Anti-respiratory syncytial virus antibodies, and methods of their generation and use
Abstract
Anti-RSV antibodies with neutralizing potency against RSV subtype A and RSV subtype B are provided, as well as methods for their identification, isolation, generation, and methods for their preparation and use are provided.
Term
11.1 yearsto projected expiry
Projected expiry 20 October 2037, counted from filing; an application has no term until it is granted.
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- Filed
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19 claims: 7 independent, 12 dependent
- 1An isolated antibody or an antigen-binding fragment thereof that specifically binds to Respiratory Syncytial Virus (RSV) F protein (F), wherein the antibody or antigen-binding fragment thereof comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs 563, 565, 567, 571, 573 and 575; SEQ ID NOs 2147, 2148, 2149, 2152, 2153 and 2154; SEQ ID NOs 2157, 2158, 2159, 2162, 2163 and 2164; SEQ ID NOs 2167, 2168, 2169, 2172, 2173 and 2174; SEQ ID NOs 2177, 2178, 2179, 2182, 2183 and 2184; SEQ ID NOs 2187, 2188, 2189, 2192, 2193 and 2194; SEQ ID NOs 2197, 2198, 2199, 2202, 2203 and 2204; SEQ ID NOs 2207, 2208, 2209, 2212, 2213 and 2214; or SEQ ID NOs 2217, 2218, 2219, 2222, 2223, 2224, and wherein said antibody or the antigen-binding fragment thereof also has one or more of the following characteristics:a) the antibody or antigen-binding fragment thereof cross-competes with said antibody or antigen-binding fragment thereof for binding to RSV-F;b) the antibody or antigen-binding fragment thereof displays better binding affinity for the PreF form of RSV-F relative to the PostF form;c) the antibody or antigen-binding fragment thereof displays a clean or low polyreactivity profile;d) the antibody or antigen-binding fragment thereof displays neutralization activity towards RSV subtype A and RSV subtype B in vitro;e) the antibody or antigen-binding fragment thereof displays antigenic site specificity for RSV-F at Site III;f) the antibody or antigen-binding fragment thereof displays antigenic site specificity for RSV-F Site III relative to RSV-F Site I, Site II, or Site IV;g) at least a portion of the epitope with which the antibody or antigen-binding fragment thereof interacts comprises the α3 helix and β3/β4 hairpin of PreF;h) the antibody or antigen-binding fragment thereof displays an in vitro neutralization potency (IC 50 ) of between about 0.5 microgram/milliliter (µg/ml) to about 5 µg/ml, between about 0.05 µg/ml to about 0.5 µg/ml, or less than about 0.05 µg/ml;i) the binding affinity and/or epitopic specificity of the antibody or antigen-binding fragment thereof for any one of the RSV-F variants designated as 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG in Figure 7A is reduced or eliminated relative to the binding affinity and/or epitopic specificity of said antibody or antigen-binding fragment thereof for the RSV-F or RSV-F DS-Cav1;j) the antibody or antigen-binding fragment thereof displays a cross-neutralization potency (IC 50 ) against human metapneumovirus (HMPV);k) the antibody or antigen-binding fragment thereof does not compete with D25, palivisumab, motavizumab, or AM-14;or l) the antibody or antigen-binding fragment thereof displays at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, greater than about 100-fold, and folds in between any of the foregoing, greater neutralization potency (IC 50 ) than antibody D25 and/or palivizumab.
- 4The isolated antibody or antigen-binding fragment thereof of any one of claims 1 through 3, wherein the antibody is selected from the group consisting antibodies that are at least 70% identical, at least 75% identical, 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99%, and/or all percentages of identity in between, to an antibody comprising an HC amino acid sequence as set forth in SEQ ID NO 562 and a LC amino acid sequence as set forth in SEQ ID NO 570;a HC amino acid sequence as set forth in SEQ ID NO 2146 and a LC amino acid sequence as set forth in SEQ ID NO 2151;a HC amino acid sequence as set forth in SEQ ID NO 2156 and a LC amino acid sequence as set forth in SEQ ID NO 2161;a HC amino acid sequence as set forth in SEQ ID NO 2166 and a LC amino acid sequence as set forth in SEQ ID NO 2171;a HC amino acid sequence as set forth in SEQ ID NO 2176 and a LC amino acid sequence as set forth in SEQ ID NO 2181;a HC amino acid sequence as set forth in SEQ ID NO 2186 and a LC amino acid sequence as set forth in SEQ ID NO 2191;a HC amino acid sequence as set forth in SEQ ID NO 2196 and a LC amino acid sequence as set forth in SEQ ID NO 2201;a HC amino acid sequence as set forth in SEQ ID NO 2206 and a LC amino acid sequence as set forth in SEQ ID NO 2211;or a HC amino acid sequence as set forth in SEQ ID NO 2216 and a LC amino acid sequence as set forth in SEQ ID NO 2221.
- 5The isolated antibody or antigen-binding fragment thereof of any one of claims 1 through 4, wherein the antibody comprises a HC amino acid sequence as set forth in SEQ ID NO 562 and a LC amino acid sequence as set forth in SEQ ID NO 570;a HC amino acid sequence as set forth in SEQ ID NO 2146 and a LC amino acid sequence as set forth in SEQ ID NO 2151;a HC amino acid sequence as set forth in SEQ ID NO 2156 and a LC amino acid sequence as set forth in SEQ ID NO 2161;a HC amino acid sequence as set forth in SEQ ID NO 2166 and a LC amino acid sequence as set forth in SEQ ID NO 2171;a HC amino acid sequence as set forth in SEQ ID NO 2176 and a LC amino acid sequence as set forth in SEQ ID NO 2181;a HC amino acid sequence as set forth in SEQ ID NO 2186 and a LC amino acid sequence as set forth in SEQ ID NO 2191;a HC amino acid sequence as set forth in SEQ ID NO 2196 and a LC amino acid sequence as set forth in SEQ ID NO 2201;a HC amino acid sequence as set forth in SEQ ID NO 2206 and a LC amino acid sequence as set forth in SEQ ID NO 2211;or a HC amino acid sequence as set forth in SEQ ID NO 2216 and a LC amino acid sequence as set forth in SEQ ID NO 2221.
- 15The one or more antibodies or antigen-binding fragments thereof, the nucleic acid sequences, the expression vector, the host cell, or the pharmaceutical composition for the use according to any one of claims 12 through 14, wherein the use further comprises administering to the patient a second therapeutic agent.
Independent claims7
290 paragraphs in 1 section, as filed
Cross Reference to Related Applications
The present application claims the benefit of <patcit id="pcit0001" dnum="US62411508"><text>United States Provisional Patent Application number 62/411,508, filed October 21, 2016</text></patcit>, the entire contents of which are incorporated herein by reference.
Sequence Listing
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 20, 2017, is named "2009186_0218_SL.TXT" and is 1,019,600 bytes in size.
Field of the Invention
The invention relates, inter alia, to anti-Respiratory Syncytial Virus (RSV) antibodies and functional fragments thereof, nucleic acid sequences encoding such antibodies and methods and reagents for their preparation and use.
Background of the Invention
All references cited herein, including without limitation patents, patent applications, and non-patent references and publications referenced throughout are hereby expressly incorporated by reference in their entireties for all purposes.
Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in young children and the elderly, is the leading cause of infant hospitalization in the United States and accounts for an estimated 64 million infections and 160,000 deaths world-wide each year. However, despite decades of research, the development of a safe and effective vaccines or therapeutic and/or prophylactic antibodies against RSV has remained elusive, highlighting the need for novel strategies that induce or provide protective immune responses. (<i>1-3</i>). Indeed, to date there are currently no approved RSV vaccines, and passive prophylaxis with the monoclonal antibody palivizumab (marketed as Synagis<sup>®</sup>) is restricted to high-risk infants in part due to its modest efficacy.
Certain populations of children are at risk for developing an RSV infection and these include preterm infants (<nplcit id="ncit0001" npl-type="s"><text>Hall et al., 1979, New Engl. J. Med. 300:393-396</text></nplcit>), children with congenital malformations of the airway, children with bronchopulmonary dysplasia (<nplcit id="ncit0002" npl-type="s"><text>Groothuis et al., 1988, Pediatrics 82:199-203</text></nplcit>), children with congenital heart disease (<nplcit id="ncit0003" npl-type="s"><text>MacDonald et al., New Engl. J. Med. 307:397-400</text></nplcit>), and children with congenital or acquired immunodeficiency (<nplcit id="ncit0004" npl-type="s"><text>Ogra et al., 1988, Pediatr. Infect. Dis. J. 7:246-249</text></nplcit>; and <nplcit id="ncit0005" npl-type="s"><text>Pohl et al., 1992, J. Infect. Dis. 165:166-169</text></nplcit>), and cystic fibrosis (<nplcit id="ncit0006" npl-type="s"><text>Abman et al., 1988, J. Pediatr. 1 13:826-830</text></nplcit>).
RSV can infect the adult population as well. In this population, RSV causes primarily an upper respiratory tract disease, although elderly patients may be at greater risk for a serious infection and pneumonia (<nplcit id="ncit0007" npl-type="b"><text>Evans, A. S., eds., 1989, Viral Infections of Humans. Epidemiology and Control, 3rd ed., Plenum Medical Book, New York at pages 525-544</text></nplcit>), as well as adults who are immunosuppressed, particularly bone marrow transplant patients (<nplcit id="ncit0008" npl-type="s"><text>Hertz et al., 1989, Medicine 68:269-281</text></nplcit>). Other at risk patients include those suffering from congestive heart failure and those suffering from chronic obstructive pulmonary disease (i.e. COPD). There have also been reports of epidemics among nursing home patients and institutionalized young adults (<nplcit id="ncit0009" npl-type="s"><text>Falsey, A. R., 1991 , Infect. Control Hosp. Epidemiol. 12:602-608</text></nplcit>; and <nplcit id="ncit0010" npl-type="s"><text>Garvie et al., 1980, Br. Med. J. 281 :1253-1254</text></nplcit>).
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 (<nplcit id="ncit0011" npl-type="b"><text>Fields et al., eds, 1990, Fields Virology, 2nd ed., Vol. 1, Raven Press, New York at pages 1045-1072</text></nplcit>).
Similar to other pneumoviruses, RSV expresses two major surface glycoproteins: the fusion protein (F) and the attachment protein (G). Although both have been shown to induce protective neutralizing antibody responses, F is less genetically variable than G, is absolutely required for infection, and is the target for the majority of neutralizing activity in human serum (<i>4-8</i>). RSV F is also the target of the monoclonal antibody palivizumab, which is used to passively protect high-risk infants from severe disease (<i>9</i>). Consequently, the RSV F protein is considered to be a highly attractive target for vaccines and antibody-based therapies.
The mature RSV F glycoprotein initially exists in a metastable prefusion conformation (preF) (<i>10</i>), before undergoing a conformational change that leads to insertion of the hydrophobic fusion peptide into the host-cell membrane. Subsequent refolding of F into a stable, elongated postfusion conformation (postF) (<i>11, 12</i>) results in fusion of the viral and host-cell membranes. Due to its inherent instability, the preF protein has the propensity to prematurely trigger into postF, both in solution and on the viral surface (<i>13</i>). Recently, stabilization of preF has been achieved by protein engineering (<i>14, 15</i>), and stabilized preF has been shown to induce higher titers of neutralizing antibodies than postF in animal models (<i>15</i>).
Despite the importance of neutralizing antibodies in protection against severe RSV disease, our understanding of the human antibody response to RSV has been limited to studies of human sera and a small number of RSV-specific human monoclonal antibodies (<i>16-19</i>). The epitopes recognized by these human antibodies, as well as several murine antibodies, have defined at least four 'antigenic sites' on RSV F (<i>1</i>, <i>10, 16, 18-20</i>) (see also, e.g, Table 1). Three of these sites-I, II, and IV-are present on both pre- and postF, whereas antigenic site Ø exists exclusively on preF. Additional preF-specific epitopes have been defined by antibodies MPE8 (<i>17</i>) and AM14 (<i>21</i>). Although serum mapping studies have shown that site Ø-directed antibodies are responsible for a large proportion of the neutralizing antibody response in most individuals (<i>8</i>), there are additional antibody specificities that contribute to serum neutralizing activity that remain to be defined. In addition, it was heretofore unknown whether certain antibody sequence features are required for recognition of certain neutralizing sites, as observed for other viral targets (22-25). Accordingly, understanding the relationship between neutralization potency and epitope specificity would be advantageous in the selection and/or design of vaccine antigens, as well as therapeutic and/or prophylactic antibodies, which induce potent neutralizing responses to RSV.
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 (<nplcit id="ncit0012" npl-type="b"><text>Fields et al., eds, 1990, Fields Virology, 2nd ed., Vol. 1 , Raven Press, New York at pages 1045-1072</text></nplcit>).
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 (<nplcit id="ncit0013" npl-type="s"><text>Smith et al., 1991, New Engl. J. Med. 325:24-29</text></nplcit>). The use of ribavirin is limited due to concerns surrounding its potential risk to pregnant women who may be exposed to the aerosolized drug while it is being administered in a hospital environment.
Similarly, while a vaccine may be useful, no commercially available vaccine has been developed to date. Several vaccine candidates have been abandoned and others are under development (<nplcit id="ncit0014" npl-type="s"><text>Murphy et al., 1994, Virus Res. 32: 13-36</text></nplcit>). The development of a vaccine has proven to be problematic. In particular, immunization would be required in the immediate neonatal period since the peak incidence of lower respiratory tract disease occurs at 2-5 months of age. 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 (<nplcit id="ncit0015" npl-type="s"><text>Murphy et al., 1988, J. Virol. 62:3907-3910</text></nplcit>; and <nplcit id="ncit0016" npl-type="s"><text>Murphy et al., 1991, Vaccine 9:185-189</text></nplcit>).
Currently, the only approved approach to prophylaxis of RSV disease is passive immunization. For example, the humanized antibody, palivizumab (SYNAGIS<sup>®</sup>), which is specific for an epitope on the F protein, is approved for intramuscular administration to pediatric patients for prevention of serious lower respiratory tract disease caused by RSV at recommended monthly doses of 15 mg/kg of body weight throughout the RSV season (November through April in the northern hemisphere). SYNAGIS<sup>®</sup> is a composite of human (95%) and murine (5%) antibody sequences. (<nplcit id="ncit0017" npl-type="s"><text>Johnson et al., (1997), J. Infect. Diseases 176:1215-1224</text></nplcit> and <patcit id="pcit0002" dnum="US5824307A"><text>U.S. Pat. No. 5,824,307</text></patcit>).
Although SYNAGIS<sup>®</sup> has been successfully used for the prevention of RSV infection in pediatric patients, multiple intramuscular doses of 15 mg/kg of SYNAGIS<sup>®</sup> are required to achieve a prophylactic 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 antibody, and this lead to the identification and development of motavizumab, also referred to as NUMAX<sup>™</sup>. 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 antibody was then discontinued.
