of one or
more of the antibodies described herein).
The term "surface plasmon resonance", as used herein, refers to an optical
enon that allows for the analysis of real-time ecular interactions by detection of
tions in protein concentrations within a biosensor matrix, for example using the
BIACORE™ system (Pharmacia Biosensor AB, a, Sweden and Piscataway, N.J.).
The term "KD ", as used herein, is intended to refer to the equilibrium dissociation
constant of a particular dy-antigen interaction.
The term “epitope” refers to an antigenic determinant that interacts with a specific
antigen binding site in the variable region of an antibody molecule known as a paratope. A
single antigen may have more than one epitope. Thus, different antibodies may bind to different
areas on an antigen and may have ent biological effects. The term “epitope” also refers to
a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen
that is bound by an antibody. es may be defined as structural or functional. Functional
es are generally a subset of the structural epitopes and have those residues that directly
contribute to the affinity of the interaction. Epitopes may also be conformational, that is,
composed of non-linear amino acids. In certain embodiments, epitopes may include
determinants that are chemically active surface groupings of molecules such as amino acids,
sugar side chains, phosphoryl , or sulfonyl , and, in certain embodiments, may
have specific three-dimensional structural characteristics, and/or specific charge characteristics.
The term "substantial ty" or "substantially cal," when referring to a nucleic acid
or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions
or deletions with another nucleic acid (or its complementary strand), there is nucleotide
sequence ty in at least about 90%, and more ably at least about 95%, 96%, 97%,
98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence
identity, such as FASTA, BLAST or GAP, as sed below. A nucleic acid molecule having
substantial identity to a reference nucleic acid molecule may, in n instances, encode a
polypeptide having the same or substantially similar amino acid sequence as the polypeptide
encoded by the reference nucleic acid molecule.
As applied to polypeptides, the term "substantial similarity" or “substantially similar”
means that two peptide sequences, when optimally aligned, such as by the programs GAP or
T using default gap weights, share at least 90% sequence identity, even more
preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions, which
16536800_1 ters) P40719NZ00
are not cal, differ by conservative amino acid substitutions. A rvative amino acid
substitution" is one in which an amino acid residue is substituted by another amino acid residue
having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
In l, a vative amino acid substitution will not substantially change the onal
properties of a protein. In cases where two or more amino acid sequences differ from each
other by conservative substitutions, the percent or degree of similarity may be adjusted s
to correct for the conservative nature of the substitution. Means for making this adjustment are
well known to those of skill in the art. (See, e.g ., Pearson (1994) Methods Mol. Biol. 24: 307-
331). Examples of groups of amino acids that have side chains with similar chemical properties
include 1) aliphatic side : glycine, alanine, valine, leucine and isoleucine; 2) aliphatichydroxyl
side chains: serine and threonine; 3) amide-containing side chains: asparagine and
glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side
: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7)
sulfur-containing side chains: cysteine and methionine. red conservative amino acids
substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,
alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative
ement is any change having a positive value in the PAM250 log-likelihood matrix
disclosed in Gonnet et al. (1992) Science 256: 1443 45. A "moderately conservative"
replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
Sequence similarity for polypeptides is typically measured using sequence analysis
software. Protein analysis software matches similar sequences using measures of similarity
assigned to various substitutions, deletions and other modifications, including conservative
amino acid tutions. For instance, GCG software contains programs such as GAP and
BESTFIT which can be used with default ters to determine sequence homology or
ce identity between closely related polypeptides, such as homologous ptides from
different species of organisms or between a wild type protein and a mutein thereof. See, e.g.,
GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or
ended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3)
provides alignments and percent sequence identity of the regions of the best overlap between
the query and search ces (Pearson (2000) supra ). Another preferred algorithm when
comparing a sequence of the invention to a database containing a large number of sequences
from different sms is the computer program BLAST, ally BLASTP or N,
using default parameters. (See, e.g., Altschul et al. (1990) J. Mol. Biol. 215: 403 410 and (1997)
Nucleic Acids Res. 25:3389 402).
In specific embodiments, the antibody or antibody fragment for use in the method of the
invention may be mono-specific, bi-specific, or multi-specific. Multi-specific antibodies may be
specific for different epitopes of one target polypeptide or may contain antigen-binding domains
16536800_1 (GHMatters) P40719NZ00
ic for epitopes of more than one target ptide. An exemplary bi-specific antibody
format that can be used in the context of the present invention involves the use of a first
immunoglobulin (Ig) CH3 domain and a second Ig CH3 domain, wherein the first and second Ig
CH3 domains differ from one another by at least one amino acid, and wherein at least one amino
acid difference reduces binding of the bi-specific antibody to Protein A as compared to a bispecific
antibody g the amino acid difference. In one embodiment, the first Ig C H3 domain
binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes
Protein A binding such as an H95R modification (by IMGT exon numbering; H435R by EU
numbering). The second CH3 may further comprise an Y96F modification (by IMGT; Y436F by
EU). Further cations that may be found within the second CH3 include: D16E, L18M,
N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I by
EU) in the case of IgG1 mAbs; N44S, K52N, and V82I (IMGT; N384S, K392N, and V422I by
EU) in the case of IgG2 mAbs; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by
IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I by EU) in the case of IgG4
mAbs. Variations on the bi-specific antibody format bed above are contemplated within
the scope of the present invention.
By the phrase “therapeutically effective amount” is meant an amount that produces the
desired effect for which it is administered. The exact amount will depend on the purpose of the
treatment, and will be ascertainable by one d in the art using known techniques (see, for
example, Lloyd (1999) The Art, Science and Technology of ceutical Compounding).
An "immunogenic composition" relates to a composition containing an
antigen/immunogen, e.g. a rganism, such as a virus or a bacterium, or a component
thereof, a protein, a ptide, a fragment of a protein or ptide, a whole cell inactivated,
subunit or attenuated virus, or a polysaccharide, or combination thereof, administered to
stimulate the recipient's humoral and/or cellular immune s to one or more of the
antigens/immunogens present in the immunogenic composition. The genic
compositions of the present invention can be used to treat a human susceptible to RSV
infection, by means of administering the immunogenic compositions via a systemic route. These
administrations can include injection via the intramuscular (i.m.), intradermal (i.d.), intranasal or
inhalation route, or subcutaneous (s.c.) routes; application by a patch or other transdermal
ry device. In one embodiment, the immunogenic composition may be used in the
manufacture of a vaccine or in the elicitation of polyclonal or monoclonal antibodies that could
be used to passively protect or treat a mammal.
The terms "vaccine" or "vaccine composition", which are used interchangeably, refer to a
ition comprising at least one immunogenic composition that induces an immune
response in an animal.
In one embodiment of the invention, the protein of interest comprises an antigen. The
16536800_1 (GHMatters) P40719NZ00
terms "antigen," "immunogen," "antigenic," "immunogenic," "antigenically active," and
ologically active" when made in reference to a molecule, refer to any substance that is
e of inducing a specific humoral and/or cell-mediated immune response. In one
embodiment, the antigen comprises an epitope, as defined above.
"Immunologically protective amount", as used herein, is an amount of an antigen
effective to induce an immunogenic se in the recipient that is adequate to t or
ameliorate signs or symptoms of disease, including adverse health effects or complications
f. Either humoral immunity or cell-mediated immunity or both can be induced. The
immunogenic response of an animal to a composition can be evaluated, e.g., indirectly through
measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring
signs and symptoms after challenge with the microorganism. The protective immunity conferred
by an immunogenic composition or vaccine can be evaluated by ing, e.g., ion of
shed of challenge organisms, reduction in clinical signs such as mortality, morbidity,
temperature, and overall physical condition, health and performance of the t. The immune
response can se, without limitation, induction of cellular and/or humoral immunity. The
amount of a ition or vaccine that is therapeutically effective can vary, depending on the
particular sm used, or the condition of the animal being treated or vaccinated.
"Immune response", or "immunological response" as used herein, in a subject refers to
the development of a humoral immune se, a cellular- immune se, or a humoral
and a cellular immune response to an antigen/immunogen. A "humoral immune response"
refers to one that is at least in part mediated by antibodies. A "cellular immune response" is one
mediated by T-!ymphocytes or other white blood cells or both, and includes the production of
cytokines, chemokines and similar molecules produced by activated T-cells, white blood cells,
or both. Immune responses can be ined using standard immunoassays and
neutralization , which are known in the art. "Immunogenicity", as used herein, refers to
the capability of a protein or polypeptide to elicit an immune response directed ically
against a bacteria or virus that causes the fied disease.
General Description
Respiratory syncytial virus (RSV) is a negative sense, single stranded RNA virus that is
the leading cause of serious respiratory tract infections in infants and children, with the primary
infection occurring in children from 6 weeks to 2 years of age and uncommonly in the first 4
weeks of life during nosocomial ics (Hall et al., 1979, New Engl. J. Med. 300:393-396).
(Feigen et al.,eds., 1987, In: Textbook of Pediatric Infectious Diseases, W B Saunders,
Philadelphia at pages 675; New Vaccine Development, Establishing Priorities, Vol. 1,
1985, National Academy Press, Washington D.C. at pages 397-409; Ruuskanen et al., 1993,
Curr. Probl. Pediatr. 23:50-79; Hall et al., 1979, New Engl. J. Med. 300:393-396). Certain
16536800_1 (GHMatters) P40719NZ00
populations of children are at risk for developing an RSV infection and these include m
infants (Hall et al., 1979, New Engl. J. Med. 300:393-396), children with congenital
mations of the airway, children with bronchopulmonary dysplasia (Groothuis et al., 1988,
Pediatrics 82:199-203), children with congenital heart disease (MacDonald et al., New Engl. J.
Med. 307:397-400), and children with ital or acquired immunodeficiency (Ogra et al.,
1988, r. 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).
RSV can infect the adult population as well. In this population, RSV causes primarily an
upper respiratory tract disease, although elderly ts may be at greater risk for a s
infection and pneumonia (Evans, A. S., eds., 1989, Viral Infections of Humans. Epidemiology
and Control, 3rd ed., Plenum Medical Book, New York at pages 525-544), as well as adults who
are suppressed, particularly bone marrow transplant ts (Hertz et al., 1989,
Medicine 68:269-281). Other at risk patients e those suffering from congestive heart
failure and those suffering from chronic obstructive pulmonary disease (ie. COPD). There have
also been reports of epidemics among nursing home patients and institutionalized young adults
(Falsey, A. R., 1991, . Control Hosp. Epidemiol. 12:602-608; and Garvie et al., 1980, Br.
Med. J. 281:1253-1254).
While treatment options for established RSV disease are limited, more severe forms of
the disease of the lower respiratory tract often require considerable supportive care, including
administration of humidified oxygen and respiratory assistance (Fields et al., eds, 1990, Fields
Virology, 2nd ed., Vol. 1, Raven Press, New York at pages 1045-1072).
Ribavirin, which is the only drug approved for treatment of infection, has been shown to
be effective in the treatment of pneumonia and bronchiolitis associated with RSV infection, and
has been shown to modify the course of severe RSV disease in immunocompetent children
(Smith et al., 1991, New Engl. J. Med. -29). However, the use of ribavirin is limited due
to concerns surrounding its potential risk to pregnant women who may be d to the
aerosolized drug while it is being administered in a hospital environment. Its use is also limited
due to its relatively high cost.
Other peptide inhibitors of RSV infection have been identified, which inhibit viral growth
in vitro, but have failed when tested in vivo, most likely due to lack of oral availability and a
relatively low half life in ation (Lambert, D.M., et al. (1996), PNAS (USA) 93:2186-2191;
Magro, M. et al., (2010), J. Virol. 84:7970-7982; Park, M. et al. (2011), Anal. Biochem. 409:195-
201).
Other small molecule inhibitors of RSV infection have also been identified, but have
been discontinued for various reasons, some of which may be due to toxic side effects (Wyde,
P.R. et al. (1998), Antiviral Res. 38:31-42; Nikitenko, A.A. et al. (2001), Bioorg Med Chem Lett
16536800_1 (GHMatters) P40719NZ00
11:1041-1044; Douglas, J.L., et al. (2003), J. Virol 77:5054-5064; Bonfanti, J.F. et al, (2008), J.
Med Chem 51:875-896).
Similarly, while a vaccine may be useful, no commercially available vaccine has been
developed to date. Several e candidates have been abandoned and others are under
development (Murphy et al., 1994, Virus Res. 32:13-36). The development of a vaccine has
proven to be problematic. In particular, immunization would be required in the ate
neonatal period since the peak incidence of lower atory tract disease occurs at 2-5 months
of age. However, it is known that the neonatal immune response is immature at that time. Plus,
the infant at that point in time still has high titers of maternally acquired RSV antibody, which
might reduce vaccine immunogenicity (Murphy et al., 1988, J. Virol. 62:3907-3910; and Murphy
et al., 1991, Vaccine 9:185-189).
Currently, passive immunization appears to be the only approved approach to
prophylaxis of RSV disease. Initial evidence that suggested a protective role for IgG was
obtained from s demonstrating al antibody in ferrets e, 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. r. 98:708-715).
Hemming et al. (Morell et al., eds., 1986, Clinical Use of enous Immunoglobulins,
Academic Press, London at pages 285-294) recognized the possible utility of RSV antibody in
treatment or prevention of RSV infection during studies involving the pharmacokinetics of an
intravenous immune in (IVIG) in newborns suspected of having neonatal sepsis. 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, . 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 an primate model.
More recent studies have concentrated on the role of two glycoproteins, designated F
and G, which are found on the surface of RSV, as targets of neutralizing antibodies, due to the
role of these roteins in viral attachment and fusion with the host cell (Fields et al., 1990,
supra; and Murphy et al., 1994, . The G protein binds to a ic cellular receptor and
the F protein promotes fusion of the virus with the cell. The F protein is also expressed on the
surface of infected cells and is sible for subsequent fusion with other cells g to
ia formation. Thus, antibodies to the F protein may directly neutralize virus, or block
fusion of the virus with the cell, or prevent cell to cell spread by preventing syncytia formation.
The first humanized antibody approved for use in pediatric ts for prevention of
serious lower respiratory tract disease caused by RSV was palivizumab (SYNAGIS®), which
16536800_1 (GHMatters) P40719NZ00
immunospecifically binds to the F protein and is administered intramuscularly at recommended
monthly doses of 15 mg/kg of body weight throughout the RSV season (November through April
in the northern hemisphere). SYNAGIS® is composed of 95% human and 5% murine antibody
sequences. (See Johnson et al., 1997, J. Infect. Diseases 176:1215-1224 and U.S. Pat. No.
,824,307).
While SYNAGIS® has been successfully used for the prevention of RSV infection in
pediatric patients, the need for multiple visits to the doctor’s office for multiple intramuscular
doses of 15 mg/kg of SYNAGIS® was not only inconvenient for the patient but could also result
in missed doses. Thus, there was a need for development of antibodies that retained the
immunospecificity for the RSV antigen, but which were more potent, with an ed
pharmacokinetic profile, and thus have an overall improved therapeutic profile. Such an
antibody is bed in U.S Patent Publication 2003/0091584 and is known as zumab
(NUMAX™). Although NUMAX™ has improved binding characteristics that may overcome the
higher dosing requirements described above for SYNAGIS®, it also had a 3 to 5 fold se in
the frequency and severity of hypersensitivity reactions compared to SYNAGIS®. NUMAX™
was then withdrawn from future development.
Accordingly, there is still a need for effective therapies against RSV infections, and in
ular, there is a need to identify a more potent antibody for preventing and ng RSV
infections, but without the adverse side effects associated with those bed above. The
antibodies described herein, while exhibiting a lower binding affinity for RSV-F (i.e. the
antibodies of the present invention do not bind as y to RSV-F as palivizumab) than that
described for palivizumab or motavizumab appears to exhibit better neutralization capabilities
and addresses those needs.
In certain embodiments, the antibodies of the invention are obtained from mice
immunized with a primary immunogen, such as a whole RSV le, either live, attenuated, or
inactivated, or with a recombinant form of the virus, or with a purified F protein (See GenBank
accession number 94.1 (SEQ ID NO: 354)), or a recombinantly ed F n (See
SEQ ID NO: 353), followed by immunization with a secondary immunogen (whole virus, or
purified F protein), or with an immunogenically active fragment of the F protein.
The immunogen may be DNA encoding the F n or an active fragment thereof.
The gen may be derived from the inal or C-terminal domain of either the
67 KDa precursor (F0), or from either of the two fragments generated from the precursor by a
furin-like protease yielding two disulfide linked ptides, designated as F2 and F1, from the
N and C terminal, respectively. The fragment may be derived from any of the known regions of
RSV-F protein (See Sun, Z. et al. (2013), Viruses 5:211-225).
The full-length amino acid sequence of RSV-F is shown as SEQ ID NO: 354 and is also
shown in GenBank accession number AAX23994.1.
A genetic construct containing the F protein of RSV is shown as SEQ ID NO: 353.
16536800_1 (GHMatters) P40719NZ00
In certain embodiments, antibodies that bind specifically to RSV-F may be prepared
using fragments of the noted regions, or peptides that extend beyond the ated
regions by about 5 to about 20 amino acid residues from either, or both, the N or C terminal
ends of the regions described herein. In n embodiments, any combination of the above-
noted regions or fragments thereof may be used in the ation of RSV-F specific antibodies.
In n embodiments, any one or more of the above-noted regions of RSV-F, or fragments
thereof may be used for preparing monospecific, bispecific, or multispecific antibodies.
Antigen-Binding Fragments of Antibodies
Unless specifically indicated otherwise, the term ody," as used herein, shall be
understood to ass antibody molecules comprising two immunoglobulin heavy chains
and two immunoglobulin light chains (i.e. , "full antibody molecules") as well as antigen-binding
fragments thereof. The terms "antigen-binding portion" of an antibody, "antigen-binding
fragment" of an antibody, and the like, as used herein, include any naturally occurring,
enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that
specifically binds an antigen to form a complex. The terms "antigen-binding portion" of an
antibody, or "antibody fragment”, as used herein, refers to one or more fragments of an antibody
that retain the ability to specifically bind to RSV-F. An antibody fragment may include a Fab
fragment, a F(ab')2 fragment, a Fv fragment, a dAb fragment, a fragment containing a CDR, or
an isolated CDR. Antigen-binding fragments of an antibody may be derived, e.g. , from full
antibody molecules using any suitable standard techniques such as proteolytic digestion or
recombinant c engineering techniques ing the manipulation and expression of DNA
ng antibody variable and (optionally) nt domains. Such DNA is known and/or is
readily available from, e.g. , commercial sources, DNA libraries (including, e.g. , phage-antibody
libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or
by using molecular biology techniques, for example, to arrange one or more variable and/or
constant domains into a suitable configuration, or to introduce , create cysteine es,
, add or delete amino acids, etc.
Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii)
F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) -chain Fv (scFv) molecules;
(vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid es that
mimic the hypervariable region of an antibody (e.g. , an isolated complementarity determining
region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other
engineered molecules, such as domain-specific dies, single domain antibodies, domaindeleted
antibodies, chimeric antibodies, CDR-grafted antibodies, ies, triabodies,
tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.),
small modular pharmaceuticals ), and shark variable IgNAR domains, are also
16536800_1 (GHMatters) P40719NZ00
encompassed within the expression "antigen-binding fragment," as used herein.
An antigen-binding fragment of an antibody will lly comprise at least one variable
domain. The variable domain may be of any size or amino acid composition and will generally
comprise at least one CDR, which is adjacent to or in frame with one or more ork
sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the
VH and VL domains may be situated relative to one another in any suitable arrangement. For
example, the variable region may be dimeric and n VH - VH, VH - VL or VL - VL dimers.
Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL
domain.
In certain embodiments, an antigen-binding nt of an antibody may contain at
least one variable domain covalently linked to at least one constant domain. Non-limiting,
exemplary configurations of variable and nt domains that may be found within an antigenbinding
fragment of an antibody of the present invention include: (i) VH -CH1; (ii) VH -CH2; (iii) VH
-CH3; (iv) VH -CH1-CH2; (v) VH -CH1-CH2-CH3; (vi) VH H3; (vii) VH -CL; (viii) VL -CH1; (ix) VL -
CH2; (x) VL -CH3; (xi) VL -CH1-CH2; (xii) VL -CH1-CH2-CH3; (xiii) VL -CH2-CH3; and (xiv) VL -CL. In
any configuration of variable and constant domains, including any of the ary
configurations listed above, the variable and constant domains may be either directly linked to
one another or may be linked by a full or partial hinge or linker region. A hinge region may
consist of at least 2 (e.g. , 5, 10, 15, 20, 40, 60 or more) amino acids, which result in a flexible or
semi-flexible e between adjacent variable and/or constant domains in a single polypeptide
le. Moreover, an antigen-binding fragment of an antibody of the present invention may
se a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant
domain configurations listed above in non-covalent association with one another and/or with one
or more monomeric VH or VL domain (e.g. , by disulfide bond(s)).
As with full antibody les, antigen-binding fragments may be mono-specific or
multi-specific (e.g. , bi-specific). A multi-specific antigen-binding fragment of an antibody will
typically se at least two different variable domains, wherein each variable domain is
e of specifically binding to a separate antigen or to a different epitope on the same
antigen. Any multi-specific antibody format, including the exemplary bi-specific antibody formats
disclosed herein, may be d for use in the context of an antigen-binding fragment of an
antibody of the present invention using routine techniques available in the art.
Preparation of Human Antibodies
s for generating human antibodies in transgenic mice are known in the art. Any
such known methods can be used in the context of the present invention to make human
antibodies that specifically bind to RSV-F.
Using VELOCIMMUNE® technology (see, for example, US 541, Regeneron
16536800_1 (GHMatters) P40719NZ00
Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal
antibodies, high affinity chimeric antibodies to RSV-F are lly isolated having a human
variable region and a mouse constant region. The MMUNE® technology involves
generation of a transgenic mouse having a genome comprising human heavy and light chain
variable regions operably linked to endogenous mouse nt region loci such that the mouse
produces an dy comprising a human variable region and a mouse nt region in
response to antigenic stimulation. The DNA encoding the variable regions of the heavy and
light chains of the antibody are isolated and operably linked to DNA encoding the human heavy
and light chain constant regions. The DNA is then expressed in a cell e of sing the
fully human antibody.
Generally, a VELOCIMMUNE® mouse is challenged with the antigen of interest, and
lymphatic cells (such as B-cells) are recovered from the mice that express antibodies. The
lymphatic cells may be fused with a a cell line to prepare immortal hybridoma cell lines,
and such hybridoma cell lines are screened and selected to fy hybridoma cell lines that
produce antibodies specific to the n of interest. DNA encoding the variable regions of the
heavy chain and light chain may be isolated and linked to desirable isotypic constant regions of
the heavy chain and light chain. Such an antibody n may be produced in a cell, such as a
CHO cell. Alternatively, DNA ng the antigen-specific chimeric antibodies or the variable
domains of the light and heavy chains may be isolated directly from antigen-specific
lymphocytes.
Initially, high affinity chimeric antibodies are isolated having a human variable region and
a mouse constant region. As in the experimental section below, the antibodies are
characterized and selected for desirable characteristics, including affinity, selectivity, epitope,
etc. The mouse constant regions are replaced with a desired human nt region to
te the fully human antibody of the invention, for example wild-type or modified IgG1 or
IgG4. While the constant region selected may vary according to specific use, high affinity
antigen-binding and target specificity characteristics reside in the variable region.
In certain embodiments, the antibodies of the instant invention possess affinities (KD)
ranging from about 1.0 x 10 -7 M to about 1.0 x 10-12 M, when ed by binding to antigen
either immobilized on solid phase or in solution phase. In certain embodiments, the antibodies
of the invention possess affinities (KD) ranging from about 1 x 10-7 M to about 6 x10-10 M, when
measured by binding to antigen either immobilized on solid phase or in solution phase. In
certain ments, the antibodies of the invention possess affinities (KD) ranging from about
1 x 10-7 M to about 9 x10-10 M, when measured by binding to antigen either immobilized on solid
phase or in solution phase. The mouse constant regions are replaced with desired human
constant s to generate the fully human antibodies of the invention. While the constant
region selected may vary according to specific use, high affinity antigen-binding and target
16536800_1 (GHMatters) P40719NZ00
specificity characteristics reside in the variable region. Surprisingly, certain antibodies of the
present invention, while demonstrating lower affinities than motavizumab, are more potent in
terms of virus neutralization.
Bioequivalents
The SV-F antibodies and antibody fragments of the present invention encompass
ns having amino acid ces that vary from those of the described antibodies, but that
retain the ability to bind RSV-F. Such variant antibodies and antibody fragments comprise one
or more additions, deletions, or substitutions of amino acids when compared to parent
sequence, but exhibit biological activity that is essentially equivalent to that of the described
antibodies. Likewise, the antibody-encoding DNA sequences of the t invention
encompass sequences that comprise one or more additions, deletions, or substitutions of
nucleotides when compared to the disclosed sequence, but that encode an antibody or antibody
fragment that is essentially bioequivalent to an antibody or antibody fragment of the ion.
Two n-binding proteins, or antibodies, are considered bioequivalent if, for
example, they are pharmaceutical equivalents or ceutical alternatives whose rate and
extent of absorption do not show a significant difference when administered at the same molar
dose under similar experimental conditions, either single does or multiple dose. Some
dies will be considered lents or pharmaceutical atives if they are equivalent in
the extent of their absorption but not in their rate of absorption and yet may be considered
bioequivalent because such differences in the rate of absorption are intentional and are
reflected in the labeling, are not essential to the attainment of ive body drug concentrations
on, e.g ., chronic use, and are considered medically insignificant for the ular drug t
studied.
In one embodiment, two antigen-binding proteins are bioequivalent if there are no
clinically meaningful differences in their safety, purity, and potency.
In one ment, two antigen-binding proteins are bioequivalent if a patient can be
switched one or more times between the reference product and the biological product without
an expected increase in the risk of adverse effects, including a clinically significant change in
immunogenicity, or diminished effectiveness, as compared to continued y without such
switching.
In one embodiment, two antigen-binding proteins are bioequivalent if they both act by a
common mechanism or mechanisms of action for the condition or conditions of use, to the
extent that such isms are known.
ivalence may be demonstrated by in vivo and/or in vitro methods. Bioequivalence
measures include, e.g ., (a) an in vivo test in humans or other mammals, in which the
concentration of the antibody or its metabolites is measured in blood, plasma, serum, or other
16536800_1 (GHMatters) P40719NZ00
biological fluid as a function of time; (b) an in vitro test that has been correlated with and is
reasonably predictive of human in vivo bioavailability data; (c) an in vivo test in humans or other
mammals in which the appropriate acute pharmacological effect of the antibody (or its target) is
measured as a function of time; and (d) in a well-controlled clinical trial that establishes safety,
efficacy, or bioavailability or bioequivalence of an antibody.
ivalent variants of the antibodies of the invention may be constructed by, for
e, making various substitutions of residues or sequences or deleting terminal or internal
residues or sequences not needed for biological activity. For example, cysteine residues not
essential for biological activity can be deleted or replaced with other amino acids to prevent
formation of unnecessary or incorrect intramolecular disulfide s upon ration. In
other contexts, bioequivalent antibodies may e antibody variants comprising amino acid
changes, which modify the glycosylation teristics of the antibodies, e.g. , ons that
eliminate or remove glycosylation.
Biological teristics of the Antibodies
In general, the antibodies of the present invention may on by binding to RSV-F
and in so doing act to block the fusion of the viral membrane with the host cell membrane. The
antibodies of the present invention may also on by binding to RSV-F and in so doing block
the cell to cell spread of the virus and block syncytia ion associated with RSV infection of
cells.
In certain embodiments, the antibodies of the present invention may function by blocking
or ting RSV fusion to the cell membrane by binding to any other region or fragment of the
full length native F protein, the amino acid sequence of which is shown in SEQ ID NO: 354, also
shown as GenBank accession number AAX23994.1. The antibodies may also bind to any
region which is found in SEQ ID NO: 353, or to a fragment found within SEQ ID NO: 353.
In one embodiment, the invention provides a fully human monoclonal antibody or
antigen-binding fragment thereof that binds to the F protein of RSV subtype A or B, wherein the
antibody or nt thereof exhibits one or more of the following characteristics: (a) is a fully
human monoclonal antibody; (b) exhibits a KD ranging from about 1 X 10-7 M to about 6 x 10-10 M;
(c) is capable of neutralizing respiratory syncytial virus subtype A and e B strains in vitro;
(d) demonstrates the ability to significantly reduce the viral load in an animal model of RSV
infection (e) demonstrates a 1-2 logs greater reduction of nasal and/or lung viral titers when
compared to palivizumab; (f) demonstrates an effective dose 99 (ED99 ) of about 0.15 mg/kg or
less when administered subcutaneously in a mouse model of RSV subtype A infection, or an
ED 99 of about 0.62 mg/kg or less when administered in a cotton rat model of RSV subtype A
infection, or an ED99 of about 2.5 mg/kg or less when administered in a cotton rat model of RSV
subtype B infection; (g) trates an ED99 that is about 2 to 3 fold lower than the ED99 for
palivizumab or motavizumab; (h) demonstrates a neutralization potency against one or more
00_1 (GHMatters) P40719NZ00
subtype A laboratory strains of RSV that is about 15 to 17 fold improvement over palivizumab,
or demonstrates a neutralization potency against one or more subtype A clinical strains of RSV
that is about 10 to 22 fold improvement over palivizumab; (i) demonstrates a neutralization
potency against a subtype B tory strain of RSV that is about a 2 to 5 fold improvement
over palivizumab (j) demonstrates a neutralization potency against a subtype A laboratory strain
or clinical strain of RSV that is about a 0.5 to 2 fold improvement over AM-22; (k) trates
a neutralization potency against one or more subtype B laboratory strains of RSV that is about a
2.5 to 17 fold improvement over AM-22; (l) comprises a HCVR having an amino acid sequence
selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162,
178, 194, 210, 226, 242, 258, 274, 290, 306, 322 and 338, or a ntially similar sequence
thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity; (m)
comprises a LCVR having an amino acid sequence selected from the group ting of SEQ
ID NO: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298,
314, 330 and 346, or a substantially similar sequence thereof having at least 90%, at least 95%,
at least 98% or at least 99% sequence ty; (n) comprises a HCDR3 domain having an
amino acid sequence selected from the group consisting of SEQ ID NO: 8, 24, 40, 56, 72, 88,
104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, 312, 328, and 344, or a
substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least
99% sequence identity and a LCDR3 domain having an amino acid sequence selected from the
group consisting of SEQ ID NO: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224,
240, 256, 272, 288, 304, 320, 336 and 352, or a substantially r sequence thereof having at
least 90%, at least 95%, at least 98% or at least 99% sequence identity; (o) comprises a
HCDR1 domain having an amino acid ce selected from the group consisting of SEQ ID
NO: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, 308,
324 and 340, or a substantially r sequence thereof having at least 90%, at least 95%, at
least 98% or at least 99% sequence identity; a HCDR2 domain having an amino acid sequence
selected from the group consisting of SEQ ID NO: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150,
166, 182, 198, 214, 230, 246, 262, 278, 294, 310, 326 and 342, or a substantially similar
ce thereof having at least 90%, at least 95%, at least 98% or at least 99% ce
identity; a LCDR1 domain having an amino acid sequence selected from the group consisting of
SEQ ID NO: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284,
300, 316, 332 and 348, or a substantially similar sequence thereof having at least 90%, at least
95%, at least 98% or at least 99% sequence identity; and a LCDR2 domain having an amino
acid sequence selected from the group consisting of SEQ ID NO: 14, 30, 46, 62, 78, 94, 110,
126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, 318, 334 and 350, or a substantially
similar ce thereof having at least 90%, at least 95%, at least 98% or at least 99%
sequence identity; (p) interacts with an amino acid sequence comprising residues ranging from
16536800_1 ters) P40719NZ00
about position 161 to about position 188 of SEQ ID NO: 354; (q) interacts with either the serine
at position 173 of SEQ ID NO: 354, or the threonine at position 174 of SEQ ID NO: 354, or both
the serine at position 173 of SEQ ID NO: 354 and the threonine at on 174 of SEQ ID NO:
354; (r) does not cross-compete for binding to RSV-F protein with zumab or motavizumab;
(s) inhibits fusion of the virus to the cell.
n anti-RSV-F antibodies of the present invention are able to bind to the F protein of
RSV and lize the infectivity of both subtypes A and B of RSV as determined by in vitro
assays. The ability of the antibodies of the invention to bind to and neutralize the infectivity of
the subtypes of RSV may be measured using any standard method known to those skilled in the
art, including binding assays, or neutralization assays, or in vivo protection assays, as bed
herein.