Other antibodies to RSV-F protein have been described and can be found in <patcit id="pcit0003" dnum="US6656467B"><text>US6656467</text></patcit>; <patcit id="pcit0004" dnum="US5824307A"><text>US5824307</text></patcit>, <patcit id="pcit0005" dnum="US7786273B"><text>US 7786273</text></patcit>; <patcit id="pcit0006" dnum="US7670600B"><text>US 7670600</text></patcit>; <patcit id="pcit0007" dnum="US7083784B"><text>US 7083784</text></patcit>; <patcit id="pcit0008" dnum="US6818216B"><text>US6818216</text></patcit>; <patcit id="pcit0009" dnum="US7700735B"><text>US7700735</text></patcit>; <patcit id="pcit0010" dnum="US7553489B"><text>US7553489</text></patcit>; <patcit id="pcit0011" dnum="US7323172B"><text>US7323172</text></patcit>; <patcit id="pcit0012" dnum="US7229619B"><text>US7229619</text></patcit>; <patcit id="pcit0013" dnum="US7425618B"><text>US7425618</text></patcit>; <patcit id="pcit0014" dnum="US7740851B"><text>US7740851</text></patcit> ; <patcit id="pcit0015" dnum="US7658921B"><text>US7658921</text></patcit> ; <patcit id="pcit0016" dnum="US7704505B"><text>US7704505</text></patcit>; <patcit id="pcit0017" dnum="US7635568B"><text>US7635568</text></patcit>; <patcit id="pcit0018" dnum="US6855493B"><text>US6855493</text></patcit>; <patcit id="pcit0019" dnum="US6565849B"><text>US6565849</text></patcit>; <patcit id="pcit0020" dnum="US7582297B"><text>US7582297</text></patcit>; <patcit id="pcit0021" dnum="US7208162B"><text>US7208162</text></patcit>; <patcit id="pcit0022" dnum="US7700720B"><text>US7700720</text></patcit>; <patcit id="pcit0023" dnum="US6413771B"><text>US6413771</text></patcit> ; <patcit id="pcit0024" dnum="US5811524A"><text>US581 1524</text></patcit>; <patcit id="pcit0025" dnum="US6537809B"><text>US6537809</text></patcit>; <patcit id="pcit0026" dnum="US5762905A"><text>US5762905</text></patcit>; <patcit id="pcit0027" dnum="US7070786B"><text>US7070786</text></patcit>; <patcit id="pcit0028" dnum="US7364742B"><text>US7364742</text></patcit>; <patcit id="pcit0029" dnum="US7879329B"><text>US7879329</text></patcit>; <patcit id="pcit0030" dnum="US7488477B"><text>US7488477</text></patcit>; <patcit id="pcit0031" dnum="US7867497B"><text>US7867497</text></patcit>; <patcit id="pcit0032" dnum="US5534411A"><text>US553441 1</text></patcit> ; <patcit id="pcit0033" dnum="US6835372B"><text>US6835372</text></patcit>; <patcit id="pcit0034" dnum="US7482024B"><text>US7482024</text></patcit>; <patcit id="pcit0035" dnum="US7691603B"><text>US7691603</text></patcit>; <patcit id="pcit0036" dnum="US8562996B"><text>US8562996</text></patcit>; <patcit id="pcit0037" dnum="US8568726B"><text>US8568726</text></patcit>; <patcit id="pcit0038" dnum="US9447173B"><text>US9447173</text></patcit>; <patcit id="pcit0039" dnum="US20100015596A"><text>US20100015596</text></patcit>; <patcit id="pcit0040" dnum="WO2009088159A1"><text>WO2009088159A1</text></patcit>; and <patcit id="pcit0041" dnum="WO2014159822A"><text>WO2014159822</text></patcit>. To date, none other than SYNAGIS<sup>®</sup> has been approved by a regulatory agency for use in preventing an RSV infection.
There remains a need for the provision of highly specific, high affinity, and highly potent neutralizing anti-RSV antibodies and antigen-binding fragments thereof with neutralize at least one, but preferably both, of subtype A and subtype B RSV viral strains, and which preferentially recognize PreF relative to PostF conformations of the F protein. There also remains a need for the provision of anti-RSV and anti-HMPV cross-neutralizing antibodies and antigen-binding fragments thereof.
Summary of the Invention
Applicant has now discovered, isolated, and characterized, inter alia, an extensive panel of RSV F-specific monoclonal antibodies from the memory B cells of a healthy adult human donor and used these antibodies to comprehensively map the antigenic topology of RSV F. A large proportion of the RSV F-specific human antibody repertoire was advantageously comprised of antibodies with greatly enhanced specificity for the PreF conformation of the F protein (relative to the PostF form), many if not most of which exhibited remarkable potency in neutralization assays against one or both of RSV subtype A and RSV subtype B strains. Indeed, a large number of these antibodies display neutralization potencies that are multiple-fold greater - some 5- to 100-fold greater or more - to previous anti-RSV therapeutic antibodies, such as D25 and pavlizumamab thus serve as attractive therapeutic and/or prophylactic candidates for treating and/or preventing RSV infection and disease.
The most potent antibodies were found to target two distinct antigenic sites that are located near the apex of the preF trimer, providing strong support for the development of therapeutic and/or prophylactic antibodies targeting these antigenic sites, as well as preF-based vaccine candidates that preserve these antigenic sites. Furthermore, the neutralizing antibodies described and disclosed herein represent new opportunities for the prevention of severe RSV disease using passive immunoprophylaxis.
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 exposed to, or at risk for acquiring an infection with RSV, or suffering from infection with RSV. The antibodies and pharmaceutical compositions described herein may also be used to prevent or to treat an RSV infection in a patient who may experience a more severe form of the RSV infection due to an underlying or pre-existing medical condition. A patient who may benefit from treatment with an antibody and/or a pharmaceutical composition of the invention may be a pre-term infant, a full-term infant born during RSV season (approximately 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 medical condition, such as congestive heart failure (CHF), or chronic obstructive pulmonary disease (COPD). A patient who may benefit from such therapy 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 compromises 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 underlying infectious disease or cancerous condition 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 patient) or radiation therapy. A patient who may benefit from the antibodies and/or pharmaceutical compositions of the invention may be a patient that suffers from chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), bronchopulmonary dysplasia, congestive heart failure (CHF), or congenital heart disease.
Because the inventive antibodies and antigen-binding fragments thereof are more effective at neutralization of RSV compared to known antibodies, lower doses of the antibodies or antibody fragments or pharmaceutical compositions of the invention could be used to achieve a greater level of protection against infection with RSV, and more effective treatment and/or amelioration of symptoms associated with an RSV infection. Accordingly, the use of lower doses of antibodies or fragments thereof which immunospecifically bind to RSV-F antigen and/or pharmaceutical compositions may result in fewer or less severe adverse events. Likewise, the use of more effective neutralizing antibodies may result in a diminished need for frequent administration of the antibodies or antibody fragments or pharmaceutical compositions 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 or pharmaceutical compositions 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 or pharmaceutical compositions may be used alone or in conjunction with a second agent useful for treating an RSV infection. In certain embodiments, the antibodies or pharmaceutical compositions 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 primary infection, or to ameliorate at least one symptom associated with the infection. In certain embodiments, the antibodies or pharmaceutical compositions may be used prophylactically as stand-alone therapy to protect patients 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 <u style="double">or</u> pharmaceutical compositions 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')<sub>2</sub> or scFv fragment), and may be modified to affect functionality, e.g., to eliminate residual effector functions (<nplcit id="ncit0018" npl-type="s"><text>Reddy et al., (2000), J. Immunol. 164:1925-1933</text></nplcit>).
Accordingly, in certain embodiments are provided isolated antibodies or antigen-binding fragments thereof that specifically bind to Respiratory Syncytial Virus (RSV) F protein (F), wherein at least one, at least two, at least three, at least four, at least five, or at least six of the CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and CDRL3 amino acid sequence such antibodies or the antigen-binding fragments thereof are at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99%; at least 100% and/or all percentages of identity in between; to at least one, at least two, at least three, at least four, at least five, or at least six of the CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and/or a CDRL3 amino acid sequences as disclosed in Table 6 of an antibody selected from Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; and wherein said antibody or the antigen-binding fragment thereof also has one or more of the following characteristics: a) the antibodies or antigen-binding fragments thereof cross-compete with said antibodies or antigen-binding fragments thereof for binding to RSV-F; b) the antibodies or antigen-binding fragments thereof display better binding affinity for the PreF form of RSV-F relative to the PostF form; c) the antibodies or antigen-binding fragments thereof display a clean or low polyreactivity profile; d) the antibodies or antigen-binding fragments thereof display neutralization activity toward RSV subtype A and RSV subtype B in vitro; e) the antibodies or antigen-binding fragments thereof display antigenic site specificity for RSV-F at Site Ø, Site I, Site II, Site III, Site IV, or Site V f) the antibodies or antigen-binding fragments thereof display antigenic site specificity for RSV-F Site Ø, Site V, or Site III relative to RSV-F Site I, Site II, or Site IV; g) at least a portion of the epitope with which the antibodies or antigen-binding fragments thereof interact comprises the α3 helix and β3/β4 hairpin of PreF; h) the antibodies or antigen-binding fragments thereof display an in vitro neutralization potency (IC<sub>50</sub>) of between about 0.5 microgram/milliliter (ug/ml) to about 5 ug/ml; between about 0.05 ug/ml to about 0.5 ug/ml; or less than about 0.05 mg/ml; i) the binding affinities and/or epitopic specificities of the antibodies or antigen-binding fragments thereof for any one of the RSV-F variants designated as 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG in <figref idref="f0010">Figure 7A</figref> is reduced or eliminated relative to the binding affinities and/or epitopic specificities of said antibodies or antigen-binding fragments thereof for the RSV-F or RSV-F DS-Cav1; j) the antibodies or antigen-binding fragments thereof display a cross-neutralization potency (IC<sub>50</sub>) against human metapneumovirus (HMPV); k) the antibodies or antigen-binding fragments thereof do not complete with D25, MPE8, palivizumab, or motavizumab; or 1) the antibodies or antigen-binding fragments thereof display at least about 2-fold; at least about 3-fold; at least about 4-fold; at least about 5-fold; at least about 6-fold; at least about 7-fold; at least about 8-fold; at least about 9-fold; at least about 10-fold; at least about 15-fold; at least about 20-fold; at least about 25-fold; at least about 30-fold; at least about 35-fold; at least about 40-fold; at least about 50-fold; at least about 55-fold; at least about 60-fold; at least about 70-fold; at least about 80-fold; at least about 90-fold; at least about 100-fold; greater than about 100-fold; and folds in between any of the foregoing; greater neutralization potency (IC50) than D25 and/or palivizumab.
In certain other embodiments, the isolated antibodies or antigen-binding fragments thereof comprise: at least two; at least three; at least 4; at least 5; at least 6; at least 7; at least 8; at least 9; at least 10; at least 11; or at least 12; of characteristics a) through 1) above.
In certain other embodiments, the isolated antibodies or antigen-binding fragments thereof comprise: a) the CDRH3 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372as disclosed in Table 6; b) the CDRH2 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; c) the CDRH1 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; d) the CDRL3 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; e) the CDRL2 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372a s disclosed in Table 6; f) the CDRL1 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; or g) any combination of two or more of a), b), c), d), e), and f).
In certain other embodiments, the isolated antibodies or antigen-binding fragments thereof comprise: a) a heavy chain (HC) amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; and/or b) a light chain (LC) amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain other embodiments, the isolated antibodies or antigen-binding fragments thereof are selected from the group consisting of antibodies that are at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99%; and/or all percentages of identity in between; to any one of the antibodies designated as Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain other embodiments, the isolated antibodies or antigen-binding fragments thereof are selected from the group consisting of the antibodies designated as Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In other embodiments are provided isolated nucleic acid sequences encoding antibodies light and/or heavy chains thereof, antigen-binding fragments thereof, or light and/or heavy chains encoding such antigen-beinding fragments according to any of the other embodiments disclosed herein.
In other embodiments are provided expression vectors comprising isolated nucleic acid sequences according to other embodiments disclosed herein.
In other embodiments are provided host cells transfected, transformed, or transduced with nucleic acid sequences or expression vectors according to other embodiments disclosed herein.
In other embodiments are provided pharmaceutical compositions comprising one or more of the isolated antibodies or antigen-binding fragments thereof according to other embodiments disclosed herein; and a pharmaceutically acceptable carrier and/or excipient.
In other embodiments are provided pharmaceutical compositions comprising a nucleic acid sequence of the invention, e.g., one or more nucleic acid sequences encoding at least one of a light or heavy chain of an antibody or both according other embodiments disclosed herein; or one or more expression vectors according to other embodiments disclosed herein; and a pharmaceutically acceptable carrier and/or excipient.
In other embodiments are provided transgenic organisms comprising nucleic acid sequences according to other embodiments disclosed herein; or expression vectors according to other embodiments disclosed herein.
In other embodiments are provided methods of treating or preventing a Respiratory Syncytial Virus (RSV) infection, or at least one symptom associated with RSV infection, comprising administering to a patient in need there of or suspected of being in need thereof: a) one or more antibodies or antigen-binding fragments thereof according to other embodiments disclosed herein; b) nucleic acid sequences according to other embodiments disclosed herein; an expression vector according to other embodiments disclosed herein; a host cell according to other embodiments disclosed herein; or e) a pharmaceutical composition according to other embodiments disclosed herein; such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity.
In other embodiments are provided methods of treating or preventing either a Respiratory Syncytial Virus (RSV) infection and/or a human metapneumovirus (HMPV) infection, or at least one symptom associated with said RSV infection or said HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof: a) one or more antibodies or antigen-binding fragments thereof according to other embodiments disclosed herein; b) a nucleic acid sequences according to other embodiments disclosed herein; c) an expression vector according to other embodiments disclosed herein; d)a host cell according to other embodiments disclosed herein; or e) a pharmaceutical composition according to other embodiments disclosed herein; such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity. In other embodiments are provided methods according to other embodiments wherein the one or more antibodies or antigen-binding fragments thereof of a) is selected from the group consisting of the antibodies designated as Antibody Number 340 as disclosed in Table 6.
In other embodiments are provided methods according to other embodiments wherein the method further comprises administering to the patient a second therapeutic agent.
In other embodiments are provided methods according to other embodiments, wherein the second therapeutic agent is selected group consisting of: an antiviral agent; a vaccine specific for RSV, a vaccine specific for influenza virus, or a vaccine specific 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.
In certain embodiments are provided pharmaceutical compositions comprising any one or more of the isolated antibodies or antigen-binding fragments thereof, or one or more nucleic acid sequences encoding at least one of a light chain or heavy chain of an antibody according to other embodiments disclosed herein or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier and/or excipient.
In certain embodiments are provided pharmaceutical compositions according to other embodiments for use in preventing a respiratory syncytial virus (RSV) infection in a patient in need thereof or suspected of being 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 prevented, ameliorated, or lessened in severity and/or duration as a result of such use.