Non-limiting, exemplary in vitro and in vivo assays for measuring binding ty and in
vitro neutralization and in vivo efficacy are illustrated in Examples 3, 4, 5, 7, 8, 9, 10, 11 and 12
herein. In Example 3, the binding affinities and kinetic constants of human anti-RSV-F
antibodies were determined by surface plasmon resonance and the measurements were
conducted on a Biacore 4000 or T200 instrument. In e 4, the potency of the antibodies
was tested in a RSV micro-neutralization assay. Example 5 demonstrates the ability of the
antibodies of the invention to neutralize an RSV infection in vivo in two ent animal models.
Examples 7 and 8 demonstrate the interaction of the antibodies of the invention with particular
binding sites on RSV-F protein. Examples 9 and 10 demonstrate the neutralization capabilities
of the antibodies with several laboratory and clinical strains of RSV subtypes A and B. Example
11 demonstrates the ability of the antibodies of the invention to inhibit fusion of the virus to cells.
Example 12 demonstrates the cross-competition of various antibodies for binding to RSV-F.
Epitope Mapping and Related Technologies
Various techniques known to s of ordinary skill in the art can be used to
ine whether an antibody acts with one or more amino acids" within a ptide or
protein. Exemplary techniques include, for example, a routine cross-blocking assay such as
that described Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)
can be performed. Other s include alanine scanning mutational analysis, peptide blot
analysis (Reineke (2004) Methods Mol Biol 248:443-63), peptide cleavage analysis
crystallographic studies and NMR analysis. In addition, methods such as epitope on,
epitope extraction and chemical modification of antigens can be employed (Tomer (2000)
Protein Science 9: 6). Another method that can be used to identify the amino acids within
a polypeptide with which an antibody interacts is hydrogen/deuterium exchange ed by
mass spectrometry. In general terms, the hydrogen/deuterium exchange method involves
deuterium-labeling the protein of interest, followed by binding the antibody to the deuteriumlabeled
protein. Next, the n/antibody x is transferred to water and exchangeable
00_1 (GHMatters) P40719NZ00
protons within amino acids that are ted by the antibody complex undergo deuterium-tohydrogen
xchange at a slower rate than exchangeable protons within amino acids that
are not part of the ace. As a result, amino acids that form part of the protein/antibody
interface may retain ium and therefore exhibit relatively higher mass compared to amino
acids not included in the interface. After dissociation of the antibody, the target protein is
subjected to protease cleavage and mass spectrometry analysis, thereby revealing the
deuterium-labeled residues that correspond to the specific amino acids with which the antibody
interacts. See, e.g., Ehring (1999) ical mistry 267(2):252-259; Engen and Smith
(2001) Anal. Chem. 73:256A-265A.
The term "epitope" refers to a site on an antigen to which B and/or T cells respond. B-
cell epitopes can be formed both from uous amino acids or noncontiguous amino acids
juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are
typically retained on exposure to denaturing solvents, s epitopes formed by ry
folding are typically lost on treatment with denaturing solvents. An epitope lly es at
least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
Modification-Assisted Profiling (MAP), also known as Antigen ure-based Antibody
Profiling (ASAP) is a method that categorizes large s of monoclonal antibodies (mAbs)
directed against the same antigen according to the similarities of the binding profile of each
antibody to chemically or enzymatically modified antigen surfaces (US 2004/0101920). Each
category may reflect a unique epitope either distinctly different from or partially overlapping with
e represented by another category. This technology allows rapid filtering of genetically
identical antibodies, such that characterization can be focused on genetically distinct antibodies.
When applied to hybridoma screening, MAP may facilitate identification of rare hybridoma
clones that e mAbs having the desired characteristics. MAP may be used to sort the
antibodies of the ion into groups of antibodies binding different epitopes.
In certain embodiments, the antibodies or antigen-binding fragments of the invention
interact with an amino acid sequence comprising amino acid residues ranging from about
position 161 to about position 188 of SEQ ID NO: 354. In certain embodiments, the antibodies
of the invention may interact with amino acid residues that extend beyond the region identified
above by about 5 to 10 amino acid residues, or by about 10 to 15 amino acid residues, or by
about 15 to 20 amino acid residues towards either the amino terminal or the carboxy terminal of
the RSV-F protein.
In one ment, the invention provides an isolated human monoclonal antibody that
specifically binds RSV-F, or an antigen-binding nt thereof, wherein the antibody or
antigen-binding fragment thereof interacts with at least one amino acid sequence selected from
the group consisting of SEQ ID NO: 355 and 356.
In one embodiment, the invention provides an isolated human monoclonal antibody that
16536800_1 (GHMatters) P40719NZ00
ically binds RSV-F, or an antigen-binding fragment thereof, wherein the antibody or
antigen-binding fragment thereof interacts with at least one amino acid residue within residues
161 through 188 of SEQ ID NO: 354.
In one embodiment, the invention provides an isolated human monoclonal antibody that
specifically binds RSV-F, or an antigen-binding fragment thereof, wherein the antibody or
n-binding fragment thereof interacts with at least one amino acid residue within SEQ ID
NO: 355 or SEQ ID NO:356.
In one embodiment, the invention provides an isolated human monoclonal antibody that
specifically binds RSV-F, or an n-binding fragment thereof, wherein the antibody or
antigen-binding fragment thereof interacts with either the serine at on 173 of SEQ ID NO:
354, or the threonine at position 174 of SEQ ID NO: 354, or both the serine at position 173 of
SEQ ID NO: 354 and the threonine at on 174 of SEQ ID NO: 354.
The t invention includes anti-RSV-F antibodies that bind to the same epitope as
any of the specific exemplary dies described herein in Table 1. Likewise, the present
invention also includes SV-F antibodies that compete for g to RSV-F fragment with
any of the specific exemplary antibodies described herein in Table 1.
In certain embodiments, the antibodies of the present invention do not cross-compete for
binding to RSV-F with palivizumab, motavizumab, or AM-22.
In certain embodiments, the antibodies of the present invention do not bind to the same
e on RSV-F protein as palivizumab or motavizumab.
In certain embodiments, the dies of the present invention do not bind to an epitope
on RSV-F ranging from amino acid residue 255 to amino acid residue 276 of SEQ ID NO: 354.
One can easily determine whether an antibody binds to the same epitope as, or
competes for binding with, a reference anti-RSV-F antibody by using e methods known in
the art. For example, to determine if a test antibody binds to the same epitope as a reference
RSV-F antibody of the invention, the reference antibody is allowed to bind to a RSV-F protein or
e under saturating conditions. Next, the y of a test antibody to bind to the RSV-F
molecule is assessed. If the test antibody is able to bind to RSV-F following saturation binding
with the reference anti- RSV-F antibody, it can be concluded that the test antibody binds to a
different epitope than the reference anti- RSV-F antibody. On the other hand, if the test
antibody is not able to bind to the RSV-F molecule following saturation binding with the
reference anti-RSV-F antibody, then the test antibody may bind to the same epitope as the
e bound by the reference anti- RSV-F antibody of the ion.
To determine if an dy competes for binding with a reference anti- RSV-F antibody,
the above-described binding methodology is performed in two orientations: In a first orientation,
the reference antibody is allowed to bind to a RSV-F molecule under saturating conditions
followed by assessment of binding of the test antibody to the RSV-F molecule. In a second
16536800_1 (GHMatters) P40719NZ00
orientation, the test antibody is allowed to bind to a RSV-F molecule under saturating conditions
followed by assessment of binding of the reference antibody to the RSV-F molecule. If, in both
ations, only the first (saturating) antibody is capable of binding to the RSV-F molecule,
then it is concluded that the test antibody and the reference antibody compete for binding to
RSV-F. As will be appreciated by a person of ordinary skill in the art, an antibody that competes
for binding with a reference antibody may not necessarily bind to the identical e as the
reference antibody, but may sterically block binding of the reference antibody by binding an
overlapping or adjacent epitope.
Two antibodies bind to the same or overlapping epitope if each competitively inhibits
(blocks) g of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one
antibody ts binding of the other by at least 50% but preferably 75%, 90% or even 99% as
measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 1990
50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid
ons in the antigen that reduce or eliminate binding of one antibody reduce or eliminate
binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations
that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.
Additional routine experimentation (e.g. , peptide mutation and binding analyses) can
then be carried out to confirm whether the observed lack of binding of the test antibody is in fact
due to g to the same epitope as the nce antibody or if steric blocking (or another
phenomenon) is responsible for the lack of observed g. Experiments of this sort can be
performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other
quantitative or qualitative antibody-binding assay available in the art.
conjugates
The invention encompasses a human RSV-F monoclonal dy conjugated to a
therapeutic moiety (“immunoconjugate”), such as an agent that is capable of reducing the
severity of primary ion with RSV, or to ameliorate at least one m associated with
RSV infection, including coughing, fever, pneumonia, or the severity thereof. Such an agent
may be a second ent antibody to RSV-F, or a vaccine. The type of therapeutic moiety that
may be ated to the anti- RSV-F antibody and will take into account the condition to be
treated and the desired therapeutic effect to be achieved. Alternatively, if the desired
eutic effect is to treat the sequelae or symptoms associated with RSV infection, or any
other condition resulting from such infection, such as, but not limited to, nia, it may be
advantageous to conjugate an agent appropriate to treat the ae or symptoms of the
ion, or to alleviate any side effects of the antibodies of the invention. Examples of suitable
agents for forming immunoconjugates are known in the art, see for example, WO 05/103081.
16536800_1 (GHMatters) P40719NZ00
Multi-specific Antibodies
The antibodies of the present invention may be mono-specific, bi-specific, or multi-
specific. Multi-specific antibodies may be specific for different epitopes of one target
polypeptide or may n antigen-binding domains specific for more than one target
polypeptide. See, e.g., Tutt et al., 1991, J. Immunol. -69; Kufer et al., 2004, Trends
Biotechnol. 22:238-244. The antibodies of the present invention can be linked to or coexpressed
with another functional molecule, e.g ., another peptide or protein. For example, an
antibody or fragment thereof can be functionally linked (e.g., by chemical ng, genetic
fusion, noncovalent association or otherwise) to one or more other molecular entities, such as
r antibody or antibody fragment to produce a bi-specific or a specific antibody with a
second binding specificity.
An exemplary bi-specific dy format that can be used in the t of the present
ion involves the use of a first immunoglobulin (Ig) CH3 domain and a second Ig CH3
, wherein the first and second Ig CH3 s differ from one r by at least one
amino acid, and wherein at least one amino acid difference reduces binding of the bi-specific
antibody to Protein A as ed to a bi-specific antibody lacking the amino acid difference.
In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain
contains a mutation that reduces or abolishes Protein A binding such as an H95R modification
(by IMGT exon ing; H435R by EU numbering). The second CH3 may further comprise a
Y96F modification (by IMGT; Y436F by EU). r modifications that may be found within the
second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I (by IMGT; D356E, L358M,
N384S, K392N, V397M, and V422I by EU) in the case of IgG1 antibodies; N44S, K52N, and
V82I (IMGT; N384S, K392N, and V422I by EU) in the case of IgG2 antibodies; and Q15R,
N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K,
E419Q, and V422I by EU) in the case of IgG4 antibodies. Variations on the cific antibody
format described above are contemplated within the scope of the present invention.
Therapeutic Administration and Formulations
The invention provides therapeutic compositions comprising the anti-RSV-F antibodies
or antigen-binding fragments thereof of the present invention. The administration of therapeutic
compositions in accordance with the invention will be administered with suitable carriers,
excipients, and other agents that are incorporated into formulations to provide improved
transfer, delivery, tolerance, and the like. A multitude of riate formulations can be found
in the formulary known to all pharmaceutical ts: Remington's Pharmaceutical Sciences,
Mack Publishing Company, Easton, PA. These formulations include, for example, powders,
pastes, nts, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such
as LIPOFECTIN™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil
emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid
16536800_1 (GHMatters) P40719NZ00
gels, and semi-solid es ning carbowax. See also Powell et al. "Compendium of
excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-311.
The dose of each of the dies of the invention may vary depending upon the age
and the size of a subject to be administered, target disease, conditions, route of administration,
and the like. When the antibodies of the present invention are used for treating a RSV infection
in a patient, or for treating one or more symptoms associated with a RSV ion, such as the
cough or pneumonia associated with a RSV infection in a patient, or for ing the severity of
the disease, it is advantageous to administer each of the antibodies of the present invention
enously or subcutaneously normally at a single dose of about 0.01 to about 30 mg/kg body
weight, more preferably about 0.1 to about 20 mg/kg body weight, or about 0.1 to about 15
mg/kg body weight, or about 0.02 to about 7 mg/kg body weight, about 0.03 to about 5 mg/kg
body weight, or about 0.05 to about 3 mg/kg body weight, , or about 1 mg/kg body weight, or
about 3.0 mg/kg body weight, or about 10 mg/kg body weight, or about 20 mg/kg body .
Multiple doses may be administered as ary. Depending on the severity of the condition,
the frequency and the duration of the treatment can be adjusted. In certain embodiments, the
antibodies or antigen-binding fragments thereof of the invention can be administered as an
initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about
300 mg, or about 10 to about 150 mg, to about 100 mg, or to about 50 mg. In certain
embodiments, the initial dose may be followed by administration of a second or a plurality of
subsequent doses of the dies or antigen-binding fragments thereof in an amount that can
be approximately the same or less than that of the initial dose, wherein the subsequent doses
are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks;
at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least
9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.
Various delivery systems are known and can be used to administer the pharmaceutical
ition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules,
recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis
(see, e.g ., Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but
are not d to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural and oral routes. The composition may be administered by
any convenient route, for example by on or bolus injection, by absorption through epithelial
or mucocutaneous linings (e.g., oral mucosa, nasal mucosa, rectal and intestinal mucosa, etc.)
and may be stered together with other biologically active agents. Administration can be
ic or local. It may be delivered as an aerosolized formulation (See /0311515 and
US2012/0128669). The delivery of agents useful for treating respiratory diseases by inhalation
is becoming more widely accepted (See A. J. Bitonti and J. A. Dumont, (2006), Adv. Drug Deliv.
Rev, 58:1106-1118). In addition to being effective at treating local pulmonary disease, such a
16536800_1 (GHMatters) P40719NZ00
delivery mechanism may also be useful for systemic delivery of antibodies (See Maillet et al.
(2008), ceutical Research, Vol. 25, No. 6, 2008).
The pharmaceutical composition can be also delivered in a vesicle, in particular a
liposome (see, for example, Langer (1990) e 249:1527-1533).
In n situations, the pharmaceutical composition can be delivered in a controlled
release system. In one embodiment, a pump may be used. In another embodiment, polymeric
materials can be used. In yet another embodiment, a controlled release system can be placed
in proximity of the composition’s , thus requiring only a fraction of the systemic dose.
The injectable preparations may include dosage forms for intravenous, subcutaneous,
intracutaneous and uscular injections, drip infusions, etc. These injectable ations
may be prepared by methods publicly known. For example, the injectable preparations may be
prepared, e.g., by dissolving, ding or emulsifying the antibody or its salt bed above
in a sterile aqueous medium or an oily medium conventionally used for injections. As the
aqueous medium for injections, there are, for example, physiological saline, an isotonic solution
ning glucose and other auxiliary agents, etc., which may be used in combination with an
appropriate solubilizing agent such as an alcohol (e.g ., ethanol), a polyalcohol (e.g., propylene
glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50
(polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there
are employed, e.g ., sesame oil, n oil, etc., which may be used in combination with a
solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus ed is
preferably filled in an appropriate ampoule.