In certain embodiments are provided pharmaceutical compositions according to other embodiments for use in treating or preventing either a Respiratory Syncytial Virus (RSV) infection and/or a human metapneumovirus (HMPV) infection, or at least one symptom associated with said RSV infection and/or said HMPV infection, in a patient in need thereof or suspected of being in need thereof, wherein the infection is either prevented, or at least one symptom or complication associated with the infection is prevented, ameliorated, or lessened in severity and/or duration as a result of such use.
In certain other embodiments are provided uses of the pharmaceutical compositions according to other embodiments in the manufacture of a medicament for 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 prevented, ameliorated, or lessened in severity and/or duration.
In certain other embodiments are provided uses of the pharmaceutical compositions according to other embodiments in the manufacture of a medicament for preventing either a Respiratory Syncytial Virus (RSV) infection and/or a human metapneumovirus (HMPV) infection, or at least one symptom associated with said RSV infection and/or said HMPV infection, in a patient in need thereof or suspected of being in need thereof, wherein the infection is either prevented, or at least one symptom or complication associated with the infection is prevented, ameliorated, or lessened in severity and/or duration as a result of such use.
Brief Description of the Figures
<ul id="ul0001" list-style="none"><li><figref idref="f0001 f0002">Figures 1A-1F</figref> illustrate the anti-RSV repertoire cloning and sequence analysis of the identified and isolated antibodies. <figref idref="f0001">Figure 1A</figref>: RSV F-specific B cell sorting. FACS plots show RSV F reactivity of IgG<sup>+</sup> and IgA<sup>+</sup> B cells from the healthy adult donor. B cells in quadrant 2 (Q2) were single cell sorted. <figref idref="f0001">Figure 1B</figref>: Isotype analysis. Index sort plots show the percentage of RSV F-specific B cells that express IgG or IgA. <figref idref="f0002">Figure 1C</figref>: Clonal lineage analysis. Each slice represents one clonal lineage; the size of the slice is proportional to the number of clones in the lineage. The total number of clones is shown in the center of the pie. Clonal lineages were assigned based on the following criteria: 1) matching of variable and joining gene segments; 2) identical CDR3 loop lengths; and 3) >80% homology in CDR3 nucleotide sequences. <figref idref="f0002">Figure 1D</figref>: VH repertoire analysis. VH germline genes were considered to be enriched in the RSV repertoire if a given gene was found to be enriched by greater than 3-fold over non-RSV-specific repertoires (33). <figref idref="f0002">Figure 1E</figref>: CDRH3 length distribution. <figref idref="f0002">Figure 1F</figref>: Somatic hypermutation in VH (excluding CDRH3). Red bars indicate the average number of nucleotide substitutions. Each clonal lineage is only represented once in Figure ID and Figure IE. Data for non-RSV reactive IgGs were derived from published sequences obtained by high-throughput sequencing of re-arranged antibody variable gene repertoires from healthy individuals (33).</li><li><figref idref="f0003">Figures 2A-2D</figref> illustrate the similar antibody preferences observed for conformational state and subtype of RSV F in the repertoire. <figref idref="f0003">Figure 2A</figref>: IgG affinities for preF and postF are plotted as shown. <figref idref="f0003">Figure 2B</figref>: Percentage of antibodies within the donor repertoire that recognized both conformations of F (green) or bind only to preF (blue) or postF (orange). <figref idref="f0003">Figure 2C</figref>: Percentage of antibodies within the donor repertoire that bind specifically to subtype A (green), subtype B (blue), or both subtypes A and B (red). N.B., non-binder. IgG KDs were calculated for antibodies with BLI responses >0.1 nm. Antibodies with BLI responses <0.05 nm were designated as N.B. <figref idref="f0003">Figure 2D</figref>: Polyreactivity analysis of anti-RSV antibodies. The polyreactivity of the isolated anti-RSV F antibodies was measured using a previously described assay (42, 43). Three panels of control antibodies were included for comparison: a group of 138 antibodies currently in clinical trials, 39 antibodies that have been approved for clinical use and 14 broadly neutralizing HIV antibodies.</li><li><figref idref="f0004 f0005">Figures 3A-3G</figref> illustrate mapping and specificities of anti-RSV antibodies for antigenic sites spanning the surface of PreF and PostF. <figref idref="f0004">Figure 3A</figref>: The previously determined structure of preF with one protomer shown as ribbons and with six antigenic sites rainbow colored from red to purple. <figref idref="f0004">Figure 3B</figref>: The percentage of antibodies targeting each antigenic site is shown. <figref idref="f0004">Figure 3C</figref>: Percentage of preF-specific antibodies targeting each antigenic site. <figref idref="f0005">Figure 3D</figref>: Apparent antibody binding affinities for subtype A PreF antigenic sites. <figref idref="f0005">Figure 3E</figref>: Apparent binding affinities for subtype A postF antigenic sites. <figref idref="f0005">Figure 3F</figref>: Apparent antibody binding affinities for subtype B PreF antigenic sites. <figref idref="f0005">Figure 3G</figref>. Apparent binding affinities for subtype B postF. Only antibodies with apparent binding affinities greater than 2 nM were included in this analysis, since antibodies with lower affinity could not be reliably mapped. Red bars show the median and the dotted grey line is at 2 nM. N.B., non-binder.</li><li><figref idref="f0006 f0007">Figures 4A-4G</figref> illustrate neutralizing potencies of anti-RSV antibodies and correlation between potency and Pref vs. PostF specificity for each of RSV subtypes A and B. <figref idref="f0006">Figure 4A</figref>: Neutralization IC<sub>50</sub>s for the antibodies isolated from the donor repertoire. Data points are colored based on neutralization potency, according to the legend on the right. Red and blue dotted lines depict motavizumab and D25 IC<sub>50</sub>s, respectively. <figref idref="f0006">Figure 4B</figref>: Percentage of neutralizing antibodies in the donor repertoire against RSV subtype A or subtype B, stratified by potency as indicated in the legend in the <i>right</i> portion of the figure. <figref idref="f0006">Figure 4C</figref>: Percentage of antibodies within the donor repertoire that neutralized both RSV subtypes A and B (red) or neutralized only RSV subtype A (green) or subtype B (blue). <figref idref="f0007">Figure 4D</figref>: Apparent binding affinities for subtype A, preF and postF, plotted for each antibody (IgG KDs were calculated for antibodies with BLI responses >0.1 nm. Antibodies with BLI responses <0.05 nm were designated as N.B.) <figref idref="f0007">Figure 4E</figref>: Neutralization IC<sub>50</sub>s plotted for RSV subtype A preF-specific, postF-specific, and cross-reactive antibodies. (Red and blue dotted lines depict motavizumab and D25 IC<sub>50</sub>s, respectively. Red bars depict median. N.B., non-binder; N.N., non-neutralizing). <figref idref="f0007">Figure 4F</figref>: Apparent antibody binding affinities for subtype B, preF and postF. <figref idref="f0007">Figure 4G</figref>: IC<sub>50</sub>s plotted for RSV subtype B preF-specific, postF-specific and cross-reactive antibodies. (Black bar depicts median. N.B., non-binder; N.N., non-neutralizing.)</li><li><figref idref="f0008">Figures 5A-5C</figref> illustrate that the most potent neutralizing antibodies bind with high affinity to preF and recognize antigenic sites Ø and V. <figref idref="f0008">Figure 5A</figref>: apparent preF <i>K</i><sub>D</sub> plotted against neutralization IC<sub>50</sub> and colored according to antigenic site, as shown in the legend <i>at right</i> of <figref idref="f0008">Figure 5C. Figure 5B</figref>: apparent postF <i>K</i><sub>D</sub> plotted against neutralization IC<sub>50</sub> and colored as in <figref idref="f0008">Figure 5A. Figure 5C</figref>: antibodies grouped according to neutralization potency and colored by antigenic site as in legend at <i>right.</i> N.B., non-binder; N.N., non-neutralizing. IgG <i>K</i><sub>D</sub>S were calculated for antibodies with BLI responses >0.1 nm. Antibodies with BLI responses <0.05 nm were designated as N.B. Statistical significance was determined using an unpaired two-tailed t test. The Pearson's correlation coefficient, r, was calculated using Prism software version 7.0. Antibodies that failed to bind or neutralize were excluded from the statistical analysis due to the inability to accurately calculate midpoint concentrations.</li><li><figref idref="f0009">Figures 6A-6C</figref> illustrate the nature and purification of pre- and postF sorting probes. <figref idref="f0009">Figure 6A</figref>: Schematic of fluorescent prefusion RSV F probe shows one PE-conjugated streptavidin molecule bound by four avi-tagged trimeric prefusion F molecules. <figref idref="f0009">Figure 6B</figref>: Coomassie-stained SDS-PAGE gel demonstrating the isolation of RSV F with a single AviTag per trimer using sequential Ni-NTA and <i>Strep-Tactin</i> purifications, as described in the Methods. <figref idref="f0009">Figure 6C</figref>: Fluorescence size-exclusion chromatography (FSEC) trace of the tetrameric probes on a Superose 6 column. Positions of molecular weight standards are indicated with arrows.</li><li><figref idref="f0010">Figures 7A-7C</figref> illustrate the generation and validation of preF patch panel mutants. <figref idref="f0010">Figure 7A</figref>: Panel of RSV F variants used for epitope mapping. <figref idref="f0010">Figure 7B</figref>: Prefusion RSV F shown as molecular surface with one protomer colored in white. The nine variants, each containing a patch of mutations, are uniquely colored according to the table in <figref idref="f0010">Figure 7A. Figure 7C</figref>: Binding of each IgG to fluorescently labeled beads coupled to each of the variants listed in <figref idref="f0010">Figure 7A</figref> was measured using PE-conjugated anti-human Fc antibody on a FLEXMAP 3D flow cytometer (Luminex). Reduced binding of D25 and motavizumab to patches 1 and 5, respectively, is consistent with their structurally defined epitopes (10, 11). AM14 binding was reduced for both patch 3 and patch 9, due to its unique protomer-spanning epitope (21). This characteristic binding profile was used to assist in the classification of other possible quaternary-specific antibodies in the panel.</li><li><figref idref="f0011">Figure 8</figref> illustrates the antigenic site V resides between the epitopes recognized by D25, MPE8 and motavizumab. Prefusion F is shown with one promoter as a cartoon colored according to antigenic site location and the other two protomers colored grey. D25 and motavizumab Fabs are shown in blue and pink, respectively. The MPE8 binding site is circled in black. Antigenic site V is located between the binding sites of D25 and MPE8 within one protomer, explaining the competition between site-V directed antibodies and these controls. Competition with motavizumab may occur across two adjacent protomers (left) or within one protomer (right), depending on the angle-of-approach of these site-V directed antibodies.</li><li><figref idref="f0012">Figure 9</figref> illustrates percentage of anti-RSV antibodies demonstrating the indicated neutralizing activities of preF-specific, postF-specific, and cross-reactive antibodies. Antibodies were stratified according to neutralization potency and the percentage of antibodies in each group that were preF-specific (pink), postF-specific (white) or cross-reactive (orange) were plotted for subtype A (<i>left panel</i>) and subtype B (<i>rightpanel</i>)<i>.</i></li><li><figref idref="f0013">Figures 10A-10C</figref> illustrate the relationship between subtype B neutralization and antigenic site specificity for anti-RSV antibodies. <figref idref="f0013">Figure 10A</figref>: Subtype B preF affinity plotted against neutralization IC<sub>50</sub> for all antibodies and colored by antigenic site according to the color scheme depicted in <figref idref="f0013">Figure 10C</figref>, <i>right portion.</i><figref idref="f0013">Figure 10B</figref>: PostF affinity plotted against IC<sub>50</sub> and colored as in <figref idref="f0013">Figure 10A. Figure 10C</figref>: Antibodies with preF affinities higher than 2 nM grouped according to neutralization potency and colored by antigenic site (<i>right portion</i>)<i>.</i></li><li><figref idref="f0014">Figure 11</figref> illustrates <i>in vitro</i> neutralization of RSV A2 for specific anti-RSV antibodies. Inhibition of RSV-replication was measured in an ELISA based neutralization Assay using Hep-2 cells. Cells, mAbs and viruses were co-incubated for 4 days at 37°C, followed by quantification of viral proteins in infected cells using a polyclonal anti-RSV antibody. % inhibition was calculated relative to control cells infected with virus in absence of neutralizing antibody. Data are expressed as half-maximal inhibitory concentration that resulted in 50% reduction in virus replication (IC50) and represent the mean +/- SEM of two independent experiments. An isotype matched control mAb (*) was included in every experiment and did not exhibit virus neutralization.</li></ul>
Detailed Description of the Invention
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.).
Definitions
"Respiratory Syncytial Virus-F protein", also referred to as "RSV-F" or "RSV F" is a type I transmembrane surface protein, which has an N terminal cleaved signal peptide and a membrane anchor near the C terminus (<nplcit id="ncit0019" npl-type="s"><text>Collins, P.L. et al., (1984), PNAS (USA) 81 :7683-7687</text></nplcit>). The RSV-F protein is synthesized as an inactive 67 KDa precursor denoted as F0 (<nplcit id="ncit0020" npl-type="s"><text>Calder, L.J.; et al., Virology (2000), 277,122-131</text></nplcit>. 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 "pep27". There are furin cleavage sites (FCS) on either side of the pep27 (<nplcit id="ncit0021" npl-type="s"><text>Collins, P.L.; Mottet, G. (1991), J. Gen. Virol., 72: 3095-3101</text></nplcit> ; <nplcit id="ncit0022" npl-type="s"><text>Sugrue, R.J, et al. (2001), J.. Gen. Virol., 82, 1375- 1386</text></nplcit>). The F2 subunit consists of the Heptad repeat C (HRC), while the F1 contains the fusion polypeptide (FP), heptad repeat A (HRA), domain I, domain II, heptad repeat B (HRB), transmembrane (TM) and cytoplasmic domain (CP) (See <nplcit id="ncit0023" npl-type="s"><text>Sun, Z. et al. Viruses (2013), 5:21 1 - 225</text></nplcit>). The RSV-F protein plays a role in fusion of the virus particle to the cell membrane, and is expressed on the surface of infected cells, thus playing a role in cell to cell transmission of the virus and syncytia formation. The amino acid sequence of the RSV-F protein is provided in GenBank as accession number AAX23994.
A stabilized variant of the PreF trimeric conformation of RSV-F, termed "RSV-DS-Cav1", or "DS-Cavl" disclosed in, inter alia, <nplcit id="ncit0024" npl-type="s"><text>Stewart-Jones et al., PLos One, Vol. 10(6)):e0128779</text></nplcit> and <patcit id="pcit0042" dnum="WO2011050168A"><text>WO 2011/050168</text></patcit> was used in the identification, isolation, and characterization of the antibodies disclosed herein.
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 strain" 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 "ED<sub>99</sub>" 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 present invention, the ED<sub>99</sub> refers to the dosage of the anti-RSV-F antibodies that will neutralize the virus infection (e.g., reduce 99% of viral load) in vivo, as described in Example 5.