A pharmaceutical composition of the present invention can be red subcutaneously
or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous
delivery, a pen delivery device readily has applications in delivering a pharmaceutical
composition of the present invention. Such a pen delivery device can be reusable or
disposable. A reusable pen delivery device generally utilizes a eable cartridge that
contains a pharmaceutical composition. Once all of the pharmaceutical composition within the
cartridge has been administered and the cartridge is empty, the empty cartridge can y be
discarded and replaced with a new cartridge that contains the pharmaceutical composition. The
pen ry device can then be reused. In a able pen delivery device, there is no
replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the
pharmaceutical composition held in a oir within the device. Once the reservoir is emptied
of the pharmaceutical composition, the entire device is discarded.
Numerous reusable pen and autoinjector delivery s have applications in the
aneous delivery of a pharmaceutical composition of the present invention. Examples
include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK),
DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX
16536800_1 (GHMatters) P40719NZ00
75/25™ pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis, IN),
NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo
Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ),
OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis,
Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having
ations in subcutaneous delivery of a pharmaceutical composition of the present invention
e, but nly are not limited to the SOLOSTAR™ pen (sanofi-aventis), the FLEXPEN™
(Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK ™ Autoinjector (Amgen,
Thousands Oaks, CA), the PENLET ™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey,
L.P.) and the HUMIRA ™ Pen (Abbott Labs, Abbott Park, IL), to name only a few.
Advantageously, the pharmaceutical compositions for oral or parenteral use described
above are prepared into dosage forms in a unit dose suited to fit a dose of the active
ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, es,
injections les), suppositories, etc. The amount of the aforesaid antibody contained is
generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of
injection, it is preferred that the aid antibody is contained in about 5 to about 100 mg and
in about 10 to about 250 mg for the other dosage forms.
Administration Regimens
According to certain embodiments of the present invention, multiple doses of an
antibody to RSV-F may be administered to a subject over a defined time course. The methods
according to this aspect of the invention comprise sequentially administering to a subject
multiple doses of an dy to RSV-F. As used herein, "sequentially administering" means
that each dose of antibody to RSV-F is stered to the subject at a different point in time,
e.g. , on different days separated by a predetermined interval (e.g. , hours, days, weeks or
months). The present invention es s which comprise sequentially administering to
the patient a single l dose of an antibody to RSV-F, followed by one or more secondary
doses of the dy to RSV-F and optionally followed by one or more tertiary doses of the
antibody to RSV-F .
The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal
sequence of administration of the dy to RSV-F. Thus, the "initial dose" is the dose which
is administered at the beginning of the treatment regimen (also referred to as the "baseline
dose"); the "secondary doses" are the doses which are administered after the initial dose; and
the "tertiary doses" are the doses which are administered after the secondary doses. The initial,
secondary, and tertiary doses may all contain the same amount of antibody to RSV-F, but
generally may differ from one r in terms of frequency of administration. In certain
embodiments, however, the amount of antibody to RSV-F contained in the initial, secondary
16536800_1 (GHMatters) P40719NZ00
and/or tertiary doses vary from one another (e.g. , adjusted up or down as appropriate) during
the course of ent. In certain embodiments, two or more (e.g. , 2, 3, 4, or 5) doses are
administered at the beginning of the treatment regimen as "loading doses" followed by
subsequent doses that are administered on a less frequent basis (e.g. , enance doses").
In one exemplary embodiment of the present invention, each secondary and/or tertiary
dose is stered 1 to 26 (e.g. , 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9,
9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19,
19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks after the
immediately ing dose. The phrase "the immediately preceding dose," as used herein,
means, in a sequence of multiple administrations, the dose of antibody to RSV-F which is
stered to a patient prior to the administration of the very next dose in the sequence with
no intervening doses.
The methods according to this aspect of the invention may comprise administering to a
patient any number of secondary and/or tertiary doses of an antibody to RSV-F. For example,
in certain ments, only a single secondary dose is administered to the patient. In other
embodiments, two or more (e.g. , 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered
to the t. Likewise, in certain embodiments, only a single tertiary dose is administered to
the patient. In other embodiments, two or more (e.g. , 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses
are administered to the patient.
In embodiments ing multiple secondary doses, each secondary dose may be
administered at the same frequency as the other secondary doses. For e, each
secondary dose may be administered to the patient 1 to 2 weeks after the immediately
preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose
may be administered at the same frequency as the other tertiary doses. For example, each
tertiary dose may be administered to the patient 2 to 4 weeks after the immediately preceding
dose. Alternatively, the frequency at which the secondary and/or tertiary doses are
administered to a patient can vary over the course of the treatment n. The frequency of
administration may also be adjusted during the course of ent by a physician depending on
the needs of the individual patient following clinical examination.
Therapeutic Uses of the Antibodies
Due to their binding to/interaction with, the RSV fusion protein ), the present
antibodies are useful for preventing fusion of the virus with the host cell membrane, for
preventing cell to cell virus spread, and for inhibition of syncytia formation. As such, the
antibodies of the present invention are useful for preventing an infection of a subject with RSV
when administered prophylactically. Alternatively, the antibodies of the present invention may
be useful for ameliorating at least one symptom associated with the infection, such as coughing,
fever, pneumonia, or for lessening the severity, duration, and/or frequency of the ion. The
16536800_1 (GHMatters) P40719NZ00
antibodies of the ion are also contemplated for prophylactic use in patients at risk for
developing or acquiring an RSV infection. These patients include pre-term s, full term
infants born during RSV season (late fall to early spring), the elderly (for example, in anyone 65
years of age or older), or patients immunocompromised due to illness or treatment with
immunosuppressive therapeutics, or patients who may have an underlying medical condition
that predisposes them to an RSV infection (for example, cystic fibrosis patients, patients with
congestive heart failure or other cardiac conditions, ts with airway impairment, patients
with COPD). It is plated that the antibodies of the ion may be used alone, or in
conjunction with a second agent, or third agent for treating RSV infection, or for alleviating at
least one m or complication associated with the RSV infection, such as the fever,
coughing, bronchiolitis, or pneumonia associated with, or resulting from such an infection. The
second or third agents may be delivered concurrently with the dies of the invention, or
they may be administered separately, either before or after the antibodies of the invention. The
second or third agent may be an anti-viral such as ribavirin, an NSAID or other agents to reduce
fever or pain, another second but different antibody that specifically binds RSV-F, an agent (e.g.
an antibody) that binds to another RSV n, such as RSV-G, a e against RSV, an
siRNA specific for an RSV antigen.
In yet a r ment of the invention the present antibodies are used for the
preparation of a pharmaceutical composition for treating patients suffering from a RSV infection.
In yet another embodiment of the invention the present antibodies are used for the preparation
of a pharmaceutical composition for reducing the severity of a primary infection with RSV, or for
reducing the duration of the infection, or for reducing at least one symptom associated with the
RSV ion. In a further embodiment of the invention the present antibodies are used as
t therapy with any other agent useful for ng an RSV infection, including an antiviral,
a toxoid, a vaccine, a second RSV-F antibody, or any other dy specific for an RSV
antigen, including an RSV-G antibody, or any other palliative therapy known to those skilled in
the art.
Combination ies
As noted above, the methods of the present invention, according to certain
embodiments, comprise administering to the subject one or more additional therapeutic agents
in combination with an antibody to RSV-F. As used herein, the expression "in combination with"
means that the additional eutic agents are administered before, after, or concurrent with
the pharmaceutical ition comprising the anti-RSV-F antibody. The term “in combination
with” also includes sequential or concomitant administration of the anti-RSV-F antibody and a
second therapeutic agent.
For example, when administered "before" the pharmaceutical composition comprising
the anti-RSV-F antibody, the additional therapeutic agent may be administered about 72 hours,
16536800_1 (GHMatters) P40719NZ00
about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10
hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30
minutes, about 15 minutes or about 10 minutes prior to the administration of the pharmaceutical
composition comprising the anti-RSV-F antibody. When administered "after" the
pharmaceutical composition comprising the anti-RSV-F antibody, the additional therapeutic
agent may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1
hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12
hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours or about 72 hours after
the administration of the pharmaceutical composition comprising the SV-F antibodies.
Administration "concurrent" or with the ceutical composition comprising the anti-RSV-F
antibody means that the additional eutic agent is administered to the subject in a separate
dosage form within less than 5 minutes (before, after, or at the same time) of administration of
the pharmaceutical composition comprising the anti-RSV-F antibody, or administered to the
subject as a single combined dosage formulation comprising both the additional eutic
agent and the anti-RSV-F antibody.
Combination therapies may include an anti-RSV-F antibody of the invention and any
additional eutic agent that may be advantageously combined with an antibody of the
invention, or with a biologically active fragment of an antibody of the invention.
For example, a second or third therapeutic agent may be employed to aid in reducing
the viral load in the lungs, such as an antiviral, for example, ribavirin. The antibodies may also
be used in ction with other therapies, as noted above, including a toxoid, a vaccine
specific for RSV, a second antibody specific for RSV-F, or an antibody specific for another RSV
antigen, such as RSV-G.
stic Uses of the Antibodies
The anti-RSV antibodies of the present invention may also be used to detect and/or
measure RSV in a sample, e.g. , for diagnostic purposes. It is oned that confirmation of an
infection thought to be caused by RSV may be made by measuring the presence of the virus
through use of any one or more of the antibodies of the ion. Exemplary diagnostic assays
for RSV may comprise, e.g. , contacting a , obtained from a patient, with an SV-F
antibody of the invention, wherein the SV-F antibody is labeled with a able label or
reporter molecule or used as a capture ligand to selectively e the virus containing the F
protein from patient samples. Alternatively, an unlabeled anti-RSV-F antibody can be used in
diagnostic ations in combination with a secondary antibody which is itself detectably
labeled. The detectable label or reporter le can be a radioisotope, such as 3H, 14 C, 32 P,
S, or 125 I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or
rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish
16536800_1 (GHMatters) P40719NZ00
peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure
RSV containing the F n in a sample include enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).
Samples that can be used in RSV diagnostic assays according to the present ion
include any tissue or fluid sample obtainable from a patient, which contains detectable
quantities of RSV-F n, or fragments thereof, under normal or pathological conditions.
Generally, levels of RSV-F in a particular sample obtained from a healthy t (e.g. , a patient
not afflicted with a disease or condition associated with the presence of RSV-F) will be
measured to initially establish a baseline, or standard, level of the F protein from RSV. This
baseline level of RSV-F can then be compared against the levels of RSV-F ed in
samples obtained from individuals suspected of having an RSV infection, or symptoms
associated with such ion.
Vaccines and Immunogenic Compositions
One aspect of the invention provides an immunogenic composition, or a vaccine, that
when administered to an individual, preferably a human, induces an immune response in such
individual to a Respiratory Syncytial Virus (RSV) n, for example, a RSV-F polypeptide,
wherein the composition may se a recombinant RSV-F protein, or a polypeptide fragment
of a RSV-F protein, or an epitope contained within and obtained from an antigen of the RSV-F
polypeptide or a fragment thereof, and/or comprises DNA and/or RNA which encodes and
expresses an epitope from an antigen of the RSV-F polypeptide, or other polypeptides of the
invention. The genic composition or vaccine may be used therapeutically or
prophylactically and may be used to elicit antibody ty and/or cellular immunity, such as
cellular immunity arising from CTL or CD4+ T cells.
In one ment of the ion, the immunogenic composition, or vaccine, may
comprise the RSV-F protein as shown in SEQ ID NO: 354. In one embodiment of the invention,
the immunogenic composition, or vaccine, may comprise a RSV-F polypeptide fragment
comprising residues 161 through 188 of SEQ ID NO: 354. In one embodiment of the invention,
the immunogenic composition, or vaccine, may comprise one or more amino acid residues
contained within SEQ ID NO: 355 and/or SEQ ID NO: 356. In one ment of the invention,
the immunogenic composition, or vaccine, may comprise SEQ ID NO: 355 and/or SEQ ID NO:
356.
In a related aspect, the invention es a method for inducing an immune response in
an individual, particularly a mammal, preferably humans, by administering to an dual an
immunogenic composition, or a vaccine, comprising a RSV-F protein, or an immunogenic
fragment thereof, or a RSV-F antigen or an immunogenic nt thereof comprising one or
more es ned within the RSV-F antigen or fragment thereof, adequate to produce an
16536800_1 (GHMatters) P40719NZ00
dy and/or a T cell immune response to protect the individual from infection, particularly
infection with Respiratory Syncytial Virus (RSV). Also provided are methods of using the
immunogenic compositions, or vaccines of the invention for ng an immune response that
results in inhibiting, or slowing the ssion of cell to cell viral spread. Methods are also
provided for rating at least one symptom associated with RSV ion by administering
an immunogenic composition, or a vaccine, comprising at least one RSV-F antigen, or one or
more epitopes contained within the RSV-F antigen, which when administered will induce an
immune response in the individual.
For example, in one ment the invention provides a method of inducing an
immune response in an individual comprising delivering to the individual an immunogenic
composition, or vaccine sing, an RSV-F antigen (e.g . the amino acid sequence shown in
SEQ ID NO: 354), or an antigenic nt thereof, ( e.g. a polypeptide comprising residues 161
through 188 of SEQ ID NO: 354), or a nucleic acid vector comprising a nucleotide sequence to
direct expression of such viral polypeptide, or a fragment or a variant thereof, in vivo in order to
induce an immune response.
In one embodiment of the invention, the polypeptide to be used in an immunogenic
composition or in a vaccine for ng an immune response in an individual comprises
residues 161 through 188 of SEQ ID NO: 354. In one embodiment of the ion, the
polypeptide to be used in an immunogenic composition or in a vaccine for inducing an immune
response in an individual comprises one or more amino acid residues ned within SEQ ID
NO: 355 and/or SEQ ID NO: 356. In one embodiment of the ion, the polypeptide to be
used in an immunogenic composition or in a vaccine for inducing an immune response in an
individual comprises SEQ ID NO: 355 and/or SEQ ID NO: 356. In one embodiment of the
invention, the genic composition, or vaccine, may elicit an antibody response specific for
the RSV-F antigen of RSV, wherein the antibodies generated interact with either the serine at
position 173 of SEQ ID NO: 354, or the threonine at position 354, or both the serine at position
173 of SEQ ID NO: 354 and the threonine at position 174 of SEQ ID NO: 354.
In certain embodiments, it is advantageous for the RSV-F antigens or fragments thereof
to be formulated into immunogenic compositions, or vaccines that se immunogenic,
preferably immunologically effective, amounts of additional antigens to elicit immunity to other
ens, preferably viruses and/or bacteria. Such additional antigens may include an
influenza virus antigen, an antigen from metapneumovirus or from a coronavirus, an antigen
from Haemophilus influenzae, Streptococcus pneumonia, or Bordetella pertussis. Other RSV
antigens may be included in the immunogenic itions, or vaccines, such as the RSV-G
glycoprotein, or genic nts thereof, the HN protein, or derivatives thereof. In
certain embodiments, influenza virus antigens to be included in the immunogenic compositions
or vaccines of the invention may include whole, live or inactivated virus, split influenza virus,
16536800_1 (GHMatters) P40719NZ00
grown in eggs or MDCK cells, or Vero cells or whole flu virosomes, or purified or recombinant
proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof.
In certain embodiments of the invention, the immunogenic composition, or vaccine
formulation may se an immunogenic recombinant polypeptide and/or polynucleotide of
the invention, or a combination thereof, together with a suitable carrier/excipient, such as a
pharmaceutically acceptable carrier/excipient. The immunogenic composition and/or vaccine is
preferably stered parenterally, ing, for example, administration that is
subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral
administration include aqueous and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostatic compounds and solutes which render the ation
isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and eous
sterile suspensions which may e suspending agents or thickening agents. The
formulations may be presented in unit-dose or multi-dose containers, for example, sealed
ampoules and vials and may be stored in a freeze-dried ion requiring only the addition of
the sterile liquid carrier immediately prior to use.