The term "IC<sub>50</sub>" refers to the "half maximal inhibitory concentration", which value measures the effectiveness of compound (e.g., anti-RSV-F antibody) inhibition towards a biological or biochemical utility. This quantitative measure indicates the quantity required for a particular inhibitor to inhibit a given biological process by half. In certain embodiments, RSV virus neutralization potencies for anti-RSV and/or anti-RSV/anti-HMPV cross-neutralizing antibodies disclosed herein are expressed as neutralization IC<sub>50</sub> values.
"Palivizumab", also referred to as "SYNAGIS<sup>®</sup>", is a humanized anti-RSV-F antibody with heavy and light chain variable domains having the amino acid sequences as set forth in <patcit id="pcit0043" dnum="US7635568B"><text>US7,635,568</text></patcit> and <patcit id="pcit0044" dnum="US5824307A"><text>US 5,824,307</text></patcit>. This antibody, which immunospecifically binds to the RSV-F protein, is currently FDA-approved for the passive immunoprophylaxis of serious RSV disease in high-risk children 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<sup>®</sup> is composed of 95% human and 5% murine antibody sequences. See also <nplcit id="ncit0025" npl-type="s"><text>Johnson et al., (1997), J. Infect. Diseases 176:1215-1224</text></nplcit>.
"Motavizumab", also referred to as "NUMAX<sup>™</sup>", is an enhanced potency RSV-F-specific humanized monoclonal antibody derived by in vitro affinity maturation of the complementarity- determining regions of the heavy and light chains of palivizumab. For reference purposes, the amino acid sequence of the NUMAX<sup>™</sup> antibody is disclosed in <patcit id="pcit0045" dnum="US20030091584A"><text>U.S Patent Publication 2003/0091584</text></patcit> and in <patcit id="pcit0046" dnum="US6818216B"><text>U.S. Pat. No. 6,818,216</text></patcit> and in <nplcit id="ncit0026" npl-type="s"><text>Wu et al., (2005) J. Mol. Bio. 350(1):126- 144</text></nplcit> and in <nplcit id="ncit0027" npl-type="s"><text>Wu, et al. (2007) J. Mol. Biol. 368:652-665</text></nplcit>. 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 amelioration of the progression, severity, and/or duration of an upper and/or lower respiratory tract RSV infection and/or human metapneumovirus (HMPV), 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 administration of one or more prophylactic or therapeutic agents). In certain embodiments, such terms refer to the reduction or inhibition of the replication of RSV and/or HMPV, the inhibition or reduction in the spread of RSV and/or HMPV 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 and/or HMPV, or the amelioration of one or more symptoms associated 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 and/or HMPV 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 and/or HMPV infection to a lower respiratory tract RSV and/or HMPV infection, otitis media or a respiratory condition 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 and/or HMPV infection, otitis media or a respiratory condition related thereto, or the administration of a combination of therapies (e.g., a combination of prophylactic or therapeutic agents). As used herein, the terms "ameliorate" and "alleviate" refer to a reduction or diminishment in the severity a condition or any symptoms thereof.
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 antigen-binding fragments thereof. Each heavy chain (HC) is comprised of a heavy chain variable region ("HCVR" or "V<sub>H</sub>") and a heavy chain constant region (comprised of domains C<sub>H</sub>1, C<sub>H</sub>2 and C<sub>H</sub>3). Each light chain (LC) is comprised of a light chain variable region ("LCVR or "V<sub>L</sub>") and a light chain constant region (C<sub>L</sub>). The V<sub>H</sub> and V<sub>L</sub> regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each V<sub>H</sub> and V<sub>L</sub> is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR, 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 naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. Accordingly, the CDRs in a heavy chain are designated "CHRH1", "CDRH2", and "CDRH3", respectively, and the CDRs in a light chain are designated "CDRL1", "CDRL2", and "CDRL3".
Substitution of one or more CDR residues or omission of one or more CDRs is also possible. Antibodies have been described in the scientific literature in which one or two CDRs can be dispensed with for binding. <nplcit id="ncit0028" npl-type="s"><text>Padlan et al. (1995 FASEB J. 9:133-139</text></nplcit>) analyzed the contact regions between antibodies and their antigens, based on published crystal structures, and concluded that only about one fifth to one third of CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs had no amino acids in contact with an antigen (see also, <nplcit id="ncit0029" npl-type="s"><text>Vajdos et al. 2002 J Mol Biol 320:415-428</text></nplcit>).
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 outside Chothia CDRs, by molecular modeling and/or empirically. If a CDR or residue(s) thereof is omitted, it is usually substituted with an amino acid occupying the corresponding position in another human antibody sequence or a consensus of such sequences. Positions for substitution within CDRs and amino acids to substitute can also be selected empirically.
The fully human monoclonal antibodies disclosed herein may comprise one or more amino acid substitutions, 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 sequence databases. The present invention includes antibodies, and antigen-binding fragments thereof, which are derived from any of the amino acid sequences disclosed herein, wherein 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 derived, 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 "germline 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 V<sub>H</sub> and/or V<sub>L</sub> 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 residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework 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 residues 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, improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed within the present invention.
The present invention also includes fully monoclonal antibodies comprising variants of any of the CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes antibodies having 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 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 germline 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 "humanized antibody" refers to human antibody in which one or more CDRs of such antibody have been replaced with one or more corresponding CDRs obtained a non-human derived (e.g., mouse, rat, rabbit, primate) antibody. Humanized antibodies may also include certain non-CDR sequences or residues derived from such non-human antibodies as well as the one or more non-human CDR sequence. Such antibodies may also be referred to as "chimeric antibodies".
The term "recombinant" generally 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 protein 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 genetic engineering methods.
The antibodies of the invention may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibody", as used herein, is intended to include all antibodies, including human or humanized antibodies, that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected 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., <nplcit id="ncit0030" npl-type="s"><text>Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295</text></nplcit>) 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 transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V<sub>H</sub> and V<sub>L</sub> regions of the recombinant antibodies are sequences that, while derived from and related to human germline V<sub>H</sub> and V<sub>L</sub> sequences, may not naturally exist within the human 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 conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1 x10<sup>-6</sup> M or less (e.g., a smaller K<sub>D</sub> denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been identified by surface plasmon resonance, e.g., BIACORE<sup>™</sup>, biolayer interferometry measurements using, e.g., a ForteBio Octet HTX instrument (Pall Life Sciences), which bind specifically to RSV-F. Moreover, multi-specific antibodies that bind to RSV-F protein and one or more additional antigens, such as an antigen expressed by HMPV, or a bi-specific that binds to two different regions of RSV-F are nonetheless considered antibodies that "specifically bind", as used herein. In certain embodiments, the antibodies disclosed herein display equilibrium dissociation constants (and hence specificities) of about 1 x 10-<sup>6</sup> M; about 1 x 10-<sup>7</sup> M; about 1 x 10-<sup>8</sup> M; about 1 x 10-<sup>9</sup> M; about 1 x 10<sup>-10</sup> M; between about 1 x 10<sup>-6</sup> M and about 1 x 10-<sup>7</sup>M; between about 1 x 10-<sup>7</sup>M and about 1 x 10-<sup>8</sup> M; between about 1 x 10-<sup>8</sup>M and about 1 x 10-<sup>9</sup> M; or between about 1 x 10-<sup>9</sup> M and about 1 x 10-<sup>10</sup> M.
The term "high affinity" antibody refers to those mAbs having a binding affinity to RSV-F and/or HMPV, expressed as K<sub>D</sub>, of at least 10-<sup>9</sup> M; more preferably 10-<sup>10</sup>M, more preferably 10<sup>-11</sup>M, more preferably 10<sup>-12</sup>M as measured by surface plasmon resonance, e.g., BIACORE<sup>™</sup>, biolayer interferometry measurements using, e.g., a ForteBio Octet HTX instrument (Pall Life Sciences), or solution- affinity ELISA.
By the term "slow off rate", "Koff" or "kd" is meant an antibody that dissociates from RSV- F, with a rate constant of 1 x 10<sup>-3</sup> s<sup>"1</sup> or less, preferably 1 x 10<sup>-4</sup> s<sup>"1</sup> or less, as determined by surface plasmon resonance, e.g., BIACORE<sup>™</sup> or a ForteBio Octet HTX instrument (Pall Life Sciences).
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. In certain embodiments, 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 and/or HMPV.
An antibody fragment may include a Fab fragment, a F(ab')<sub>2</sub>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 genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and (optionally) constant 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 codons, create cysteine residues, modify, 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) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues 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 antibodies, single domain antibodies, domain- deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.
An antigen-binding fragment of an antibody will typically 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 framework sequences. In antigen-binding fragments having a V<sub>H</sub> domain associated with a V<sub>L</sub> domain, the V<sub>H</sub> and V<sub>L</sub> domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain V<sub>H</sub> - V<sub>H</sub>, V<sub>H</sub> - V<sub>L</sub> or V<sub>L</sub> - V<sub>L</sub> dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric V<sub>H</sub> or V<sub>L</sub> domain.
In certain embodiments, an antigen-binding fragment 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 constant domains that may be found within an antigen- binding fragment of an antibody of the present invention include: (i) V<sub>H</sub> -C<sub>H</sub>1 ; (ii) V<sub>H</sub> -C<sub>H</sub>2; (iii) V<sub>H</sub> -C<sub>H</sub>3; (iv) V<sub>H</sub> -C<sub>h</sub>1 - C<sub>h</sub>2; (v) V<sub>H</sub> -C<sub>h</sub>1-C<sub>h</sub>2-C<sub>h</sub>3; (vi) V<sub>H</sub> -C<sub>H</sub>2-C<sub>H</sub>3; (vii) V<sub>H</sub> -C<sub>L</sub>; (viii) V<sub>L</sub> -C<sub>H</sub>1; (ix) V<sub>L</sub> - C<sub>H</sub>2; (X) V<sub>L</sub> -C<sub>H</sub>3; (xi) V<sub>L</sub> -C<sub>H</sub>1-C<sub>H</sub>2; (xii) V<sub>L</sub> -C<sub>H</sub>1-C<sub>H</sub>2-C<sub>H</sub>3; (xiii) V<sub>L</sub> -C<sub>H</sub>2-C<sub>H</sub>3; and (xiv) V<sub>L</sub> -C<sub>L</sub>. In any configuration of variable and constant domains, including any of the exemplary 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 linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the present invention may comprise 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 V<sub>H</sub> or V<sub>L</sub> domain (e.g., by disulfide bond(s)).
As with full antibody molecules, 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 comprise at least two different variable domains, wherein each variable domain is capable 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 adapted for use in the context of an antigen-binding fragment of an antibody of the present invention using routine techniques available in the art.
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-F antibody, an anti-HMPV antibody, a vaccine, or a toxoid, or any other therapeutic moiety useful for treating an RSV infection and/or an HMPV infection.
An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies (Abs) having different antigenic specificities (e.g., an isolated antibody that specifically binds RSV-F and/or HMPV, or a fragment thereof, is substantially free of Abs that specifically bind antigens other than RSV-F and/or HMPV.
A "blocking antibody" or a "neutralizing antibody", as used herein (or an "antibody that neutralizes RSV-F and/or HMPV activity"), is intended to refer to an antibody whose binding to RSV-F or to an HMPV antigen, as the case may be as disclosed herein, results in inhibition of at least one biological activity of RSV-F and/or HMPV. For example, an antibody of the invention may aid in blocking the fusion of RSV and/or HMPV to a host cell, or prevent syncytia formation, or prevent the primary disease caused by RSV and/or HMPV. Alternatively, an antibody of the invention may demonstrate the ability to ameliorate at least one symptom of the RSV infection and or HMPV infection. This inhibition of the biological activity of RSV-F and/or HMPV can be assessed by measuring one or more indicators of RSV-F and/or HMPV 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 and/or HMPV following administration of one or more of the antibodies described herein).
The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE<sup>™</sup> system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).
The term "K<sub>D</sub> ", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-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 different 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. Epitopes may be defined as structural or functional. Functional epitopes 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 groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
The term "substantial identity", or "substantially identical," 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 identity in at least about 90%, and more preferably 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 discussed below. Accordingly, nucleic acid sequences that display a certain percentage "identity" share that percentage identity, and/or are that percentage "identical" to one another. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.
In certain embodiments, the disclosed antibody nucleic acid sequences are, e.g,: at least 70% identical; at least 75% identical; at least 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99% identical; or 100% identical; and/or all percentages of identity in between; to other sequences and/or share such percentage identities with one another (or with certain subsets of the herein-disclosed antibody sequences).
As applied to polypeptides, the term "substantial identity" or "substantially identical" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Accordingly, amino acid sequences that display a certain percentage "identity" share that percentage identity, and/or are that percentage "identical" to one another. Accordingly, amino acid sequences that display a certain percentage "identity" share that percentage identity, and/or are that percentage "identical" to one another.
In certain embodiments, the disclosed antibody amino acid sequences are, e.g., at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99%; and/or all percentages of identity in between; to other sequences and/or share such percentage identities with one another (or with certain subsets of the herein-disclosed antibody sequences).
Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. A "conservative 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 general, a conservative amino acid substitution will not substantially change the functional 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 upwards 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., <nplcit id="ncit0031" npl-type="s"><text>Pearson (1994) Methods Mol. Biol. 24: 307- 331</text></nplcit>). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in <nplcit id="ncit0032" npl-type="s"><text>Gonnet et al. (1992) Science 256: 1443 45</text></nplcit>. 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 substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides 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 recommended 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 sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. (See, e.g., <nplcit id="ncit0033" npl-type="s"><text>Altschul et al. (1990) J. Mol. Biol. 215: 403 410</text></nplcit> and (<nplcit id="ncit0034" npl-type="s"><text>1997) Nucleic Acids Res. 25:3389 402</text></nplcit>).
In certain 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 specific for epitopes of more than one target polypeptide. 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) C<sub>H</sub>3 domain and a second Ig C<sub>H</sub>3 domain, wherein the first and second Ig C<sub>H</sub>3 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 bi- specific antibody lacking the amino acid difference. In one embodiment, the first Ig C<sub>H</sub>3 domain binds Protein A and the second Ig C<sub>H</sub>3 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 C<sub>H</sub>3 may further comprise an Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second C<sub>H</sub>3 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 described 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 skilled in the art using known techniques (see, for example, <nplcit id="ncit0035" npl-type="s"><text>Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding</text></nplcit>).
An "immunogenic composition" relates to a composition containing an antigen/immunogen, e.g., a microorganism, such as a virus or a bacterium, or a component thereof, a protein, a polypeptide, a fragment of a protein or polypeptide, 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 systems to one or more of the antigens/immunogens present in the immunogenic composition. The immunogenic compositions of the present invention can be used to treat a human susceptible to RSV and/or HMPV infection or suspected of having or being susceptible to RSV and/or HMPV 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 delivery 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 composition comprising at least one immunogenic composition that induces an immune response in an animal.
In certain embodiments, a protein of interest comprises an antigen. The terms "antigen," "immunogen," "antigenic," "immunogenic," "antigenically active," and "immunologically active" when made in reference to a molecule, refer to any substance that is capable 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 response in the recipient that is adequate to prevent or ameliorate signs or symptoms of disease, including adverse health effects or complications thereof. 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 measuring, e.g., reduction of shed of challenge organisms, reduction in clinical signs such as mortality, morbidity, temperature, and overall physical condition, health and performance of the subject. The immune response can comprise, without limitation, induction of cellular and/or humoral immunity. The amount of a composition or vaccine that is therapeutically effective can vary, depending on the particular organism used, or the condition of the animal being treated or vaccinated.
An "immune response", or "immunological response" as used herein, in a subject refers to the development of a humoral immune response, a cellular- immune response, 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-lymphocytes 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 determined using standard immunoassays and neutralization assays, 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 specifically against a bacteria or virus that causes the identified disease.
Unless specifically indicated otherwise, the term "antibody," as used herein, shall be understood to encompass 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.
Preparation of Human Antibodies
As disclosed herein, anti-RSV and or anti-RSV/ anti-HMPF cross neutralizing antibodies by be obtained through B cell sorting techniques available to the artisan, and, for example, as described in the EXAMPLES below. Methods for generating human antibodies in transgenic mice are also known in the art and may be employed in order to derive antibodies in accordance with the present disclosure. Any such known methods can be used in the context of the present invention to make human antibodies that specifically bind to RSV-F (see, for example, <patcit id="pcit0047" dnum="US6596541B"><text>U.S. Pat. No. 6,596,541</text></patcit>).
In certain embodiments, the antibodies of the instant invention possess affinities (K<sub>D</sub>) ranging from about 1.0 x 10-<sup>7</sup>M to about 1.0 x 10<sup>-12</sup> M, when measured by binding to antigen either immobilized on solid phase or in solution phase. In certain embodiments, the antibodies of the invention possess affinities (K<sub>D</sub>) ranging from about 1 x 10<sup>-7</sup> M to about 6 x10<sup>-10</sup>M, when measured by binding to antigen either immobilized on solid phase or in solution phase. In certain embodiments, the antibodies of the invention possess affinities (K<sub>D</sub>) ranging from about 1 x 10<sup>-7</sup> M to about 9 x10<sup>-10</sup>M, when measured by binding to antigen either immobilized on solid phase or in solution phase.
The anti-RSV-F and/or anti-HMPV antibodies and antibody fragments disclosed herein encompass proteins having amino acid sequences 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 present 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 invention.
Two antigen-binding proteins, or antibodies, are considered bioequivalent if, for example, they are pharmaceutical equivalents or pharmaceutical 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 antibodies will be considered equivalents or pharmaceutical alternatives 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 effective body drug concentrations on, e.g., chronic use, and are considered medically insignificant for the particular drug product 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 embodiment, 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 therapy 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 mechanisms are known.
Bioequivalence 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 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.
Bioequivalent variants of the antibodies of the invention may be constructed by, for example, 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 bridges upon renaturation. In other contexts, bioequivalent antibodies may include antibody variants comprising amino acid changes, which modify the glycosylation characteristics of the antibodies, e.g., mutations that eliminate or remove glycosylation.
Biological and Biophysical Characteristics of the Antibodies
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof specifically bind to Respiratory Syncytial Virus (RSV) F protein (F), wherein at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3 amino acid sequences of such antibody or the antigen-binding fragment thereof is at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99% identical; or 100% identical, and/or all percentages of identity in between; to at least one of the CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and/or a CDRL3 amino acid sequences as disclosed in Table 6 of an antibody selected from Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, such antibodies also possess at least one, two, three, four, five, six, seven, eight, nine, ten, or more characteristics disclosed in the immediately following eleven paragraphs.
Without wishing to be bound by any theory, it is believed that the inventive antibodies and antigen-binding fragments thereof may function by binding to RSV-F, preferably in the PreF conformation, 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 function by binding to RSV-F and in so doing block the cell to cell spread of the virus and block syncytia formation associated with RSV infection of cells. Advantageously, both RSV subtype A and RSV subtype B are effectively blocked, or neutralized, by the majority of the anti-RSV antibodies disclosed herein.
In certain embodiments, the inventive antibodies and antigen-binding fragment thereof display better binding affinity for the PreF form of RSV-F relative to the PostF form of RSV-F.
In certain other embodiments, the inventive antibodies and antigen-binding fragments thereof advantageously display a clean or low polyreactivity profile (see, e.g., <patcit id="pcit0048" dnum="WO2014179363A"><text>WO 2014/179363</text></patcit> and <nplcit id="ncit0036" npl-type="s"><text>Xu et al., Protein Eng Des Sel, Oct;26(10):663-70</text></nplcit>), and are thus particularly amenable to development as safe, efficacious, and developable therapeutic and/or prophylactic anti-RSV and/or HMPV treatments.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof, without wishing to be bound by any theory, may function by blocking or inhibiting RSV fusion to the cell membrane by binding to any one or more of, e.g., antigenic Sites Ø, I, II, III, IV, or Site V of the PreF conformation of the F protein. In certain embodiments, the inventive antibodies display antigenic site specificity for Site Ø, Site V, or Site III of PreF relative to RSV-F Site I, Site II, or Site IV.
In certain embodiments, at least a portion of the epitope with which the inventive antibodies and antigen-binding fragments thereof interacts comprises a portion of the α3 helix and β3/β4 hairpin of PreF. In certain embodiments, substantially all of the epitope of such antibodies comprises the α3 helix and β3/β4 hairpin of PreF. In still further embodiments, the inventive antibodies cross-compete with antibodies that recognize a portion or substantially all of the α3 helix and β3/β4 hairpin of PreF.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof display an in vitro neutralization potency (IC<sub>50</sub>) of between about 0.5 microgram/milliliter (ug/ml) to about 5 ug/ml; between about 0.05 ug/ml to about 0.5 ug/ml; or less than about 0.05 mg/ml.
In certain embodiments, the binding affinity and/or epitopic specificity of the inventive antibodies and antigen-binding fragments thereof for any one of the RSV-F variants designated as 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG in <figref idref="f0010">Figure 7A</figref> is reduced or eliminated relative to the binding affinity and/or epitopic specificity of said antibody or antigen-binding fragment thereof for the RSV-F or RSV-F DS-Cav1.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof display a cross-neutralization potency (IC<sub>50</sub>) against human metapneumovirus (HMPV) as well as RSV. In certain such embodiments, the inventive antibodies and antigen-binding fragments thereof comprise at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3 amino acid sequences of such antibody or the antigen-binding fragment thereof is at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99% identical; or 100% identical; and/or all percentages of identity in between; to at least one of the CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and/or a CDRL3 amino acid sequences as disclosed in Table 6 of Antibody Number 340 as disclosed in Table 6.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof do not complete with D25, MPE8, palivisumab, motavizumab, or AM-14. In certain embodiments, the inventive antibodies and antigen-binding fragments thereof do not complete with D25, MPE8, palivisumab, or motavizumab. In certain embodiments, the inventive antibodies and antigen-binding fragments thereof do not complete with MPE8, palivisumab, or motavizumab. In certain embodiments, the inventive antibodies and antigen-binding fragments thereof do not complete with D25, palivisumab, or motavizumab. In certain embodiments, the inventive antibodies and antigen-binding fragments thereof do not complete with D25. In certain embodiments, the inventive antibodies and antigen-binding fragments thereof do not complete with MPE8. In certain embodiments, the inventive antibodies and antigen-binding fragments thereof do not complete with palivisumab. In certain embodiments, the inventive antibodies and antigen-binding fragments thereof do not complete with motavizumab.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof complete with one or more of D25, MPE8, palivisumab, motavizumab, and/or AM-14.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof display at least about 2-fold; at least about 3-fold; at least about 4-fold; at least about 5-fold; at least about 6-fold; at least about 7-fold; at least about 8-fold; at least about 9-fold; at least about 10-fold; at least about 15-fold; at least about 20-fold; at least about 25-fold; at least about 30-fold; at least about 35-fold; at least about 40-fold; at least about 50-fold; at least about 55-fold; at least about 60-fold; at least about 70-fold; at least about 80-fold; at least about 90-fold; at least about 100-fold; greater than about 100-fold; and folds in between any of the foregoing; greater neutralization potency (IC50) than D25 and/or palivizumab.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise the CDRH3 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise the CDRH2 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise the CDRH1 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise the CDRL3 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise the CDRL2 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise the CDRL1 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise any combination of two, three, four, five, or six characteristics disclosed in the immediately preceeding six paragraphs.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise a heavy chain (HC) amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise a light chain (LC) amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof are each selected from the group consisting antibodies that are at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99% identical; or 100% identical; and/or all percentages of identity in between; to any one of the antibodies designated as Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain embodiments, the inventive antibodies and antigen-binding fragments thereof comprise are each selected from the group consisting of the antibodies designated as Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
In certain embodiments, isolated nucleic acid sequences are provided that encode antibodies or antigen binding fragments thereof that specifically bind to Respiratory Syncytial Virus (RSV) F protein and antigen-binding fragments thereof, wherein at least one of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3 amino acid sequences of the antibody or the antigen-binding fragment thereof is at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99% identical; or at least 100% identical and/or all percentages of identity in between; to at least one the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3 amino acid sequences as disclosed in Table 6 of an antibody selected from Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences that are disclosed in Table 6, and compliments thereof.
In certain embodiments, isolated nucleic acid sequences are provided that encode the inventive antibodies and antigen-binding fragments thereof, wherein such nucleic acid sequences comprise sequences that encode the CDRH3 amino acid sequence of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences that are disclosed in Table 6, and compliments thereof.
In certain embodiments, isolated nucleic acid sequences are provided that encode the inventive antibodies and antigen-binding fragments thereof, wherein such nucleic acid sequences comprise sequences that encode the CDRH2 amino acid sequences of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences that are disclosed in Table 6, and compliments thereof.
In certain embodiments, isolated nucleic acid sequences are provided that encode the inventive antibodies and antigen-binding fragments thereof, wherein such nucleic acid sequences comprise sequences that encode the CDRH1 amino acid sequences of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences that are disclosed in Table 6, and compliments thereof.
In certain embodiments, isolated nucleic acid sequences are provided that encode the inventive antibodies and antigen-binding fragments thereof, wherein such nucleic acid sequences comprise sequences that encode the CDRL3 amino acid sequences of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences that are disclosed in Table 6, and compliments thereof.
In certain embodiments, isolated nucleic acid sequences are provided that encode the inventive antibodies and antigen-binding fragments thereof, wherein such nucleic acid sequences comprise sequences that encode the CDRL2 amino acid sequences of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences that are disclosed in Table 6, and compliments thereof.
In certain embodiments, isolated nucleic acid sequences are provided that encode the inventive antibodies and antigen-binding fragments thereof, wherein such nucleic acid sequences comprise sequences that encode the CDRL1 amino acid sequences of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences that are disclosed in Table 6, and compliments thereof.
In certain embodiments, isolated nucleic acid sequences are provided that encode the inventive antibodies and antigen-binding fragments thereof, wherein such nucleic acid sequences comprise sequences that encode the heavy chain (HC) amino acid sequences of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences that are disclosed in Table 6, and compliments thereof.
In certain embodiments, isolated nucleic acid sequences are provided that encode the inventive antibodies and antigen-binding fragments thereof, wherein such nucleic acid sequences comprise sequences that encode the heavy chain (LC) amino acid sequences of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6. In certain embodiments, such nucleic acid sequences are selected from those nucleic acid sequences that are disclosed in Table 6, and compliments thereof.
In certain embodiments, isolated nucleic acid sequences are provided that encode the inventive antibodies and antigen-binding fragments thereof, wherein such nucleic acid sequences comprise sequences are each selected from the group consisting of sequences that are at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99% identical; at least 100% identical, and/or all percentages of identity in between; to any one of the nucleic acid sequences that are disclosed in Table 6, and compliments thereof.
In certain embodiments, expression vectors are provided comprising the isolated nucleic acid sequences disclose herein and throughout, and in particular in the immediately preceding ten paragraphs.
In certain embodiments, host cells transfected, transformed, or transduced with the nucleic acid sequences and/or the expression vectors disclosed immediately above are provided.
Epitope Mapping and Related Technologies
As described above and as demonstrated in the EXAMPLES, Applicant has characterized the epitopic specificities, bin assignments, and antigenic site assignments of the inventive antibodies and antigen-binding fragments thereof. In addition to the methods for conducting such characterization, various other techniques are available to the artisan that can be used to carry out such characterization or to otherwise ascertain whether an antibody "interacts with one or more amino acids" within a polypeptide or protein. Exemplary techniques include, for example, a routine cross-blocking assay such as that described <nplcit id="ncit0037" npl-type="b"><text>Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY</text></nplcit>) can be performed. Other methods include alanine scanning mutational analysis, peptide blot analysis (<nplcit id="ncit0038" npl-type="s"><text>Reineke (2004) Methods Mol Biol 248:443-63</text></nplcit>), peptide cleavage analysis crystallographic studies and NMR analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (<nplcit id="ncit0039" npl-type="s"><text>Tomer (2000) Protein Science 9: 487-496</text></nplcit>). Another method that can be used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen/deuterium exchange detected 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 deuterium- labeled protein. Next, the protein/antibody complex is transferred to water and exchangeable protons within amino acids that are protected by the antibody complex undergo deuterium-to- hydrogen back-exchange at a slower rate than exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein/antibody interface may retain deuterium 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., <nplcit id="ncit0040" npl-type="s"><text>Ehring (1999) Analytical Biochemistry 267 (2):252-259</text></nplcit>; <nplcit id="ncit0041" npl-type="s"><text>Engen and Smith (2001) Anal. Chem. 73:256A-265A</text></nplcit>.
As the artisan will understand, an epitope can be formed both from contiguous 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, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes 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 Structure-based Antibody Profiling (ASAP) is a method that categorizes large numbers 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 (<patcit id="pcit0049" dnum="US20040101920A"><text>U.S. Publ. No. 2004/0101920</text></patcit>). Each category may reflect a unique epitope either distinctly different from or partially overlapping with epitope 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 produce mAbs having the desired characteristics. MAP may be used to sort the antibodies of the invention into groups of antibodies binding different epitopes.
In certain embodiments, the inventive antibodies and/or antigen-binding fragments thereof interact with an amino acid sequence comprising the amino acid residues that are altered in one or more of the F protein patch variants disclosed, e.g., in the EXAMPLES and which are depicted in, e.g., <figref idref="f0010">Figure 7A</figref> and which are designated as RSV F Variants 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG. In certain embodiments, such inventive antibodies and antigen-binding fragments thereof interact with an amino acid sequence comprising the amino acid residues that are altered in RSV F Variant 2. In certain embodiments, the inventive antibodies and/or antigen-binding fragments thereof interact with amino acid residues that extend beyond the region(s) 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 certain embodiments, the antibodies of the present invention do not bind to the same epitope on RSV-F protein as palivizumab, motavizumab, MPE8, or AM-14.