The immunogenic composition, or vaccine formulation of the invention may also include
adjuvants for enhancing the immunogenicity of the formulation. At this time, the only adjuvant
widely used in humans has been alum (aluminum phosphate or aluminum hydroxide) and
calcium ate gels. Freund's complete adjuvant and other adjuvants used in research and
veterinary applications have toxicities, which limit their potential use in human vaccines.
However, ally defined preparations such as oil emulsions and tant based
formulations, e.g., MF59 (microfluidized ent stabilized oil-in-water emulsion), QS21
(purified saponin), AS02 [SBAS2] (oil-in-water emulsion + MPL + QS-21), Montanide ISA-51
and ISA-720 (stabilized water-in-oil emulsion), are also in development. Furthermore, microbial
derivatives (natural and synthetic), e.g., muramyl dipeptide, monophosphoryl lipid A (e.g. 3 De-
O-acylated monophosphoryl lipid A, also known as 3D-MPL, which is manufactured by Ribi
chem, Montana), Detox (MPL + M. Phleicell wall skeleton), AGP [RC-529] etic
acylated ccharide), DCChol (lipoidal stimulators able to self organize into
liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing
immunostimulatory CpG motifs), modified LT and CT (genetically modified bacterial toxins to
e non-toxic adjuvant effects), and QS21, an Hplc purified non-toxic on d from
the bark of Quillaja Saponaria Molina, have all been in development for human use..
A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European
Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA).
Other particulate adjuvants include, e.g ., virosomes (unilamellar liposomal es
incorporating a viral antigen), AS04 ([SBAS4] Al salt with MPL), ISCOMS (structured complex of
saponins and lipids), polylactide co-glycolide (PLG).
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Other suitable adjuvants include all acceptable immunostimulatory compounds, such as
cytokines, chemokines, or colony stimulating factors. For example, these may include the
interleukins IL-1, IL-2, IL-4, IL-7, IL-12, gamma-interferon, and hGM-CSF.
It is to be understood that the adjuvant and/or immunostimulatory compound to be used
will depend on the subject to which the vaccine or immunogenic ition will be
administered, the route of injection and the number of injections to be given.
While the invention has been bed with reference to certain RSV-F polypeptides, it
is to be understood that this covers nts of the naturally occurring polypeptides, and
similar polypeptides with additions, deletions or substitutions which do not substantially affect
the immunogenic properties of the recombinant ptides or polynucleotides.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art
with a complete disclosure and description of how to make and use the methods and
compositions of the invention, and are not intended to limit the scope of what the inventors
regard as their ion. Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by , molecular weight is
average molecular weight, temperature is in s Centigrade, and pressure is at or near
atmospheric.
Example 1. Generation of Human dies to RSV-F Protein
An immunogen comprising any one of the following can be used to generate antibodies
to RSV-F protein. In certain embodiments, the antibodies of the invention are obtained from
mice immunized with a primary immunogen, such as a whole respiratory syncytial virus e,
either live, ated or killed/inactivated. The mice may be given one or more booster shots
ning either the same virus isolate, or they may be boosted with the RSV-F protein itself.
In n ments, the mice are ed with live virus, followed by boosting with the
construct shown as SEQ ID NO: 353, or with isolated RSV-F protein, obtained from a virus
isolate or prepared recombinantly. (See also GenBank accession number AAX23994.1)
In certain embodiments, the dies of the invention are obtained from mice
immunized with a primary immunogen, such as a biologically active RSV, subtype A or B,
and/or the RSV fusion (F) protein, or an immunogenic fragment of the RSV fusion (RSV-F)
protein, or DNA encoding the full length protein or the active fragment thereof. The immunogen
may be delivered to the animal via any route including but not limited to intramuscularly,
subcutaneously, intravenously or intranasally.
In certain embodiments, whole virus, or the RSV-F protein or fragments thereof may be
used for preparing monospecific, bispecific, or multispecific antibodies.
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The whole virus, or full length proteins, or fragments thereof, that were used as
immunogens, as noted above, were administered directly, with an adjuvant to stimulate the
immune response, to a VELOCIMMUNE® mouse comprising DNA encoding human
Immunoglobulin heavy and kappa light chain variable regions. The antibody immune response
was monitored by a RSV-F immunoassay. When a d immune response was achieved,
cytes were ted and fused with mouse myeloma cells to preserve their viability and
form hybridoma cell lines. The oma cell lines were screened and selected to fy cell
lines that produce RSV-F-specific antibodies. Using this technique, and the various
immunogens bed above, several chimeric dies (i.e. , antibodies sing human
variable s and mouse nt domains) were obtained; certain exemplary antibodies
generated in this manner were designated as H1M3621N, H1M3622N, H1M2634N and
H1M3627N.
Anti-RSV-F antibodies were also isolated directly from antigen-positive B cells without
fusion to myeloma cells, as described in U.S. 2007/0280945A1. Using this method, several fully
human anti-RSV-F antibodies (i.e ., antibodies possessing human variable domains and human
constant domains) were ed; exemplary antibodies generated in this manner were
designated as follows: H1H3564P, H1H3565P, H1H3566P, H1H3567P, 1P, H1H3583P,
H1H3589P, H1H3591P, H1H3592P, H1H3597P, H1H3598P, H1H3603P, H1H3604P,
H1H3605P, H1H3607P, H1H3608P2, H1H3592P2 and H1H3592P3.
The biological properties of the exemplary antibodies generated in accordance with the
methods of this Example are described in detail in the Examples set forth below.
Example 2. Heavy and Light Chain Variable Region Amino Acid ces
Table 1 sets forth the heavy and light chain variable region amino acid sequence pairs of
selected antibodies specific for RSV-F protein and their corresponding antibody identifiers.
Antibodies are lly referred to herein ing to the following nomenclature: Fc prefix
(e.g. “H4H”, “H1M, “H2M”), followed by a numerical identifier (e.g. “3117” as shown in Table 1),
followed by a “P” or “N” suffix. Thus, according to this nomenclature, an antibody may be
referred to as, e.g. “H1H3117”. The H4H, H1M, and H2M prefixes on the antibody designations
used herein indicate the particular Fc region of the dy. For e, an “H2M” antibody
has a mouse IgG2 Fc, whereas an “H4H” antibody has a human IgG4 Fc. As will be appreciated
by a person of ordinary skill in the art, an H1M or H2M antibody can be converted to an H4H
antibody, and vice versa, but in any event, the variable domains (including the CDRs), which are
indicated by the numerical identifiers shown in Table 1, will remain the same. Antibodies having
the same numerical antibody designation, but differing by a letter suffix of N, B or P refer to
antibodies having heavy and light chains with identical CDR sequences but with sequence
variations in regions that fall outside of the CDR sequences (i.e., in the framework regions).
16536800_1 (GHMatters) P40719NZ00
Thus, N, B and P variants of a particular antibody have identical CDR sequences within their
heavy and light chain variable regions but differ from one r within their framework
regions.
Antibody Comparators
Anti-RSV-F antibody ls were included in the ing Examples for comparative
purposes. Isotype matched negative controls were also used in the Examples. One anti-RSV-F
control antibody is designated herein as Control I and is a humanized anti-RSV-F antibody with
heavy and light chain le domain sequences of the palivizumab (SYNAGIS®) humanized
antibody as set forth in US7635568 and US5824307. The variable light and heavy chains were
sed with human kappa and gamma-1 constants, respectively. One anti-RSV-F antibody
is designated herein as Control II and is a humanized anti-RSV-F antibody variant of
palivizumab, with heavy and light chain variable domain sequences of the motavizumab
(NUMAX™) humanized antibody described in US2003/0091584 and by Wu et al, (2007), J. Mol.
Biol. 368:652-665. The variable light and heavy chains were expressed with human kappa and
gamma-1 constants, tively. Another anti-RSV-F antibody is designated as Control III (also
referred to as AM-22) and is described in US patent No. 8568726. The amino acid sequence of
the heavy and light chain of AM-22 is shown in SEQ ID NO: 357 (for the heavy chain of the
antibody) and SEQ ID NO: 358 (for the light chain of the antibody).
Table 1
SEQ ID NOs:
Antibody
Designation HCVR HCDR1 HCDR2 HCDR3 LCVR LCDR1 LCDR2 LCDR3
2 4 6 8 10 12 14 16
H1H3564P
18 20 22 24 26 28 30 32
34 36 38 40 42 44 46 48
H1H3566P
50 52 54 56 58 60 62 64
H1H3567P
66 68 70 72 74 76 78 80
H1H3581P
82 84 86 88 90 92 94 96
H1H3583P
98 100 102 104 106 108 110 112
H1H3589P
114 116 118 120 122 124 126 128
130 132 134 136 138 140 142 144
H1H3592P
146 148 150 152 154 156 158 160
H1H3597P
162 164 166 168 170 172 174 176
178 180 182 184 186 188 190 192
H1H3603P
194 196 198 200 202 204 206 208
H1H3604P
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210 212 214 216 218 220 222 224
H1H3605P
226 228 230 232 234 236 238 240
H1H3607P
242 244 246 248 250 252 254 256
H1H3608P2
258 260 262 264 266 268 270 272
H1H3592P2
274 276 278 280 282 284 286 288
H1H3592P3
290 292 294 296 298 300 302 304
H1M3621N
306 308 310 312 314 316 318 320
H1M3622N
322 324 326 328 330 332 334 336
H1M2634N
338 340 342 344 346 348 350 352
H1M3627N
Example 3. Antibody Binding Affinities and Kinetic Constants of Human Monoclonal Anti-
RSV-F Antibodies as Determined by Surface Plasmon Resonance
Binding affinities and kinetic constants of human monoclonal anti-RSV-F dies were
determined by surface plasmon resonance at 25oC (Tables 2-3). ements were
conducted on a Biacore 4000 or T-200 instrument. Antibodies, expressed with either mouse Fc
(AbPID prefix H1M; H2M) or human IgG1 Fc (AbPID prefix H1H), were captured onto an antimouse
or uman Fc sensor surface (Mab capture format), and soluble monomeric (RSVF.mmh
; SEQ ID NO: 353) protein was injected over the surface. All Biacore binding studies
were med in HBST running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,
0.005% v/v surfactant P20). Different concentrations of RSV-F.mmh prepared in HBST running
buffer were injected over the anti-RSV-F monoclonal antibody captured surface at a flow rate of
30µl/min re 4000) or at a flow rate of 50µl/min (Biacore T-200) and the association of
RSV-F.mmh to captured monoclonal antibody was monitored for 6min or 3min respectively. The
dissociation of mmh from the monoclonal dy in HBST running buffer was
monitored for n at 25°C. Kinetic association (ka) and dissociation (k d) rate constants were
determined by processing and fitting the data to a 1:1 binding model using Scrubber 2.0 curve
fitting software. Binding dissociation equilibrium constants (KD) and dissociative half-lives (t½)
were calculated from the kinetic rate constants as: KD (M) = kd / ka; and t1/2 (min) = (ln2/(60*kd).
Anti-RSV-F antibodies of the invention displayed a broad range of affinities for mh.
l I, ed based on the public sequence of palivizumab set forth in US
7,635,568, and Control II, produced on the public sequence of motavizumab as described in Wu
et al, (2007), (J. Mol. Biol. 2-665) displayed the approximately ~70-fold ence (control
1; 38nM vs control II; 0.43nM) in affinity that has been previously reported.
Table 2: Biacore Binding Affinities of Hybridoma mAbs at 25oC
Binding at 25ºC / Mab Capture Format
AbPID ka (1/Ms) kd (1/s) KD (M) t½ (min)
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H1M3621N 2.05E+05 2.08E-04 1.01E-09 56
H1M3622N 3.84E+04 9.13E-05 2.38E-09 127
4N 1.79E+05 1.83E-04 1.02E-09 63
H1M3627N 2.59E+05 04 2.02E-09 22
Table 3: e g affinities of human Fc mAbs at 25°C
Binding at 25ºC / Mab Capture Format
AbPID ka (1/Ms) kd (1/s) KD (M) t½ (min)
4P 3.10E+03 05 2.50E-08 148
H1H3565P 04 5.80E-05 3.01E-09 199
H1H3566P 2.04E+04 4.20E-05 09 275
H1H3567P 6.05E+04 2.63E-03 4.34E-08 4
H1H3581P NB NB NB NB
H1H3583P 8.94E+04 3.08E-03 3.44E-08 4
H1H3589P 3.77E+04 9.14E-03 2.43E-07 1
H1H3591P 4.46E+04 1.53E-03 3.42E-08 8
H1H3592P 1.06E+05 4.66E-04 4.39E-09 25
H1H3592P2 9.93E+04 1.46E-03 1.47E-08 8
H1H3592P3 8.86E+04 7.47E-04 8.43E-09 15
H1H3597P NB NB NB NB
H1H3598P NB NB NB NB
H1H3603P 3.00E+03 1.23E-04 4.10E-08 94
H1H3604P 3.10E+03 9.27E-05 3.00E-08 125
H1H3605P 2.80E+03 1.68E-04 5.90E-08 69
H1H3607P 4.20E+03 1.48E-04 3.50E-08 78
H1H3608P2 4.85E+03 2.60E-05 5.35E-09 445
H1H3627N 2.56E+05 1.49E-04 5.81E-10 78
Control I 04 2.57E-03 08 4
Control II 1.89E+05 8.13E-05 4.29E-10 142
NB: No binding observed under the conditions of the experiment
Example 4. Respiratory Syncytial Virus Fusion (RSV-F) Protein Antibodies Display Potent
Neutralization Capabilities Across RSV Subtype A and Subtype B strains
Purified dies were tested in a RSV micro-neutralization assay to determine
potency. Briefly, 104 HEp-2 cells cultured in MEM high glucose medium, supplemented with 5%
Hyclone FBS, L-glutamine and antibiotics, were seeded into 96-well clear -black
microplates and incubated for 16-18 hours (37°C, 5% CO2). Next, various concentrations of
16536800_1 ters) P40719NZ00
antibodies, starting at 666 nM with subsequent 1:5 dilutions in media, were incubated with the
RSV 1540 (A2) strain at an MOI of 0.04 for 2 hours (37C, 5% CO2). Virus-free and vant
isotype controls were included.
Post incubation, the antibody:virus mixture was added to the HEp-2 cells and infection
was maintained for 3 days. The degree of infection was determined by fixing cells in 2% PFA
and performing an ELISA with Goat anti-RSV/anti-Goat HRP antibodies. Luminescence
reagents were added to the wells and signal was detected using a plate reader (Victor X3,
Perkin . Luminescence values were analyzed by a three-parameter logistic equation over
an 11-point response curve (GraphPad Prism).
The antibodies of the invention displayed a broad range of neutralization activities
against the RSV A2 (1540) strain (Table 4-5). Several antibodies displayed lower IC50 values
then control I while only a few exemplary dies H1H3627N, H1H3591P, H1H3592P and
H1H3592P3 showed better neutralization then control II. Select antibodies 27N,
H1H3592P3) were also tested for their y to neutralization RSV subtype B strains (Table 6).
This example demonstrates the efficacy of the antibodies of this invention to neutralize
several strains of RSV-F, across two es, in vitro, with greater potency than previously
demonstrated for ished controls.
Table 4. Neutralization potency for ed mAbs against RSV A2 (1540)
IC50 [pM] for RSV A2 Neutralization:
AbPID Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Trial 7
H1M3621N 582 180 - - - - -
H1M3622N 320 82 - - - - -
H1M3624N 540 270 92 - - - -
H1M3627N 4 4 5 -
H1H3564P >10000 - - - - - -
H1H3565P >10000 - - - - - -
H1H3566P >10000 - - - - - -
H1H3567P - - 390 -
H1H3581P >10000 - - - - - -
H1H3583P - - - -
H1H3589P - - - -
H1H3591P - - 8 6
H1H3592P - - 5 4
H1H3592P3 10
H1H3597P >10000 - - - - - -
H1H3598P >10000 - - - - - -
3P >10000 - - - - - -
H1H3604P >10000 - - - - - -
H1H3605P >10000 - - - - - -
H1H3607P >10000 - - - - - -
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H1H3608P2 >10000 - - - - - -
H1H3570P >10000 - - - - - -
H1H3627N - - - - - - 3
Control 1 1820 950 290 530 160 500 250
Control 2 50 30 23 20 12 12 12
Table 5. lization potency for selected mAbs against RSV subtype A
e A Neutralization: IC50 & Fold Improvement Relative to
Control 1
RSV–Long
RSV-A2 (1540)
AbPID Fold Neutral. IC50 Fold
Neutral. IC50 [pM]
[pM]
H1H3627N 2.6 138 7.3 73
H1H3592P3 10 36 15 35
Control I 360 --
Control II 14 25 65 8.2
Table 6. Neutralization y for selected mAbs against RSV subtype B
Subtype B Neutralization: IC50 & Fold Improvement Relative to
Control 1
RSV – 1580 20
AbPID Neutral. IC50 Fold Neutral. IC50 Fold
[pM] [pM]
H1H3627N 6.7 55 11 42
H1H3592P3 31 12 100 4.6
Control I 375 --
Control II 43 8.7 56 8.2
e 5. ed Anti-RSV-F Antibodies Display Potent Neutralization of RSV
Infection in vivo
A. Mouse model
The exemplary antibodies H1H3627N and H1H3592P3 were selected for in vivo RSV
neutralization studies using Balb/c mice. Briefly, 7 week old Balb/c mice (n=4-5) were injected
SC at two doses (0.15 or 0.05 mg/kg) using either H1H3627N, H1H3592P3, control I, control II
or isotype-matched antibody. The use of carrier antibody (1 mg/kg) was utilized in all
experiments to minimize the loss of anti-RSV-F antibody.
One day post-injection, mice were challenged intranasally with 50 ul (106 pfu) of RSV
A2 (1540) strain. Four days post-infection, sera was drawn, mice were sacrificed, and lungs
were extracted and homogenized in 1mL of PBS using an OmniGLH homogenizer. Lung
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homogenates were centrifuged to remove cellular debris and a portion of supernatant was used
to determine anti-RSV-F mAb concentration in the lung. The remaining supernatant was used
to make serial dilutions which were incubated with HEp-2 cells for 2 hours, to allow viral entry.
Subsequently, supernatant was removed and the cells were overlaid with 1% methylcellulose.
Six days later, cells were stained with crystal violet and plaques were counted and the log10 viral
reduction was calculated relative to isotype control.
Exemplary antibodies H1H3627N and H1H3592P3 were more efficacious in reducing
the viral load in vivo than control I or control II anti-RSV-F antibodies (Tables 7a-7e).
Specifically, at the 0.15 mg/kg dose, antibodies 7N, H1H3592P3 and control II all
effectively d RSV infection in the lung to near undetectable levels compared to control I
(viral reduction ) fold change ≥ 2.10). Total human IgG measurements in the lungs and
serum confirmed that antibody levels were relatively consistent between groups.
At a lower administrated dose, r differentiation in neutralization efficacy n
the three antibodies compared to control I was evident. At 0.05 mg/kg, H1H3592P3 showed the
greatest reduction in viral load, with fold changes ranging from 1.49 to > 2.07 logs, ed
with viral load reduction fold changes of 1.08 to 1.36 logs for 7N and 0.01 to 0.65 logs
for control II. Control I at this lower dose was only moderately effective with viral load ion
changes of 0.03 to 1.03 logs.
The results indicate that both H1H3627N and H1H3592P3 are potent RSV neutralizing
antibodies in vivo, with the latter showing a trend of being a more effective neutralizer of RSV
infection at lower doses.
A dosing range experiment was performed following the same protocol described
above, ing SC 4 different doses of l I dy (0.6, 0.3, 0.15 and 0.05mg/kg), and
two doses (0.15 and 0.05mg/kg) of H1H3592P3 and control II. Viral reduction in the lungs was
calculated as a percentage of isotype l (Exp M4, Tables 7d-e).
Exemplary dy H1H3592P3 was more efficacious in reducing the viral load in vivo
(in mouse) than control I or control II anti-RSV-F antibodies. In addition, the dose of control I
required to reach a 99% viral reduction in the lungs was 3-4 fold higher than the dose of
H1H3592P3.
Tables 7(a-e): RSV viral reduction (log (10)) in mice after administration of Anti-RSV-F
antibodies
Table 7a
Exp M1 Dose: 0.15 mg/kg Dose: 0.05 mg/kg
Mice Viral mAb mAb Viral mAb mAb
PID per Reduction [ng/ml] [ng/ml] Reduction [ng/ml] [ng/ml]
group (log10) Lungs Serum (log10) Lungs Serum
1041 ±
H1H3627N 5 >2.10 35 ± 18 1.20 7 ± 4 274 ± 38
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1731 ±
H1H3592P3 5 >2.10 44 ± 14 >2.07 17 ± 4 438 ± 51
895 ± 365 ±
Control I 5 1.02 33 ± 11 1.03 9 ± 5
132 111
1948 ±
Control II 5 >2.10 82 ± 24 0.65 7 ± 4 555 ± 80
2180 ± 1287 ±
Isotype Ctrl 5 NA 76 ± 28 NA 25 ± 2
197 120
Table 7b
Exp M2 Dose: 0.15 mg/kg Dose: 0.05 mg/kg
Mice Viral mAb mAb Viral mAb mAb
PID per Reduction [ng/ml] [ng/ml] Reduction [ng/ml] [ng/ml]
group (log10) Lungs Serum (log10) Lungs Serum
724 ±
H1H3627N 5 >2.51 23 ± 8 1.08 3 ± 3 300 ± 35
2P 1261 ±
>2.51 27 ± 5 1.49 10 ± 2 333 ± 55
3 74
611 ±
Control I 5 0.79 9 ± 2 0.15 1 ± 1 221 ± 35
587 ±
Control II 5 2.31 13 ± 8 0.01 1 ± 3 237 ± 22
1389 ±
Isotype Ctrl 5 NA 46 ± 12 NA 15 ± 4 498 ± 92
Table 7c
Exp M3 Dose: 0.15 mg/kg Dose: 0.05 mg/kg
Mice Viral mAb mAb Viral mAb mAb
PID per Reduction [ng/ml] ] Reduction [ng/ml] [ng/ml]
group (log10) Lungs Serum (log10) Lungs Serum
1143 ±
H1H3627N 4 2.7 26 ± 6 1.36 7 ± 1 394 ± 16
H1H3592P 947 ±
4 >2.83 31 ± 12 1.66 13 ± 4 371 ± 21
3 105
1426 ±
Control I 4 1.00 58 ± 14 0.03 6 ± 5 442 ± 27
1152 ±
Control II 4 2.35 20 ± 6 0.54 BDL 373 ± 21
808 ±
e Ctrl 4 NA 41 ± 3 NA 37 ± 8 326 ± 26
Table 7d
Exp M4 (ED99) Dose: 0.6 mg/kg Dose: 0.3 mg/kg
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Mice Viral mAb Viral
mAb [ng/ml]
PID per Reduction [ng/ml] Reduction
Serum
group (%) Serum (%)
8451.9 ±
Control I 5 >99 96.9 3129.7 ± 403
2562
ND: Not determined
Table 7e
Exp M4 (ED99) Dose: 0.15 mg/kg Dose: 0.05 mg/kg
Mice Viral Viral mAb
mAb [ng/ml]
PID per Reduction Reduction [ng/ml]
Serum
group (%) (%) Serum
H1H3592P3 5 >99 1578.9 ± 256 90.6 524.0 ± 42
Control I 5 57.9 1561.2 ± 282 24.2 547.5 ± 59
Control II 5 96.7 1566.0 ± 354 48.5 465.7 ± 85
e Ctrl 5 NA 1406.0 ± 196 NA 375.3 ± 86
ND: Not determined
B. Cotton rat model
The exemplary dies H1H3627N and 2P3 were selected for in vivo RSV
neutralization studies using cotton rats. Briefly, 6-8 week old cotton rats (n=5) were injected IM
at two doses (5 or 0.6 mg/kg) using either H1H3627N, H1H3592P3, control I, control II or
isotype-matched antibody.
One day post-injection, rats were challenged intranasally with 100 ul (105 pfu) of RSV
A2 strain. Four days nfection, sera was drawn, rats were sacrificed, and lung and nasal
tissues were extracted for viral titration. Lung homogenates were centrifuged to remove ar
debris and a portion of supernatant was used to determine anti-RSV-F mAb concentration in the
lung. The remaining supernatant was used to make serial dilutions, which were incubated with
HEp-2 cells to allow viral entry. Subsequently, supernatant was removed and the cells were
overlaid with 1% methylcellulose. Six days later, cells were d and plaques were counted
and the log10 viral ion was calculated ve to isotype control.
Exemplary dy H1H3592P3 was more efficacious in reducing the viral load in the
lungs and nose than control I, and as efficacious as control II in lungs and better in the nose.
Exemplary antibody H1H3627N was only better than control I and as efficacious as control II in
the nose (Table 8). Specifically, at the 5 mg/kg dose, antibodies H1H3627N, H1H3592P3,
control I and control II all effectively d RSV infection in the lung to near undetectable
levels compared to isotype control (viral reduction log(10) fold change ≥ 2.33). However, in the
nose, greater differentiation in neutralization efficacy between H1H3627N, H1H3592P3, control
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II compared to l I was evident. H1H3592P3 showed the r reduction in viral load
(2.65 logs) compared to H1H3627N (1.46 logs) or control II (1.33 logs).
At a lower strated dose, r differentiation in neutralization efficacy between
the three antibodies compared to control I was evident in the lungs. At 0.6 mg/kg, H1H3592P3
showed similar reduction in viral load than control II (1.5 logs) and they were both more
cious than control I (0.624 logs). H1H3627N showed less efficacy than the other three
antibodies.
Exemplary anti-RSV-F antibody H1H3592P3 was next selected for testing its ability to
neutralize RSV subtype B in vivo using the cotton rat model. As with RSV/A, 6- to 8-week old
cotton rats (n= 4-6/group/experiment) were intramuscularly administered either 5 or 0.6 mg/kg
of H1H3592P3, Control I or Control II. The next day, animals were challenged with 10^5 pfu of
RSV/B strain 18537. Four days post-challenge, viral titers in the lungs and nose were
ined along with serum antibody titers. The s shown in table 9 were data pooled
from two independent experiments.
H1H3592P3 showed efficacy in reducing RSV/B viral load in lungs at both high and low
doses (Table 9). At 5.0 mg/kg, RSV/B viral load in the lungs was reduced by 2.21 logs with
H1H3592P3, ed with a reduction of 2.11 logs by Control I and 2.18 logs by Control II. At
0.6 mg/kg, RSV/B viral load in the lungs was reduced by 1.29 logs with H1H3592P3, compared
with a ion of 0.75 logs by Control I and 0.83 logs by Control II.
Overall, H1H3592P3 showed superiority in neutralization of RSV Subtype B in the lungs
over both Control I and II at 0.6 mg/kg. At 5 mg/kg, H1H3592P3 showed comparable
lizing ability than Control I and Control II in reducing viral load in the lungs.
The results indicate that H1H3592P3 is a potent neutralizer of RSV subtype strains A
and B in vivo in cotton rats, being a more effective neutralizer of RSV infection at high doses in
the nose and at lower doses in the lungs. The efficacy at low doses indicates the possibility of a
lower dose regimen in the clinic.
Table 8: RSV-A viral reduction (log (10)) in cotton rats after administration of Anti-RSV-F
antibodies
Exp R1 Dose: 0.6 mg/kg Dose: 5.0 mg/kg
Viral Viral mAb Viral Viral mAb
Rats
Reduction Reduction [ng/ml] Reduction Reduction [ng/ml]
PID per
lung nose Serum lung nose Serum
group
(log10) (log10) Day 4 (log10) (log10) Day 4
H1H362 3.43 21.52
0.34 0.22 2.33 1.46
7N ± 0.25 ± 5.47
H1H359 3.49 46.28
1.66 0.19 2.56 2.66
2P3 ± 0.55 ± 7.69
3.04 39.95
Control I 5 0.62 0.21 2.37 1.07
± 0.29 ± 5.23
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Control 4.26 24.06
1.50 0.20 2.55 1.33
II ± 0.66 ± 2.96
Isotype 3.78 30.43
4 NA NA NA NA
Ctrl ± 0.99 ± 6.66
Table 9: RSV-B viral reduction (log (10)) in cotton rats after administration of Anti-RSV-F
antibodies
Exp R2 Dose: 0.6 mg/kg Dose: 5.0 mg/kg
Viral Viral mAb Viral Viral mAb
Rats
Reduction Reduction [ng/ml] Reduction Reduction ]
PID per
lung nose Serum lung nose Serum
group
(log10) ) Day 4 (log10) (log10) Day 4
H1H359 3.89 42.31 ±
1.29 0.21 2.21 0.86
2P3 ± 0.99 13.5
3.87 35.28
Control I 11 0.75 0.15 2.11 0.79
± 0.73 ±11.8
3.75 27.65
Control II 11 0.83 0.10 2.18 1.24
± 0.49 ±7.49
Isotype 3.56 34.28
NA NA NA NA
Control ± 1.17 ±9.24
C. Cotton rat model - Determination of the ED99 of an ary Antibody H1H3592P3
Dose-ranging studies using the cotton rat were performed to determine at which dose an
exemplary antibody H1H3592P3 would reduce viral load by >99% (i.e. the ED99). Cotton rats
were prophylactically administered an IM dose of H1H3592P3 or Control 1 antibody at either 10,
, 2.5, 1.25 or 0.62 mg/kg. Additionally an isotype control antibody was dosed in at either 10 or
0.62 mg/kg to bracket the active agents in this study. Following antibody treatments an
asal RSV challenge of either subtype A (RSV A2 strain) or subtype B (RSV B strain
18537) was performed. Four days nfection, sera was drawn, rats were iced, and lung
tissue was extracted for viral titration. H1H3592P3 at a dose of 0.62 mg/kg achieved >99% viral
load reduction in the lungs as compared to Control 1 which required a dose of 2.5 mg/kg to
reach the same >99% viral reduction (Table 10). The mean al Control 1 tration
(27 μg/mL) at the calculated ED99 correlated well with usly published work (Scott and
Lamb, 1999), which indicated that a serum palivizumab tration (i.e. Control 1) of 30-40
µg/mL, at the time of RSV infection, was associated with a 99% reduction in lung viral load. The
mean terminal H1H3592P3 concentration (4.9 μg/mL) correlated well with the 4-fold lower dose
delivered at its ED99. Results against subtype B challenge were similar (Table 11) in that an
ED99 for H1H3592P3 was achieved at 2.5 mg/kg while Control 1 required roughly a 4x greater
dose (10 mg/kg) to obtain that same >99% viral lung reduction.
In summary these studies support that less frequent dosing of H1H3592P3 may confer
the same level of protection as the current monthly dosing paradigm used with palivizumab.