As the artisan understands, 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 routine methods available 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 peptide under saturating conditions. Next, the ability 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 epitope bound by the reference anti- RSV-F antibody of the invention.
To determine if an antibody 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 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 orientations, 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 epitope 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) binding of the other to the antigen. That is, a 1 -, 5-, 10-, 20- or 100-fold excess of one antibody inhibits 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., <nplcit id="ncit0042" npl-type="s"><text>Junghans et al., Cancer Res. (1990) 50:1495-1502</text></nplcit>). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations 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 binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. 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.
Immunoconjugates
The invention encompasses a human RSV-F monoclonal antibody conjugated to a therapeutic moiety ("immunoconjugate"), such as an agent that is capable of reducing the severity of primary infection with RSV and/or HMPV, or to ameliorate at least one symptom associated with RSV infection and/or HMPV infection, including coughing, fever, pneumonia, or the severity thereof. Such an agent may be a second different antibody to RSV-F and/or HMPV, or a vaccine. The type of therapeutic moiety that may be conjugated to the anti- RSV-F antibody and/or anti-HMPV antibody and will take into account the condition to be treated and the desired therapeutic effect to be achieved. Alternatively, if the desired therapeutic effect is to treat the sequelae or symptoms associated with RSV and/or HMPV infection, or any other condition resulting from such infection, such as, but not limited to, pneumonia, it may be advantageous to conjugate an agent appropriate to treat the sequelae or symptoms of the condition, 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, <patcit id="pcit0050" dnum="WO05103081A"><text>WO 05/103081</text></patcit>.
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 contain antigen-binding domains specific for more than one target polypeptide. See, e.g., <nplcit id="ncit0043" npl-type="s"><text>Tutt et al., 1991, J. Immunol. 147:60-69</text></nplcit>; <nplcit id="ncit0044" npl-type="s"><text>Kufer et al., 2004, Trends Biotechnol. 22:238-244</text></nplcit>. The antibodies of the present invention can be linked to or co- expressed 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 coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bi-specific or a multi-specific antibody with a second binding specificity.
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) C<sub>H</sub>3 domain and a second Ig C<sub>H</sub>3 domain, wherein the first and second Ig C<sub>H</sub>3 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 bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig C<sub>H</sub>3 domain binds Protein A and the second Ig C<sub>H</sub>3 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 C<sub>H</sub>3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second C<sub>H3</sub> 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 bi-specific antibody format described above are contemplated within the scope of the present invention.
Therapeutic Administration and Formulations
The invention provides therapeutic compositions (including pharmaceutical compositions) comprising the inventive anti-RSV-F and/or HMPV antibodies or antigen-binding fragments thereof. 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 appropriate formulations can be found in the formulary known to all pharmaceutical chemists: <nplcit id="ncit0045" npl-type="b"><text>Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA</text></nplcit>. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN<sup>™</sup>), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also <nplcit id="ncit0046" npl-type="s"><text>Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Pharm Sci Technol 52:238-31 1</text></nplcit>.
The dose of each of the antibodies 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 and/or HMPV infection in a patient, or for treating one or more symptoms associated with a RSV infection and/or HMPV infection, such as the cough or pneumonia associated with a RSV infection and/or HMPV in a patient, or for lessening the severity of the disease, it is advantageous to administer each of the antibodies of the present invention intravenously 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 weight. Multiple doses may be administered as necessary. 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 mg to about 600 mg, about 5 mg to about 300 mg, or about 10 mg 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 antibodies 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 composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., <nplcit id="ncit0047" npl-type="s"><text>Wu et al. (1987) J. Biol. Chem. 262:4429-4432</text></nplcit>). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings {e.g., oral mucosa, nasal mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. It may be delivered as an aerosolized formulation (See <patcit id="pcit0051" dnum="US20110311515A"><text>U.S. Publ. No. 2011/0311515</text></patcit> and <patcit id="pcit0052" dnum="US20120128669A"><text>U.S. Publ. No. 2012/0128669</text></patcit>). The delivery of agents useful for treating respiratory diseases by inhalation is becoming more widely accepted (See <nplcit id="ncit0048" npl-type="s"><text>A. J. Bitonti and J. A. Dumont, (2006), Adv. Drug Deliv. Rev, 58:1 106-1118</text></nplcit>). In addition to being effective at treating local pulmonary disease, such a delivery mechanism may also be useful for systemic delivery of antibodies (See <nplcit id="ncit0049" npl-type="s"><text>Maillet et al. (2008), Pharmaceutical Research, Vol. 25, No. 6, 2008</text></nplcit>).
The pharmaceutical composition can be also delivered in a vesicle, in particular a liposome (see, for example, <nplcit id="ncit0050" npl-type="s"><text>Langer (1990) Science 249:1527-1533</text></nplcit>).
In certain 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 target, thus requiring only a fraction of the systemic dose.
The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described 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 containing 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, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.
A pharmaceutical composition of the present invention can be delivered 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 replaceable 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 readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN<sup>™</sup> (Owen Mumford, Inc., Woodstock, UK), DISETRONIC<sup>™</sup> pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75/25<sup>™</sup> pen, HUMALOG<sup>™</sup> pen, HUMALIN 70/30<sup>™</sup> pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN<sup>™</sup> I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR<sup>™</sup> (Novo Nordisk, Copenhagen, Denmark), BD<sup>™</sup> pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN<sup>™</sup>, OPTIPEN PRO<sup>™</sup>, OPTIPEN STARLET<sup>™</sup>, and OPTICLIK<sup>™</sup> (Sanofi-Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to the SOLOSTAR<sup>™</sup> pen (Sanofi-Aventis), the FLEXPEN<sup>™</sup> (Novo Nordisk), and the KWIKPEN<sup>™</sup> (Eli Lilly), the SURECLICK<sup>™</sup> Autoinjector (Amgen, Thousand Oaks, CA), the PENLET<sup>™</sup> (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.) and the HUMIRA<sup>™</sup> 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, capsules, injections (ampoules), 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 aforesaid 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, multiple doses of an antibody to RSV-F and/or HMPV, or a pharmaceutical composition comprising or encoding for these antibodies, 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 antibody to RSV-F and/or HMPV, or sequentially administering to a subject multiple doses of a pharmaceutical composition comprising or encoding for an antibody of the invention or antigen binding fragment therof. As used herein, "sequentially administering" means that each dose of antibody to RSV-F and/or HMPV, or the pharmaceutical composition, is administered 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 includes methods which comprise sequentially administering to the patient a single initial dose of an antibody to RSV-F and/or HMPV, or a composition comprising or encoding for the antibodies, followed by one or more secondary doses of the antibody to RSV-F and/or HMPV, or the composition, and optionally followed by one or more tertiary doses of the antibody to RSV-F and/or HMPV, or the composition.
The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal sequence of administration of the antibody to RSV-F and/or HMPV or the compositions of the invention. 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 and/or HMPV, but generally may differ from one another in terms of frequency of administration. In certain embodiments, however, the amount of antibody to RSV-F and/or HMPV contained in the initial, secondary and/or tertiary doses vary from one another (e.g., adjusted up or down as appropriate) during the course of treatment. 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., "maintenance doses").
In one exemplary embodiment of the present invention, each secondary and/or tertiary dose is administered 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 preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose of antibody to RSV-F and/or HMPV which is administered 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 and/or HMPV. For example, in certain embodiments, 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 patient. 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 involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, 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 regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
Accordingly, in certain embodiments are provided pharmaceutical compositions comprising: one or more of the inventive antibodies or antigen-binding fragments thereof disclosed herein and throughout and a pharmaceutically acceptable carrier and/or one or more excipients. In certain other embodiments are provided pharmaceutical compositions comprising: one or more nucleic acid sequences encoding one or more inventive antibodies or antigen-binding fragments thereof; or one or more the expression vectors harbouring such nucleic acid sequences; and a pharmaceutically acceptable carrier and/or one or more excipients.
Therapeutic Uses of the Antibodies and Pharmaceutical Compositions
Due to their binding to and interaction with the RSV fusion protein (RSV-F), it is believed that the inventive antibodies and antigen-binding fragments thereof are useful - without wishing to be bound to any theory - for preventing fusion of the virus with the host cell membrane, for preventing cell to cell virus spread, and for inhibition of syncytia formation. Additionally, as Applicant has demonstrated herein that, surprisingly, a subset of the inventive anti-RSV antibodies and antigen-binding fragment thereof display crass-neutralizing potency agains HMPV, the inventive antibodies and antigen-binding fragments thereof are advantageous for preventing an infection of a subject with RSV and/or HMPV 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 infection. The antibodies of the invention are also contemplated for prophylactic use in patients at risk for developing or acquiring an RSV infection and/or HMPV infection. These patients include pre-term infants, full term infants born during RSV season (late fall to early spring), the elderly (for example, in anyone 65 years of age or older) and/or HMPV season, 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, patients with airway impairment, patients with COPD) and/or HMPV infection. It is contemplated that the antibodies of the invention may be used alone, or in conjunction with a second agent, or third agent for treating RSV infection and/or HMPV infection, or for alleviating at least one symptom or complication associated with the RSV infection and/or HMPV 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 antibodies 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 antigen, such as RSV-G, a vaccine against RSV, an siRNA specific for an RSV antigen.
In yet a further embodiment of the invention the present antibodies are used for the preparation of a pharmaceutical composition for treating patients suffering from a RSV infection and/or HMPV 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 and/or HMPV, or for reducing the duration of the infection, or for reducing at least one symptom associated with the RSV infection and/or the HMPV infection. In a further embodiment of the invention the present antibodies are used as adjunct therapy with any other agent useful for treating an RSV infection and/or and HMPV infectin, including an antiviral, a toxoid, a vaccine, a second RSV-F antibody, or any other antibody specific for an RSV antigen, including an RSV-G antibody, or any other palliative therapy known to those skilled in the art.
Accordingly, in certain embodiments are provided methods of treating or preventing a Respiratory Syncytial Virus (RSV) infection, or at least one symptom associated with RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof one or more of the inventive antibodies or antigen-binding fragments thereof disclosed herein and throughout (or pharmaceutical composition of the invention), such as, e.g., one or more of the anti-RSV antibodies disclosed in Table 6, such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity.
In certain other embodiments are provided methods of treating or preventing a Respiratory Syncytial Virus (RSV) infection, or at least one symptom associated with RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof a nucleic acid sequence encoding one or more of the inventive antibodies or antigen-binding fragments thereof, such nucleic acid sequenced disclosed in Table 6 and compliments thereof, such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity.
In additional embodiments are provided methods of treating or preventing a Respiratory Syncytial Virus (RSV) infection, ar at least one symptom associated with RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof a host cell harboring a nucleic acid sequence or an expression vector comprising such a nucleic aci sequence, wherein such nucleic acid sequences is selected from the group consisiting of sequences disclosed in Table 6 and compliments thereof, such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity.
In additional embodiments are provided methods of treating or preventing a Respiratory Syncytial Virus (RSV) infection, ar at least one symptom associated with RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof a pharmaceutical composition comprising either: one or more of the inventive antibodies or antigen-binding fragments thereof as disclosed in Table 6; one or more nucleic acid sequences or an expression vectors comprising such a nucleic acid sequence, wherein such nucleic acid sequences are selected from the group consisting of sequences disclosed in Table 6 and compliments thereof; one or more host cells harboring one or more nucleic acid sequences or an expression vectors comprising suchone or more nucleic acid sequences, wherein such nucleic acid sequences are selected from the group consisting of sequences disclosed in Table 6 and compliments thereof; and a pharmaceutically acceptable carrier and/or one or more excipients, such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity.
In certain embodiments ase provided methods of treating or preventing either a Respiratory Syncytial Virus (RSV) infection or a human metapneumovirus (HMPV) infection, ar at least one symptom associated with said RSV infection or said HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof one or more of the inventive antibodies or antigen-binding fragments thereof disclosed herein and throughout, such as, e.g., one or more of the anti-RSV antibodies disclosed in Table 6, such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity. In certain embodiments, the one or more antibodies or antigen-binding fragments thereof comprises Antibody Number 340 as disclosed in Table 6.
In certain other embodiments are provided methods of treating or preventing either a Respiratory Syncytial Virus (RSV) infection or a human metapneumovirus (HMPV) infection, ar at least one symptom associated with said RSV infection or said HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereo a nucleic acid sequence encoding one or more of the inventive antibodies or antigen-binding fragments thereof, such nucleic acid sequenced disclosed in Table 6 and compliments thereof, such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity. In certain embodiments, the one or more antibodies or antigen-binding fragments thereof comprises Antibody Number 340 as disclosed in Table 6.
In additional embodiments are provided methods of treating or preventing either a Respiratory Syncytial Virus (RSV) infection or a human metapneumovirus (HMPV) infection, ar at least one symptom associated with said RSV infection or said HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof a host cell harboring a nucleic acid sequence or an expression vector comprising such a nucleic acid sequence, wherein such nucleic acid sequences is selected from the group consisiting of sequences disclosed in Table 6 and compliments thereof, such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity. In certain embodiments, the one or more antibodies or antigen-binding fragments thereof comprises Antibody Number 340 as disclosed in Table 6.
In additional embodiments are provided methods of treating or preventing either a Respiratory Syncytial Virus (RSV) infection or a human metapneumovirus (HMPV) infection, ar at least one symptom associated with said RSV infection or said HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof a pharmaceutical composition comprising either: one or more of the inventive antibodies or antigen-binding fragments thereof as disclosed in Table 6; one or more nucleic acid sequences or an expression vectors comprising such a nucleic acid sequence, wherein such nucleic acid sequences are selected from the group consisting of sequences disclosed in Table 6 and compliments thereof; one or more host cells harboring one or more nucleic acid sequences or an expression vectors comprising suchone or more nucleic acid sequences, wherein such nucleic acid sequences are selected from the group consisiting of sequences disclosed in Table 6 and compliments thereof; and a pharmaceutically acceptable carrier and/or one or more excipients, such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity. In certain embodiments, the one or more antibodies or antigen-binding fragments thereof comprises Antibody Number 340 as disclosed in Table 6.
Combination Therapies
As noted above, according to certain embodiments, the disclosed methods comprise administering to the subject one or more additional therapeutic agents in combination with an antibody to RSV-F and or HMPV. As used herein, the expression "in combination with" means that the additional therapeutic agents are administered before, after, or concurrent with the pharmaceutical composition 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, 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 anti-RSV-F antibodies. Administration "concurrent" or with the pharmaceutical composition comprising the anti-RSV-F antibody means that the additional therapeutic 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 therapeutic agent and the anti-RSV-F antibody.
Combination therapies may include an anti-RSV-F antibody of the invention and any additional therapeutic 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 conjunction 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.