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Table 10. Determination of the ED99 for Anti RSV-F Antibodies After RSV Subtype A
Challenge
ED99 Determination with RSV Subtype A
% Viral Lung Reduction
PID 10 mg/kg 5 mg/kg 2.5 mg/kg 1.25 mg/kg 0.62 mg/kg
H1H3592P3 >99 >99 >99 >99 >99
Control I >99 >99 >99 98.9 95.9
Isotype Ctrl NA NA NA NA NA
Antibody Serum Concentration (ug/ml)
H1H3592P3 107.2 ±3.4 48.44 ±6.1 20.15 ±1.8 10.55 ±1.5 4.91 ±0.7
Control I 89.16 ±6.5 58.07 ±6.3 26.93 ±3.3 12.72 ±2.2 6.65 ±0.5
Isotype Ctrl 90.57 ±12.6 -- -- -- 5.39 ±0.5
Table 11. Determination of the ED99 for Anti RSV-F Antibodies After RSV Subtype B
Challenge
ED99 Determination with RSV e B
% Viral Lung Reduction
PID 10 mg/kg 5 mg/kg 2.5 mg/kg 1.25 mg/kg 0.62 mg/kg
H1H3592P3 >99 >99 >99 98.4 96.7
Control I >99 97.7 98.4 96.3 88.2
Isotype Ctrl NA NA NA NA NA
Antibody Serum tration (ug/ml)
H1H3592P3 98.04 ±18.4 50.99 ±7.8 27.82 ±4.9 10.49 ±1.7 7 ±0.3
Control I 98.89 ±10.9 42.74 ±8.9 26.46 ±3.3 16.06 ±2.2 7.58 ±1.1
Isotype Ctrl 99.72 ±17.4 NA NA NA 5.38 ±0.5
Example 6. Generation of a cific Antibody
Various bi-specific antibodies are generated for use in practicing the methods of the
invention. For example, RSV-F specific antibodies are generated in a bi-specific format (a "bispecific"
) in which variable regions binding to distinct domains of the RSV- F protein are linked
together to confer dual-domain specificity within a single binding le. riately
designed bi-specifics may enhance overall virus neutralization cy through sing both
specificity and g avidity. le regions with specificity for individual domains are paired
on a structural scaffold that allows each region to bind simultaneously to separate epitopes, or
to different regions within one domain. In one example for a bi-specific, heavy chain variable
regions (VH) from a binder with specificity for one domain are recombined with light chain
variable regions (VL) from a series of binders with specificity for a second domain to identify
non-cognate VL partners that can be paired with an original VH without disrupting the original
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specificity for that VH. In this way, a single V L segment (e.g., VL1) can be ed with two
different VH domains (e.g., VH1 and VH2) to generate a bi-specific comprised of two binding
"arms" (VH1- VL1 and VH2- VL1). Use of a single VL segment reduces the xity of the
system and thereby simplifies and increases efficiency in cloning, expression, and purification
processes used to generate the bi-specific (See, for example, USSN13/022759 and
US2010/0331527).
Alternatively, antibodies that bind RSV-F and a second target, such as, but not limited to,
for example, a second different anti-RSV-F antibody, or a , or a vaccine, may be prepared
in a bi-specific format using techniques described , or other techniques known to those
skilled in the art. Antibody le regions binding to distinct regions may be linked together
with variable regions that bind to relevant sites on, for example, a different viral antigen to confer
dual-antigen icity within a single binding molecule. Appropriately designed bi-specifics of
this nature serve a dual function. For example, in the case of a bi-specific antibody that binds
ie. RSV-F and RSV-G one may be able to better neutralize the virus, without the need for
administration of
a composition containing two separate antibodies. Variable regions with specificity for RSV-F,
are combined with a variable region with specificity for RSV-G and are paired on a structural
scaffold that allows each le region to bind to the separate antigens.
The cific binders are tested for binding and functional blocking of the target
antigens, for example, RSV-F and RSV-G, in any of the assays described above for antibodies.
For example, standard methods to measure soluble protein binding are used to assess the
bispecific interaction, such as Biacore, ELISA, size exclusion chromatography, multi-angle laser
light scattering, direct scanning calorimetry, and other methods. Binding of bi-specific
antibodies to both RSV-F and RSV-G is determined h use of an ELISA binding assay in
which synthetic peptides enting the different antigens are coated onto the wells of
microtiter plates, and binding of a bi-specific is determined through use of a secondary detection
antibody. Binding experiments can also be conducted using surface n resonance
experiments, in which real-time binding interaction of peptide to antibody is measured by flowing
a peptide or bi-specific across a sensor surface on which bi-specific or peptide, respectively, is
captured. Functional in vitro blocking of both RSV-F and RSV-G by a bi-specific is ined
using any bioassay such as the neutralization assay described herein, or by in vivo tion
studies in appropriate animal models, such as those described herein, or in an in vivo model of
lung inflammation.
Example 7: In vitro Generation of RSV Escape s to ine the Binding Epitope
of H1H3592P3
tion of escape mutants to H1H3592P3
3x10 5 Hep-2 cells/well were plated in a 6-well plate for 24 h. trations of
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H1H3592P3, ranging from 50 ug/mL to 0.016 ug/mL were mixed with RSV subtype A strain
1540 or RSV subtype B strain 1580 for 1h at 37°C. After coincubation, the RSV/antibody
mixture was added to the usly seeded HEp-2 cells at a multiplicity of infection (MOI) of 10
-forming units cell. Cells were incubated for 6 days, and cytopathic effects were
monitored daily using light microscopy. At day 6, contents of each well were harvested, ed
to initial concentration of antibody and used to infect freshly seeded HEp-2 cells. This serial
passage was repeated until obvious cytopathic effects were observed at high trations of
2P3 (50 ug/mL), which is approximately 2 logs greater than the IC50 of the antibody,
suggesting the presence of viral mutants. Supernatants from these wells were confirmed from
the presence of resistant virus via a micro-neutralization assay (described below) and plaque
isolation was performed in 10 cm tissue culture dishes. 10 individual plaques were expanded in
6-well plates and virus were re-tested for resistance via eutralization. Sequencing was
then performed on these viral mutants.
Microneutralization assay
To confirm whether escape mutants generated under the pressure of H1H3592P3 were
resistant to neutralization, a microneutralization assay in Hep-2 cells was performed. Briefly, 105
Hep2 cells cultured in DMEM 1x medium, supplemented with 5% Hyclone FBS, L-glutamine and
antibiotics, were seeded into 96-well clear bottom-black lates and incubated for 16-18
hours (37C, 5% CO2).
Next, various concentrations of antibodies, ng at 666 nM and diluted 1:5 in media,
were incubated for 2 hours (37C, 5% CO2) with RSV wild-type (subtype A or B) or escape
mutants from both subtype A and B, at an MOI from 0.04 to 0.4. Controls not containing virus or
controls containing virus but no antibodies were included. All dilutions of antibody were
conducted in duplicates. After incubation, the antibody/virus e was added to cells and
infection was allowed for 3 days. Infection was determined by fixing the cells in 2% PFA and an
ELISA with Goat anti-RSV/anti-Goat HRP antibodies was med. Luminescence reagents
were added to the wells and signal was detected using a plate reader r X3, Perkin Elmer).
Luminescence values were analyzed by a three-parameter logistic equation over an 11-point
response curve (GraphPad Prism).
Results
Respiratory syncytial virus escape mutants were generated to map the specific binding
region of H1H3592P3 to RSV-F. Briefly, HEp-2 cells, ed with RSV strains 1540 (subtype
A) or 1580 (subtype B) were subjected to H1H3592P3 treatment ranging from 50 ug/mL to
0.016 ug/mL. After 6 days, contents from each well were used to infect freshly seeded HEp-2
cells. This serial passage continued until cytopathic effects were ed in HEp-2 cells even
in the presence of the highest antibody dose, indicating the presence of RSV viral s
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generated under selection pressure. Overall, viral mutants were isolated from ten distinct
plaques, confirmed for neutralization resistance in the presence of H1H3592P3 and
subsequently ced.
ce analysis confirmed that escape mutations for H1H3592P3 were found at
amino acid positions 173 and 174 (S173Y and T174K) of RSV-F (SEQ ID NO: 354), indicating
that these amino acids play an important role in antibody binding and viral neutralization. Prior
reports have determined that the binding epitopes for anti-RSV Control I and Control II
antibodies are located between S255 – N276. The data from these studies suggest a binding
site for H1H3592P3 on RSV-F that plays a major role in viral lization (see table 12) and is
distinct from that required for previously established Control antibodies.
Table 12: Neutralization Efficacy of H1H3592P3 and anti-RSV l Antibodies on RSV
subtype A and B Strains and Associated Escape Mutants
H1H3592P3 Control I l II
Virus (IC50, pM) (IC50, pM) (IC50, pM)
wt e A (RSV/A) 177 1140 108
RSV/A S173Y Resistant 1710 170
Wt subtype B (RSV/B) 290 1900 260
RSV/B S173T Resistant 1900 177
RSV/B T174K Resistant 640 108
RSV/B S173T/T174K Resistant 980 218
Example 8: Determination of the Binding Epitope of H1H3592P3 to RSV-F using
en-Deuterium Exchange & Mass Spectrometry
Hydrogen/Deuterium Exchange (H/D exchange) in ation with peptic digests and
mass spectrometry was conducted to determine the binding epitope of the anti-RSV-F antibody
H1H3592P3 to recombinant RSV-F. Two H/D exchange formats (described in detail below)
were employed: An ‘on-solution/off-beads’ method in which RSV-F peptide fragments that are
protected by H1H3592P3 from back-exchange retain D20 and yield higher molecule weights
(m/z values) by mass spectrometry and an ‘on-beads/off-beads’ control method which
ishes the baseline m/z values for all RSV-F peptides. Subtraction of the control m/z values
from the m/z values obtained using the ‘on-solution/off beads’ method yields certain amino acids
regions that show non-zero delta m/z values i.e residual D20 that correspond to the binding
epitope between H1H3592P3 and RSV-F.
Methods
On solution/ off beads format
In the ‘on-solution/off-beads’ (on-exchange in solution followed by off-exchange on
beads) , RSV-F.mmh n (SEQ ID NO: 353) was deuterated for 5 min or 10 min in
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PBS buffer prepared with D2O, and then bound to H1H3592P3 covalently attached to N-
hydroxysuccinimide (NHS) agarose beads (GE Lifescience) via a 2 min incubation. The RSV-F
/ H1H3592P3 bead complex was washed with PBS buffer (prepared with non-deuterated H2O)
and incubated in PBS buffer for half of the on-exchange time. After the off-exchange, the bound
RSV-F was eluted from beads with an ld low pH TFA solution. The eluted RSV-F was
then digested with immobilized pepsin (Thermo Scientific) for 5 min. The resulting peptides
were desalted using ZipTip chromatographic pipette tips and immediately analyzed by
UltrafleXtreme matrix assisted laser tion ionization time of flight (MALDI-TOF)-TOF mass
spectrometry (MS).
On-beads/off beads format
In the ads/off-beads’ (on-exchange on beads followed by off-exchange on beads)
format, RSV-F.mmh (SEQ ID NO: 353) was first bound to H1H3592P3 e beads and then
incubated for 5 min or 10 min in D2O for on-exchange. The RSV-F / H1H3592P3 bead complex
was washed with PBS buffer (prepared with non-deuterated H2O) and incubated in PBS buffer
for half of the on-exchange time. After the off-exchange, the bound RSV-F was eluted from
beads with an ice-cold low pH TFA on. The eluted RSV-F was then digested with
immobilized pepsin (Thermo Scientific) for 5 min. The resulting peptides were ed using
ZipTip tographic pipette tips and immediately analyzed by MALDI-TOF-TOF mass
spectrometry. The id values or average mass-to-charge ratios (m/z) of all the detected
peptides were calculated and compared between this and the ‘on-solution/off-beads’
experiment.
Peptide identification
The identification of the peptides was d out using liquid chromatography-Orbitrap
Elite (Thermo Scientific).
Results
Table 13 is a detailed comparison of the delta id m/z values for all the RSV-F
peptides detected by MALDI-TOF mass ometry following H/D exchange and peptic digest.
Two segments corresponding to amino acids 161-171 (EGEVNKIKSAL, (SEQ ID NO: 355)) and
8 (LSTNKAVVSLSNGVSVL, (SEQ ID NO: 356)) of SEQ ID NO: 354 had delta centroid
values higher than 0.20, a threshold observed in-house to be considered indicative of antibodyprotein
contact and thus an epitope region. It should also be noted that the peptide signal
corresponding to amino acids 161-171 was not quantified in the 10 min on-exchange
experiment due to low signal to noise. However, the delta value of 0.88, detected at the 5 min
on-exchange experiment, is far above the 0.2 threshold and can be attributed to the significant
alteration in H/D exchange rate upon RSV-F binding to H1H3592P3.
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Furthermore the peptide t corresponding to amino acids 172-188 contains the
amino acids of the two RSV escape mutants (S173Y and T174K; see example 7), which were
resistant to 2P3 treatment, indicating that these two amino acids play a role in dy
binding and viral neutralization. Thus the combination of sequencing escape RSV mutants
along with H/D exchange support amino acids 161-188 of SEQ ID NO: 354 defining at least in
part the binding region in RSV-F for antibody H1H3592P3.
Table 13. Centroid (m/z) Values of RSV-F Peptic es After Back-exchange following
deuteration in the Absence (on-solution/off-beads) and Presence (on-beads/off-beads) of
H1H3592P3
ment I Experiment II
min on-/2.5 min off-exchange 10 min on-/5 min off-exchange
Residues on-beads / on-solution on-beads / on-solution /
off beads / off-beads delta off beads off-beads delta
(m/z) (m/z) (m/z) (m/z)
46-52 791.06 791.10 0.04 791.06 791.15 0.09
48-56 1083.32 1083.37 0.05 1083.32 1083.35 0.03
48-58 1297.42 1297.44 0.02 1297.40 1297.44 0.04
79-92 1665.81 1665.96 0.15 1665.86 1665.89 0.03
94-107 1519.93 1520.00 0.06 1520.01 1520.09 0.07
96-107 1278.64 1278.61 -0.03 1278.61 1278.73 0.12
96-108 1434.61 1434.60 -0.01 1434.50 1434.63 0.13
148-160 1308.97 1309.12 0.16 N.A. N.A. N.A.
161-171 1188.72 0 0.88 N.A. N.A. N.A.
172-188 1689.44 1691.68 2.24 1689.60 1691.07 1.47
220-230 1390.02 1390.06 0.04 1389.98 1389.93 -0.05
220-232 1632.30 1632.34 0.04 1632.29 1632.37 0.08
223-230 1048.49 1048.54 0.05 1048.44 1048.55 0.11
223-232 1291.16 1 0.05 1291.12 1291.18 0.07
231-236 760.95 760.95 0.00 761.02 760.95 -0.06
233-240 966.29 966.33 0.04 966.20 966.30 0.09
233-249 1780.20 1780.39 0.19 1780.38 1780.38 0.00
7 1 1977.91 0.10 2 1977.80 -0.13
261-279 2205.05 2205.12 0.07 2205.10 2205.20 0.10
278-285 958.20 958.34 0.14 958.15 958.29 0.14
278-286 1121.50 1121.57 0.07 1121.54 1121.59 0.05
278-289 1453.19 1453.16 -0.03 1453.14 1453.08 -0.06
280-286 894.20 894.22 0.02 894.29 894.28 -0.02
280-289 1225.75 1225.80 0.05 1225.79 1225.81 0.02
280-290 1312.70 0 -0.01 1312.86 1312.74 -0.13
457-467 1329.73 1329.82 0.09 1329.73 1329.76 0.03
468-477 7 1180.67 0.10 0 1180.42 -0.18
527-545 2132.30 2132.32 0.02 2132.39 8 -0.01
534-545 4 1318.54 0.00 1318.64 0 -0.13
537-545 988.92 988.87 -0.05 988.93 988.84 -0.08
546-557 1528.62 1528.68 0.07 1528.64 1528.64 0.00
No ID 743.16 743.06 -0.10 743.10 742.99 -0.11
No ID 844.01 843.98 -0.03 844.03 843.96 -0.07
No ID 901.26 901.40 0.13 901.36 901.40 0.04
16536800_1 (GHMatters) P40719NZ00
No ID 943.15 943.19 0.04 943.24 943.20 -0.04
No ID 1090.41 1090.45 0.04 1090.48 1090.51 0.03
No ID 1143.51 1143.61 0.10 1143.53 1143.57 0.04
No ID 1325.52 1325.56 0.04 4 1325.66 0.12
No ID 1353.69 4 -0.06 1353.77 1353.61 -0.16
No ID 1550.39 1550.44 0.05 5 0 -0.05
No ID 2074.49 2074.41 -0.08 2074.52 2074.36 -0.15
No ID 2257.71 2257.70 -0.01 2257.89 2257.85 -0.04
No ID 2365.83 2365.72 -0.12 2365.94 2365.87 -0.07
No ID 2385.18 2385.17 -0.01 2385.23 2385.25 0.02
No ID 2405.22 2405.09 -0.12 2405.17 2405.15 -0.02
No ID 2456.18 2456.24 0.07 2456.14 2456.09 -0.05
No ID 2513.28 2513.26 -0.01 2513.32 2513.19 -0.14
Example 9. Respiratory Syncytial Virus Fusion (RSV-F) Protein Antibodies Display Potent
Neutralization Capabilities Across RSV Subtype A and B laboratory strains
H1H3592P3 and controls I and II dies were tested in a RSV micro-neutralization
assay to determine potency. Briefly, 104 HEp-2 cells cultured in DMEM 1x medium,
supplemented with 5% Hyclone FBS, amine and antibiotics, were seeded into 96-well
clear bottom-black microplates and incubated for 16-18 hours (37°C, 5% CO2). Next, various
concentrations of antibodies, starting at 666 nM with subsequent 1:5 dilutions in media, were
incubated with various RSV subtype A lab s provided by ATCC at an MOI of 0.042 for 2
hours (37C, 5% CO2). free and irrelevant isotype controls were included.
Post incubation, the antibody:virus mixture was added to the HEp-2 cells and ion
was maintained for 3 days. The degree of infection was determined by fixing cells in 2% PFA
and ming an ELISA with Goat anti-RSV/anti-Goat HRP antibodies. scence
reagents were added to the wells and signal was detected using a plate reader (Victor X3,
Perkin Elmer). Luminescence values were analyzed by a three-parameter logistic equation over
an 11-point response curve (GraphPad Prism).
The antibodies of the invention displayed a broad range of neutralization activities
against the RSV lab strains (Table 14). Antibodies H1H3592P3 and AM22 showed similar
potency than control II for RSV subtype A lab strains. Compared to control I, H1H3592P3
showed 15-17 fold more potency (IC50 44-140 pM), while AM22 showed 9-23 fold more
y (IC50 86-91 pM) (Table 14). For subtype B, antibody H1H3592P3 showed similar
potency than control II, but or than AM22 and l I. Compared to control I,
H1H3592P3 showed 2-5 fold more potency (IC50 33-230 pM), while AM22 showed 0.13-2 fold
more potency (IC50 190-2508 pM).
This example demonstrates the efficacy of the antibodies of this ion to neutralize
several lab strains of RSV from both subtype A and B, in vitro, with greater potency than
previously demonstrated for established controls.
16536800_1 (GHMatters) P40719NZ00
Table 14
Subtype/strain H1H3592P3 Control I Control II l III
IC50 (pM) IC50 (pM) IC50 (pM) IC50 (pM)
A/A2 140 2080 202 91
A/Long 44 752 83 86
B/18537 230 1190 187 660
B/1400 33 113 38 190
B/1A2 48 223 40 580
B/9320 151 338 76 2508
Example 10. Respiratory Syncytial Virus Fusion ) Protein Antibodies Display
Potent Neutralization Capabilities Across RSV e A clinical isolates
H1H3592P3 and controls I, II and III antibodies were tested in a RSV micro-
neutralization assay to determine potency. Briefly, 104 HEp-2 cells ed in DMEM 1x
medium, supplemented with 5% Hyclone FBS, L-glutamine and antibiotics, were seeded into
96-well clear bottom-black microplates and incubated for 16-18 hours (37°C, 5% CO2). Next,
various concentrations of antibodies, starting at 666 nM with subsequent 1:5 ons in media,
were incubated with various RSV subtype A clinical isolates provided by Dr. Moore (Emory
University) at a range of MOIs from 0.015 to 0.128 for 2 hours (37C, 5% CO2). Virus-free and
irrelevant isotype controls were included.
Post incubation, the antibody:virus mixture was added to the HEp-2 cells and infection
was maintained for 3 days. The degree of ion was determined by fixing cells in 2% PFA
and performing an ELISA with Goat anti-RSV/anti-Goat HRP antibodies. Luminescence
reagents were added to the wells and signal was detected using a plate reader (Victor X3,
Perkin Elmer). Luminescence values were analyzed by a three-parameter logistic equation over
an 11-point response curve (GraphPad Prism).
The antibodies of the invention displayed a broad range of neutralization activities
against the RSV clinical isolates (Table 15). Antibody H1H3592P3 showed similar potency to
controls II and III for most al es. Compared to control I, H1H3592P3 showed 10-22
fold more y (IC50 34-66 pM) (Table 15).
This example demonstrates the efficacy of the antibodies of this invention to neutralize
several clinical isolates of RSV, in vitro, with greater potency than previously demonstrated for
established controls.
Table 15. RSV-F Antibodies Display Potent lization Capabilities Across RSV
Subtype A clinical isolates
MOI H1H3592P3 Control I Control II Control III k
IC50 (pM) IC50 (pM) IC50 (pM) IC50 (pM)
A2001/2-20 0.016 43 935 74 72 98.1
A2001/3-12 0.018 66 1259 129 60 JX069799.1
A1997/12-35 0.015 40 478 41 20 JX069800.1
16536800_1 (GHMatters) P40719NZ00
A1998/3-2 0.128 35 344 36 31 JX069801.1
A1998/12-21 0.026 34 580 68 43 JX069802.1
A2000/3-4 0.040 50 899 88 55 JX069803.1
Example 11. H1H3592P3 Blocks Viral Entry by Inhibiting Fusion of Virus and Cell
A study was done to determine the mechanism by which the antibodies of the invention
block respiratory syncytial virus (RSV) infection. One exemplary antibody of the invention,
H1H3592P3, was tested to determine whether it acted to prevent/inhibit RSV fusion with host
cells (Figure 2A and 2B). The mechanism of action for control I (the positive control mAb which
is based on the sequence of palivizumab) was previously described as inhibition of viral fusion
to the host cell (Huang et al., J. of Virol., (2010), Aug. :8132-40). Because RSV-F is
involved in both attachment to the cell via the interaction of the host receptor lin, and
fusion of the viral and plasma membranes, assays were performed to determine the mechanism
of H1H3592P3.
The attachment assay (Figure 2A) was performed by incubating RSV (subtype A, strain
A2) in the presence of either H1H3592P3 or the positive control antibody (control I), then
incubating the mixture with HEp-2 cells at 4°C for one hour to allow binding of the virus to the
cells. Unbound virus was washed out, cells were fixed and the percentage of attached virus
was ed by ELISA. Heparin, which blocks RSV attachment, was used as a control.
Viral fusion was detected by allowing viral attachment at 4°C, washing out unbound
virus, then incubating with H1H3592P3, ve Control I, or an isotype negative control
antibody at 4°C and moving cells to 37°C to promote viral fusion and entry. Viral infection was
measured 3 days later by ELISA (Figure 2B). RLU: ve Luminescence Units.
2P3, like control I, blocks RSV fusion and not the ment of RSV to the cell
surface, while the isotype (negative) control mAb had no effect on viral fusion (Figure 2B).
Heparin ively blocked RSV ment to cells (Hallack et al., Virology (2000), :264-
75), whereas r antibody inhibited RSV attachment e 2A). H1H3592P3 blocked viral
fusion in this assay format with an IC50 of 230pM, while the positive control mAb (control I)
blocked viral fusion with an IC50 1nM (Figure 2B). Similar results were observed with an RSV
subtype B strain (data not shown).
Example 12. Octet Cross Competition of anti-RSV-F Antibodies for Binding to RSV-F
Binding competition between a panel of anti-RSV-F mAbs was determined using a
real time, label-free bio-layer interferometry assay on an Octet® HTX biosensor (Pall ForteBio
Corp.). The entire experiment was performed at 25°C in HBST kinetics buffer (0.01 M HEPES
pH7.4, 0.15M NaCl, 3mM EDTA, 0.05% v/v Surfactant Tween-20, 0.1mg/mL BSA) with the plate
00_1 (GHMatters) P40719NZ00
shaking at the speed of 1000 rpm. To assess whether two antibodies are able to compete with
one another for binding to their respective epitopes on the recombinant RSV-F protein
expressed with a C-terminal myc-myc-hexahistidine tag (RSV-F-mmH), around 0.36nm of RSVF-mmH
was first captured onto anti-Penta-His antibody coated Octet biosensor (Fortebio Inc,
Cat# 18-5079) by submerging the biosensors for 3 minutes into wells containing 10µg/mL
solution of recombinant RSV-F-mmH. The n ed biosensors were then saturated
with the first anti-RSV-F monoclonal antibody (subsequently referred to as mAb-1) by dipping
into wells containing 100-200µg/mL solution of mAb-1 for 10 minutes. The biosensors were then
subsequently dipped into wells containing 100-200 µg/mL solution of second anti-RSV-F
monoclonal antibody (subsequently referred to as mAb-2) for 5 minutes to check for mAb-2
g to RSV-F-mmH, which is pre-bound to mAb-1. The biosensors were washed in HBST
kinetics buffer in between every step of the experiment. The real-time binding response was
monitored throughout the course of the experiment and the maximum binding response for all
the steps was recorded. The response of mAb-2 binding to RSV-F-mmH pre-bound with mAb-1
was measured and itive/non-competitive or of different anti-RSV-F monoclonal
antibodies was determined.
Results
Sequential binding studies performed on Octet® HTX demonstrate that none of the anti-
RSV-F monoclonal antibodies compete with each other and are able to bind non-competitively
to RSV-F-mmH. As shown in Table 16, dark grey boxes with black font indicate the g
se for self-competition. No competition between antibodies that suggest a distinct binding
epitope is represented as a white box with black font. Binding of the first anti-RSV-F monoclonal
antibody (mAb-1) to the is-captured mmH n does not prevent the binding of
the second SV-F monoclonal antibody (mAb-2). For all the anti-RSV-F onal
antibodies in this study, the observed mAb-2 binding signal was found to be comparable to that
observed in the absence of mAb-1 (No mAb). Moreover, the ed binding of mAb-2 for all
the anti-RSV-F monoclonal antibodies was found to be independent of the order of binding of
anti-RSV-F antibody; suggesting that all the anti-RSV-F antibodies under investigation have
distinct binding epitopes.
Table 16. competition between anti-RSV-F monoclonal dies.
Binding of mAb-2 to the Precomplex
of Captured RSV-F-
mmH & mAb-1
Amount of
Amount of
100-
10µg/mL of
200µg/mL of
mAb-1 RSV_F.mmh mAb# 1 2 3 4
mAb-1
Captured ±
Binding
Std Dev (nm)
Level (nm)
16536800_1 ters) P40719NZ00
ator III 0.36 ± 0.01 0.33 ± 0.01 1 0.01 0.34 0.44 0.00
(AM-22)
H1H3592P3
0.36 ± 0.01 0.35 ± 0.01 2 0.26 0.00 0.30 0.00
0.39 ± 0.01 0.45 ± 0.02 3 0.29 0.23 0.01 -0.01
Comparator I
(Palivizumab)
No mAb 0.36 ± 0.01 -0.01 ± 0.01 4 0.20 0.17 0.36 0.00
16536800_1 (GHMatters) P40719NZ00
Claims (24)
1. An isolated antibody or antigen-binding fragment thereof that specifically binds to Respiratory Syncytial Virus F protein (RSV-F), wherein the antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 274; and three light chain CDRs (LCDR1, LCDR2 and LCDR3) ned within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 282, wherein the CDRs are identified by the Kabat definition, the Chothia definition, or the AbM definition.
2. The isolated antibody or antigen-binding fragment thereof of claim 1, comprising: (a) a HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 276; (b) a HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 278; (c) a HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 280; (d) a LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 284; (e) a LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 286; and (f) a LCDR3 domain sing the amino acid sequence of SEQ ID NO: 288.
3. The isolated antibody or antigen-binding fragment thereof of claim 2, comprising a HCVR comprising the amino acid sequence of SEQ ID NO: 274 and a LCVR comprising the amino acid sequence of SEQ ID NO: 282.
4. The isolated antibody or antigen-binding fragment f of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof binds to at least one amino acid residue within residues 161 through 188 of SEQ ID NO: 354.
5. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the antibody or n-binding fragment thereof binds to at least one amino acid e within SEQ ID NO: 355 or SEQ ID .
6. The ed dy or n-binding fragment thereof of any one of claims 1-3, wherein the antibody or antigen-binding fragment thereof binds to either the serine at position 173 of SEQ ID NO: 354, or the threonine at position 174 of SEQ ID NO: 354, or both the serine at position 173 of SEQ ID NO: 354 and the threonine at position 174 of SEQ ID NO: 354.
7. An isolated nucleic acid molecule encoding an antibody or n-binding fragment of any of claims 1 -6.
8. An expression vector comprising the nucleic acid molecule of claim 7 16536800_1 (GHMatters) P40719NZ00
9. An isolated host cell comprising the expression vector of claim 8.
10. A pharmaceutical composition comprising the isolated antibody or antigenbinding fragment thereof of any one of claims 1-6, and a pharmaceutically acceptable carrier.
11. Use of the isolated antibody or antigen-binding fragment f of any one of claims 1-6, or the pharmaceutical composition of claim 10, in the manufacture of a medicament for preventing a respiratory syncytial virus (RSV) infection in a t in need thereof, or for treating a patient suffering from an RSV infection, or for rating at least one symptom or complication associated with the infection.
12. The use according to claim 11, wherein the RSV infection is caused by a subtype A or a subtype B atory syncytial virus.
13. The use according to claim 11, wherein the patient in need thereof is a patient at high risk of acquiring an RSV infection, or a patient who may experience a more severe form of the RSV infection due to an underlying or pre-existing medical condition.
14. The use according to any one of claims 11-13, wherein the t in need f is a pre-term infant, a full term infant, a child r than or equal to one year of age with or without an underlying medical condition an institutionalized or alized patient, or an elderly adult aged 65 years or older with or without an underlying medical condition.
15. The use according to claim 14, wherein the patient suffers from a condition resulting from a compromised ary, cardiovascular, uscular, or immune system.
16. The use according to claim 15, n the condition is selected from the group consisting of an abnormality of the , a chronic lung disease, a chronic heart disease, a neuromuscular disease that compromises the handling of respiratory secretions and immunosuppression.
17. The use according to claim 16, wherein the chronic lung disease is chronic obstructive pulmonary disease (COPD), cystic fibrosis, or bronchopulmonary dysplasia.
18. The use according to claim 16, wherein the chronic heart disease is congestive heart failure (CHF), or congenital heart disease.
19. The use according to claim 16, wherein the immunosuppression is a result of severe combined immunodeficiency or severe acquired immunodeficiency, or is a result of any other infectious disease or cancerous condition that leads to suppression, or is a result of treatment with immunosuppressant drug therapy or radiation therapy. 16536800_1 (GHMatters) P40719NZ00
20. The use according to claim 11, wherein the at least one symptom is selected from the group consisting of fever, nasal congestion, cough, hypoxia, breathing ulties wheezing, apnea, and ation.
21. The use according to claim 11, wherein the medicament is formulated for delivery as a lactic, or as a therapeutic.
22. The use according to claim 11, wherein the antibody or antigen-binding fragment thereof, or the pharmaceutical composition is formulated to be administered via a route ed from the group consisting of intravenously, intramuscularly, and subcutaneously.
23. The use according to claim 11, n the antibody or antigen-binding fragment thereof, or the pharmaceutical composition is formulated to be administered to the patient in ation with a second therapeutic agent.
24. The use according to claim 23, wherein the second therapeutic agent is selected from the group consisting of an antiviral agent; a vaccine specific for RSV, a e specific for influenza virus, or a vaccine specific for metapneumovirus (MPV); an siRNA specific for an RSV antigen or a eumovirus (MPV) antigen; a second antibody specific for an RSV antigen or a metapneumovirus (MPV) antigen; an anti-IL4R antibody, an antibody specific for an influenza virus antigen, an anti-RSV-G antibody and a NSAID.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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US201361782215P | 2013-03-14 | 2013-03-14 | |
US61/782,215 | 2013-03-14 | ||
US201361911093P | 2013-12-03 | 2013-12-03 | |
US61/911,093 | 2013-12-03 | ||
PCT/US2014/025259 WO2014159822A2 (en) | 2013-03-14 | 2014-03-13 | Human antibodies to respiratory syncytial virus f protein and methods of use thereof |
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NZ710829A NZ710829A (en) | 2021-01-29 |
NZ710829B2 true NZ710829B2 (en) | 2021-04-30 |
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