Diagnostic Uses of the Antibodies
The inventive anti-RSV antibodies and antigen-binding fragments thereof may also be used to detect and/or measure RSV and/or HMPV in a sample, e.g., for diagnostic purposes. It is envisioned that confirmation of an infection thought to be caused by RSV and/or HMPV may be made by measuring the presence of the virus through use of any one or more of the antibodies of the invention. Exemplary diagnostic assays for RSV and/or HMPV may comprise, e.g., contacting a sample, obtained from a patient, with an anti-RSV-F and/or HMPV antibody of the invention, wherein the anti-RSV-F and/or HMPV antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate the virus containing the F protein from patient samples. Alternatively, an unlabeled anti-RSV-F and/or HMPV antibody can be used in diagnostic applications in combination with a secondary antibody which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as <sup>3</sup>H, <sup>14</sup>C, <sup>32</sup>P, <sup>35</sup>S, or <sup>125</sup>l; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure RSV containing the F protein and/or HMPV in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).
Samples that can be used in RSV and/or HMPV diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient, which contains detectable quantities of RSV-F protein and/or HMPV, or fragments thereof, under normal or pathological conditions. Generally, levels of RSV-F and/or HMPV in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease or condition associated with the presence of RSV-F and/or HMPV) will be measured to initially establish a baseline, or standard, level of the F protein from RSV and/or HMPV. This baseline level of RSV-F and/or HMPV can then be compared against the levels of RSV-F and/or HMPV measured in samples obtained from individuals suspected of having an RSV and/or HMPV infection, or symptoms associated with such infection.
EXAMPLES
Applicant has comprehensively profiled the human antibody response to RSV fusion protein (F) by isolating and characterizing 133 RSV F-specific monoclonal antibodies from the memory B cells of a healthy adult donor, and used these antibodies to comprehensively map the antigenic topology of RSV F. The antibody response to RSV F was determined to be comprised of a broad diversity of clones that target several antigenic sites. Nearly half of the most potent antibodies target a previously undefined site of vulnerability near the apex of the prefusion conformation of RSV F (preF), providing strong support for the development of RSV antibodies that target this region, as well as vaccine candidates that preserve the membrane-distal hemisphere of the preF protein. Additionally, this class of antibodies displayed convergent sequence features, thus providing a future means to rapidly detect these types of antibodies in human samples. Many of the antibodies that bound preF-specific surfaces from this donor were over 100 times more potent than palivizumab, and one antibody cross-neutralized human metapneumovirus (HMPV). Taken together, the results have implications for the design and evaluation of RSV vaccine and antibody-based therapeutic candidates, and offer new options for passive prophylaxis.
Large-scale isolation of RSV F-specific monoclonal antibodies from healthy adult human donors
In order to comprehensively profile the human antibody response to RSV F, Applicant isolated and characterized 133 monoclonal antibodies from the memory B cells of a healthy adult donor ("donor 006"). Although this donor did not have a documented history of RSV infection, healthy adults are expected to have had multiple RSV infections throughout life <i>(26).</i>
The magnitude of the memory B cell response in this donor to RSV F was assessed by staining peripheral B cells with a mixture of fluorescently labeled pre- and postfusion RSV F sorting probes (<figref idref="f0009">Figure 6A through 6B</figref>) <i>(11, 15).</i> Both proteins were dual-labeled in order to eliminate background due to non-specific fluorochrome binding (27). Flow cytometric analysis revealed that 0.04-0.18% of class-switched (IgG<sup>+</sup> and IgA<sup>+</sup>) peripheral B cells were specific for RSV F (<figref idref="f0001">Figure 1A and Figure B</figref>), which is significantly lower than the percentage of RSV F-specific cells observed after experimental RSV infection and suggests that this donor was probably not recently exposed to RSV <i>(28).</i> Notably, index sorting showed that 17-38% of circulating RSV F-specific B cells express IgA, indicating that IgA memory B cells to RSV F are present in peripheral blood (<figref idref="f0001">Figure 1B</figref>).
Approximately 200 RSV F-specific B cells were single-cell sorted from the donor sample, and antibody variable heavy (VH) and variable light (VL) chain genes were rescued by single-cell PCR <i>(29).</i> One hundred thirty-three (133) cognate heavy and light chain pairs were subsequently cloned and expressed as full-length IgGs in an engineered strain of <i>Saccharomyces cerevisiae</i> for further characterization <i>(30).</i> Preliminary binding studies showed that approximately 80% of antibodies cloned from RSV F glycoprotein (F)-specific B cells bound to recombinant RSV F proteins.
Sequence analysis of RSV F-specific antibody repertoires
Sequence analysis of the isolated monoclonal antibodies revealed that the RSV-F specific repertoire was highly diverse, containing over 70 unique lineages (<figref idref="f0002">Fig. 1C</figref> and Table 2). This result is in stark contrast to the relatively restricted repertoires observed in HIV-infected patients (<i>31</i>), or in healthy donors after influenza vaccination <i>(32).</i> Compared to non-RSV-reactive antibodies <i>(33),</i> the RSV F-specific repertoires were skewed, generally, toward certain VH germline genes (VH1-18, VH1-2, VH1-69, VH2-70, VH4-304, and VH5-51) (<figref idref="f0002">Fig. 1D</figref> and Table 2) and longer heavy chain third complementarity-determining region (CDRH3) lengths (generally, approximately 14-18 amino acids in length; Fig. IE and Table 2). Interestingly, a bias toward VH1-69 has also been observed in anti-HIV-1, anti-influenza, and anti-HCV repertoires <i>(34-36),</i> and recent studies have shown that there is a significant increase in the relative usage of VH1-18, VH1-2, and VH1-69 during acute dengue infection <i>(37).</i> Hence, it appears that these particular germline gene segments may have inherent properties that facilitate recognition of viral envelope proteins.
The average level of somatic hypermutation (SHM) ranged generally between 16 and 30 nucleotide substitutions per VH gene (excluding CDRH3) (<figref idref="f0002">Fig. 1F</figref> and Table 2), which is comparable to the average level of SHM observed in anti-influenza antibody repertoires <i>(32, 38)</i> and consistent with the recurrent nature of RSV infection <i>(26).</i> Interestingly, several antibodies contained 40 or greater VH gene nucleotide substitutions, suggesting that multiple rounds of RSV infection can result in antibodies with very high levels of somatic hypermutation (SHM).
A large proportion of antibodies bind exclusively to preF
We next measured the apparent binding affinities of the IgGs to furin-cleaved RSV F ectodomains stabilized in the prefusion (DS-Cav1) or postfusion (F ΔFP) conformation using biolayer interferometry (<i>11</i>, <i>15</i>). A relatively large proportion of the antibodies (36-67%) bound exclusively to preF (<figref idref="f0003">Figure 2A and Figure B</figref>; Table 3). The vast majority of remaining antibodies bound to both pre- and postF, with only 5-7% of antibodies showing exclusive postF specificity (<figref idref="f0003">Figure 2A and Figure B</figref>; Table 3). The low prevalence of postF-specific antibodies in these donor repertoires is consistent with the observation that less than 10% of anti-RSV F serum-binding activity specifically targets postF <i>(8).</i> Interestingly, however, the majority of cross-reactive antibodies bound with higher apparent affinity to postF (<figref idref="f0003">Figure 2A</figref>; Table 3), suggesting that these antibodies were probably elicited by and/or affinity matured against postF in vivo. Hence, the significantly higher proportion of preF- versus postF-specific antibodies is likely due to the higher immunogenicity of the unique surfaces on preF compared to postF, rather than an increased abundance of preF in vivo. Finally, as expected based on the relatively high degree of sequence conservation between RSV subtypes, most of the antibodies showed binding reactivity to F proteins derived from both subtypes A and B (<figref idref="f0003">Figure 2C</figref>; Table 3).
Since certain antiviral antibody specificities have been associated with poly- and autoreactivity <i>(39-41),</i> we also tested the RSV antibodies for polyreactivity using a previously described high-throughput assay that correlates with down-stream behaviors such as serum clearance <i>(42, 43).</i> One hundred and seventy-seven clinical antibodies, as well as several broadly neutralizing HIV antibodies, were also included for comparison. Interestingly, in contrast to many previously described HIV broadly neutralizing antibodies, the vast majority of RSV F-specific antibodies lacked significant polyreactivity in this assay (<figref idref="f0003">Figure 2D</figref>).
RSV F-specific antibodies target six major antigenic sites
To map the antigenic specificities of the RSV F-specific antibodies, Applicant first performed competitive binding experiments using a previously described yeast-based assay <i>(44).</i> Antibodies were initially tested for competition with D25, AM14 and MPE8-three previously described preF-specific antibodies (<i>10, 17</i>, <i>21</i>)-and motavizumab, an affinity-matured variant of palivizumab that binds to both pre- and postF (<i>10, 11, 45</i>). Non-competing antibodies were then tested for competition with a site IV-directed mAb (101F) <i>(46),</i> a site I-directed antibody (Site I Ab), and two high affinity antibodies (High Affinity Ab I and High Affinity Ab 2, respectively) that did not strongly compete with each other or any of the control antibodies. Each antibody was assigned a bin based on the results of this competition assay (see, e.g., Table 4).
In order to confirm and increase the resolution of our epitope assignments, the binding of each antibody to a panel of preF variants was measured using a luminex-based assay. Each variant contained 2-4 mutations clustered together to form a patch on the surface of preF. A total of nine patches that uniformly covered the surface of preF were generated (<figref idref="f0010">Figure 7A through Figure 7C</figref>). Deglycosylated preF was also included to identify antibodies targeting glycan-dependent epitopes. Binding of each antibody to the 10 preF variants was compared to that of wild-type preF and used to assign a patch (see, e.g., Table 4). Previously characterized antibodies D25, AM14 and motavizumab were used to validate the assay (se, e.g., <figref idref="f0010">Figure 7C</figref> and Table 4). The combined bin and patch data were then used to assign each antibody to a single antigenic site (<figref idref="f0004 f0005">Figure 3A through Figure 3G</figref>), which were defined based on previously determined structures, resistance mutations, and secondary structure elements of the F protein. Overall, these data show that the large majority of isolated antibodies target six dominant antigenic sites on prefusion RSV F (Ø, I, II, III, IV, and V). Interestingly, only a small proportion of the isolated antibodies had binding profiles similar to that of AM14, suggesting that antibodies targeting this quaternary epitope are not commonly elicited during natural infection. None of the antibodies were sensitive to deglycosylation of F, demonstrating that glycan-dependent antibodies are also rarely elicited by natural RSV infection.
Analysis of the preF- and postF-binding activities of the antibodies targeting each antigenic site (see, e.g., <figref idref="f0004 f0005">Figure 3C through Figure 3G</figref>; Table 4) revealed that three sites are primarily found on preF (Ø, III, and V). Antibodies targeting site Ø and site III have been previously described (<i>10, 17</i>), and these sites are located on the top and side of the preF spike, respectively. Approximately 18% of the antibodies from this donor recognized site Ø and approximately 20% recognized site III. A relatively large proportion of antibodies from this donor (approximately 26%) recognized the third preF-specific site, which has not been previously described and therefore has been designated herein as region site V (See, e.g., <figref idref="f0004 f0005">Figure 3C through Figure 3G</figref>; Table 4). The majority of site V antibodies competed with D25, MPE8 and motavizumab, which was unexpected given the distance between the epitopes recognized by these three antibodies. The patch mutant analysis revealed that these antibodies interact with the α3 helix and β3/β4 hairpin of preF. This region is located between the epitopes recognized by D25, MPE8, and motavizumab, explaining the unusual competition profile observed for this class of antibodies (See, e.g., <figref idref="f0011">Figure 8</figref>). In addition to the three primarily preF-specific sites, a large number of the antibodies that recognized antigenic site IV were preF-specific, likely due to contacts with β22, which dramatically rearranges during the transition from pre- to postF. In summary, the epitope mapping data show that the large majority of isolated antibodies target six dominant antigenic sites, approximately half of which are exclusively expressed on preF.
Highly potent neutralizing antibodies target preF-specific epitopes
The antibodies were next tested for neutralizing activity against RSV subtypes A and B using a previously described high-throughput neutralization assay <i>(15).</i> Greater than 70% of the isolated antibodies showed neutralizing activity, and approximately 35% - 40% neutralized with high potency (IC<sub>50</sub> ≤ 0.05 µg/ml) (see, e.g., <figref idref="f0006">Figure 4A and Figure 4B</figref>; Table 3). Notably, several clonally unrelated antibodies were ≥ 5.0-fold more potent than D25 and ≥ 100-fold more potent than palivizumab (see, e.g., <figref idref="f0006">Figure 4A</figref>; Table 3). Interestingly, there was no correlation between neutralization potency and level of SHM, suggesting that extensive SHM is not required for potent neutralization of RSV. Consistent with the binding cross-reactivity data, the majority of neutralizing antibodies showed activity against both subtype A and B (<figref idref="f0006">Figure 4A through Figure 4C</figref>; Table 3).
The relationship between preF- and postF-binding affinity and neutralization potency was next investigated, which clearly demonstrated that the majority of highly potent antibodies bound preferentially or exclusively to preF (see, e.g., <figref idref="f0007">Figure 4D through Figure 4G</figref>; Table 3). Quantifying this difference revealed that more than 80% of highly potent antibodies (IC<sub>50</sub> < 0.05 µg/ml) were specific for preF (See, e.g., <figref idref="f0012">Figure 9</figref>; Table 3) and that the median IC<sub>50</sub> for preF-specific antibodies was more than 8-fold lower than for pre- and postF cross-reactive antibodies and 80-fold lower than antibodies that specifically recognized postF (see, e.g., <figref idref="f0007">Figure 4E</figref>; Table 3). Importantly, there was a positive correlation between preF binding and neutralization (<i>P</i><0.001, r=0.24), and the apparent preF <i>K</i><sub>D</sub>s generally corresponded well with the neutralization IC<sub>50</sub>s (see, e.g., <figref idref="f0008">Figure 5A</figref>; Table 3). In contrast, there was no correlation between neutralization potency and postF affinity (<i>P</i>=0.44, r=-0.07) (see, e.g., <figref idref="f0008">Figure 5B</figref>; Table 3). This result is compatible with the occupancy model of antibody-mediated neutralization <i>(47),</i> and suggests that DS-Cav1 is a faithful antigenic mimic of the native preF trimer. Notably, very few antibodies neutralized with IC<sub>50</sub>s lower than 100 pM, which is consistent with the previously proposed ceiling to affinity maturation <i>(48, 49).</i>
The relationship between neutralization potency and antigenic site was next analyzed. The results, provided in, e.g., <figref idref="f0008">Figure 5C</figref>, Table 3, and Table 4, collectively, indicated that over 60% of the highly potent neutralizing antibodies targeted antigenic sites Ø and V, which are two of the three prefusion-F specific sites. In contrast, antibodies targeting sites III and IV showed a wide range of neutralization potencies, and antibodies targeting sites I and II were generally moderate to non-neutralizing. Similar results were obtained using binding affinities and neutralization potencies measured for subtype B (See, e.g., <figref idref="f0013">Figure 10A through Figure 10C</figref>; Table 3 and Table 4). Interestingly, a subset of site IV-directed antibodies neutralized with substantially lower potency than would be expected based on preF binding affinity (see, e.g., <figref idref="f0008">Figure 5A</figref>; Table 3). This result may suggest that certain epitopes within site IV are less exposed in the context of the native envelope spike expressed on the crowded surface of the virion than on recombinant preF.
Several antibodies cross-neutralize RSV and HMPV
Given that the RSV and human metapneumovirus (HMPV) F proteins share 33% amino acid identity, and certain RSV F-specific antibodies cross-neutralize HMPV (<i>17</i>, <i>50</i>), the antibodies from this donor were tested for neutralizing activity against HMPV. Of the 133 antibodies tested, one neutralized HMPV (see, e.g., Table 5). Sequence analysis revealed that this antibody represents the VH1-46 germline gene and contains a significant degree of somatic hypermutation (See, e.g., Table 2 and sequence listing). This cross-neutralizing antibody bound to both the preF and PostF and competed with MPE8 (See, e.g., Table 5), in agreement with previous studies indicating that MPE8 cross-neutralizes four pneumoviruses, including RSV and HMPV <i>(17).</i> This result suggests, inter alia, that highly conserved epitopes are relatively immunogenic in the context of natural RSV and/or HMPV infection.
Affinity maturation of RSV F-specific antibodies:
Some embodiments refer to affinity matured antibodies of any of the antibodies listed in Table 6 (each understood as a "parent" antibody" for producing an affinity matured variant). Affinty matured antibodies may be produced by mutagenesis of any one or more of the CDRs of the parent antibody. According to a specific embodiment, the invention provides for affinity matured variants comprising one or more point mutations e.g., 0, 1, 2, or 3 point mutations in each of the CDR sequences, of any of the antibodies listed in Table 6, or of an antibody comprising the six CDR sequences of any of the antibodies listed in Table 6. Affinity matured variants can be produced by any affinity maturation method employing standard mutagenesis techniques, <i>e.g.,</i> for optimizing the binding characteristics, such as increasing affinity of binding, or increasing Kon, or decreasing Koff, and can be characterized by a K<sub>D</sub> difference of at least 2 fold, 5 fold, 1 log, or 2 logs, or 3 logs, as compared to the parent antibody. Such affinity matured antibodies still have the same binding specificity as the parent antibody and <i>e.g.,</i> an optimized affinity of binding the target epitope.
Selected anti RSV antibodies were identified for affinity maturation. Oligos were ordered which comprised CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences that were variegated via NNK diversity. The NNK oligos were incorporated into the parent HC or LC via DNA shuffling, as described previously (<nplcit id="ncit0051" npl-type="s"><text>Stemmer WP et al., DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22): 10747-51</text></nplcit>). The library was then created by transforming the VH and VL PCR products into yeast already containing either the light chain or heavy chain plasmid of the parent. The diversified libraries were then selected using flow cytometry. For each FACS round, the libraries were affinity pressured using decreasing amounts of antigen and clones with improved binding affinities were sorted and propagated. Once improved binding populations were observed by flow cytometry (typically two rounds of selection), single yeast clones were be picked for sequencing and characterization (Table 6).
A specific embodiment refers to affinity matured variants of the antibody 267 in Table 6. Notably, each of the antibodies numbered 365-372 is an affinity matured variant of the antibody numbered 267 in Table 6.
Antibody production and purification of affinity matured antibodies
Yeast clones were grown to saturation and then induced for 48 h at 30°C with shaking. After induction, yeast cells were pelleted and the supernatants were harvested for purification. IgGs were purified using a Protein A column and eluted with acetic acid, pH 2.0. Fab fragments were generated by papain digestion and purified over KappaSelect (GE Healthcare LifeSciences).
RSV in vitro neutralization in ELISA based Microneutralization Assays
<i>In vitro</i> RSV neutralization was tested in ELISA based Microneutralization Assays using RSV-A strain A2 (ATCC, VR1540P). Virus (at a final multiplicity of infection of approximately 0.25) was added to 96-well plates containing serially diluted mAbs in serum-free MEM and pre-incubated for 30 min at 4°C. Freshly trypsinized Hep-2 cells (1.5 x 10E<sup>4</sup> cells/well) were then added to each well in MEM supplemented with 5% FCS. Following incubation for 4 days at 37°C and 5% CO<sub>2</sub>, medium was aspirated and cells were washed twice with 200 µl PBS/well, air-dried and fixed with 100 µl Acetone (80%). RSV replication was measured by quantification of expressed viral proteins by ELISA. For this purpose, fixed cells were washed 2x times with PBS-0.1% Tween-20, blocked with 1% skimmed milk in PBS for 1 hour at RT and then stained with a polyclonal goat-anti RSV antibody preparation (BioRad, #7950-0004) for 1 hour at RT in blocking buffer. A donkey anti-goat IgG HRP conjugate was used as detection reagent and 1 step-Ultra TMB (Thermo Fisher Scientific, #34209) as substrate. % inhibition of virus replication was calculated relative to control cells infected with virus in absence of neutralizing antibodies. An isotype matched control mAb was included in all experiments. mAb potency is expressed as half-maximal inhibitory concentration that resulted in 50% reduction in virus replication (IC<sub>50</sub>). Results are provided in <figref idref="f0014">Figure 11</figref> and demonstrate that all mAbs were able to neutralize RSV-A2 in this setting, with a broad range of IC<sub>50</sub> values.
Discussion
An in-depth understanding of the human antibody response to RSV infection will aid the development and evaluation of RSV vaccine and therapeutic and/or prophylactic antibody candidates for the treatment and/or prevention of RSV infection. Although previous studies have coarsely mapped the epitopes targeted by RSV-specific neutralizing antibodies in human sera <i>(4, 8),</i> the specificities and functional properties of antibodies induced by natural RSV infection have remained largely undefined. As disclosed herein, preF- and postF-stabilized proteins (<i>11, 15</i>), a high-throughput antibody isolation platform, and a structure-guided collection of prefusion F mutants, were used to clonally dissect the human memory B cell response to RSV F in a naturally infected adult donor, and highly potent and selective RSV-neutralizing - as well as highly potent anti-RSV/anti-HMPV cross-selective and cross-neutralizing - were isolated and characterized.
In the repertoire analyzed, the ratio of preF-specific antibodies to those that recognize both pre- and postF was slightly greater than 1:1 (See, e.g., <figref idref="f0003">Figure 2B</figref>). These values are somewhat lower than those reported for human sera, which showed approximately 70% of anti-F serum binding is specific for preF <i>(8).</i> This discrepancy may be the result of differences between the levels of individual antibodies in serum, differences in the B cell phenotypes achieved for a particular specificity, or variation between donors. Despite these minor differences, the results of both studies suggest that preF-specific epitopes and epitopes shared by pre- and postF are immunogenic during natural RSV infection, whereas the unique surfaces on postF are significantly less immunogenic.
The repertoire analysis disclosed herein revealed that the large majority of RSV F-specific antibodies target six dominant antigenic sites on prefusion RSV F: Ø, I, II, III, IV, and V. These these sites were defined based on previously determined structures, epitope binning/competition assays, resistance mutations, and secondary structure elements of the preF protein. It is important to note that the nomenclature for describing RSV F antigenic sites has evolved over time (<i>6</i>, <i>51-57</i>), and previous mapping efforts were based on the postfusion conformation of F and did not include surfaces present exclusively on preF. The crystal structure of preF has provided critical information about F structure and function as well as new reagents to map antibody binding sites on the unique surfaces of preF and surfaces shared with postF. To a first approximation, each antibody can be assigned primarily to one of these sites. However, it is likely that antibody epitopes cover the entire surface of F and that there are antibodies that bind two or more adjacent antigenic sites within a protomer and quaternary antibodies that bind across protomers.
Importantly, the results disclosed herein show that the most potently neutralizing antibodies target antigenic sites Ø and V, both of which are located near the apex of the preF trimer. These findings are consistent with results obtained from human sera mapping, which determined that the majority of neutralizing activity can be removed by pre-incubation with preF <i>(4, 8)</i> and that preF-specific sites other than site Ø make up a considerable fraction of preF-specific neutralizing antibodies <i>(8).</i> Although antigenic site Ø has been shown to be a target of potently neutralizing antibodies (<i>8</i>, <i>10</i>), the interaction of antibodies with site V is less well understood. Interestingly, it was found that the majority of site V-directed antibodies share several convergent sequence features, suggesting that it may be possible to rapidly detect these types of antibodies in human samples using high-throughput sequencing technology <i>(58).</i> Applicant anticipates this finding to be particularly advantageous in profiling antibody responses to RSV vaccine candidates that aim to preserve the apex of the preF trimer.
The extensive panel of antibodies described here provides new opportunities for passive prophylaxis, as well as for treatment of RSV infection. A large number of these antibodies neutralize RSV more potently than D25, which serves as the basis for MEDI8897-a monoclonal antibody that is currently in clinical trials for the prevention of RSV in young, at risk children (59). Additionally, a subset of these antibodies were demonstrated to cross-neutralize HMPV.
The development of an effective RSV vaccine has presented a number of unique challenges, and selection of the optimal vaccination strategy will be of the utmost importance. The in-depth analysis of the human antibody response to natural RSV infection presented here provides insights for the development of such a vaccine. Importantly, the results suggest that immunization of pre-immune donors with preF immunogens would be expected to boost neutralizing responses, whereas the use of postF immunogens would likely expand B cell clones with moderate or weak neutralizing activity. Similarly, immunization of RSV naive infants with preF immunogens would be expected to activate naive B cells targeting epitopes associated with substantially more potent neutralizing activity compared to postF immunogens. In addition, the ideal RSV vaccine should preserve antigenic sites Ø and V, since these sites are targeted by the most highly potent antibodies elicited in response to natural RSV infection.
Accordingly, disclosed herein are highly selective and potent anti-RSV antibodies, as well as highly potent cross-neutralizing anti-RSV and anti-HMPV antibodies, as well as vaccine candidates, for the treatment and or prophylaxis of RSV and/or HMPV infection. Additionally, the reagents disclosed here provide a useful set of tools for the evaluation of clinical trials, which will be critical for selecting the optimal RSV vaccination or antibody-based therapeutic strategy from the many currently under investigation (<i>60</i>). <tables id="tabl0001" num="0001"><table frame="all"><title><b>Table 1: Antigenic sites targeted by prototypic RSV antibodies</b></title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="45mm" /><colspec colnum="2" colname="col2" colwidth="55mm" /><thead valign="top"><row><entry>Antigenic site</entry><entry>Prototypic antibodies</entry></row></thead><tbody><row><entry>Ø</entry><entry>D25, 5C4, AM22 (<i>10,16</i>)</entry></row><row><entry>I</entry><entry>131-2a, 2F</entry></row><row><entry>II</entry><entry>1129, palivizumab, motavisumab (<i>6</i>)</entry></row><row><entry>III</entry><entry>MPE8 (<i>17</i>)</entry></row><row><entry>IV</entry><entry>101F (<i>57</i>), mAb 19 (<i>19</i>)</entry></row></tbody></tgroup></table></tables><tables id="tabl0002" num="0002"><table frame="all"><title><b>Table 2: Germline usage and sequence information of anti-RSV antibodies</b></title><tgroup cols="9"><colspec colnum="1" colname="col1" colwidth="15mm" align="center" /><colspec colnum="2" colname="col2" colwidth="27mm" align="center" /><colspec colnum="3" colname="col3" colwidth="27mm" align="center" /><colspec colnum="4" colname="col4" colwidth="27mm" align="center" /><colspec colnum="5" colname="col5" colwidth="31mm" align="center" /><colspec colnum="6" colname="col6" colwidth="23mm" align="center" /><colspec colnum="7" colname="col7" colwidth="18mm" align="center" /><colspec colnum="8" colname="col8" colwidth="39mm" align="center" /><colspec colnum="9" colname="col9" colwidth="39mm" align="center" /><thead valign="middle"><row><entry><b>Name</b></entry><entry><b>Antibody number (Ab #)</b></entry><entry><b>VH germline gene usage</b></entry><entry><b>LC germline gene usage</b></entry><entry><b>CDR H3 Sequence</b></entry><entry><b>CDR L3 Sequence</b></entry><entry><b>Lineage number</b></entry><entry><b>Number of nucleotide substitutions in VH</b></entry><entry><b>Number of nucleotide substitutions in VL</b></entry></row></thead><tbody><row><entry>ADI-18875</entry><entry>232</entry><entry>VH4-34</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0001.tif" /></entry><entry><img file="EP3974447A2_D0002.tif" /></entry><entry>4</entry><entry>10</entry><entry>8</entry></row><row rowsep="0"><entry>ADI-18876</entry><entry>233</entry><entry>VH4-304</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0003.tif" /></entry><entry><img file="EP3974447A2_D0004.tif" /></entry><entry>44</entry><entry>12</entry><entry>4</entry></row><row><entry /><entry /><entry /><entry /><entry><img file="EP3974447A2_D0005.tif" /></entry><entry /><entry /><entry /><entry /></row><row><entry>ADI-18877</entry><entry>234</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0006.tif" /></entry><entry><img file="EP3974447A2_D0007.tif" /></entry><entry>52</entry><entry>9</entry><entry>8</entry></row><row><entry>ADI-18878</entry><entry>235</entry><entry>VH4-304</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0008.tif" /></entry><entry><img file="EP3974447A2_D0009.tif" /></entry><entry>23</entry><entry>12</entry><entry>6</entry></row><row><entry>ADI-18879</entry><entry>236</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0010.tif" /></entry><entry><img file="EP3974447A2_D0011.tif" /></entry><entry>46</entry><entry>9</entry><entry>12</entry></row><row><entry>ADI-18880</entry><entry>237</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0012.tif" /></entry><entry><img file="EP3974447A2_D0013.tif" /></entry><entry>36</entry><entry>8</entry><entry>7</entry></row><row><entry>ADI-18882</entry><entry>238</entry><entry>VH1-69</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0014.tif" /></entry><entry><img file="EP3974447A2_D0015.tif" /></entry><entry>21</entry><entry>19</entry><entry>9</entry></row><row><entry>ADI-18883</entry><entry>239</entry><entry>VH1-69</entry><entry>VK1-12</entry><entry><img file="EP3974447A2_D0016.tif" /></entry><entry><img file="EP3974447A2_D0017.tif" /></entry><entry>79</entry><entry>30</entry><entry>6</entry></row><row><entry>ADI-18884</entry><entry>240</entry><entry>VH1-3</entry><entry>VK2-28</entry><entry><img file="EP3974447A2_D0018.tif" /></entry><entry><img file="EP3974447A2_D0019.tif" /></entry><entry>57</entry><entry>8</entry><entry>1</entry></row><row><entry>ADI-18885</entry><entry>241</entry><entry>VH4-304</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0020.tif" /></entry><entry><img file="EP3974447A2_D0021.tif" /></entry><entry>22</entry><entry>13</entry><entry>8</entry></row><row><entry>ADI-18887</entry><entry>242</entry><entry>VH1-69</entry><entry>VK1-9</entry><entry><img file="EP3974447A2_D0022.tif" /></entry><entry><img file="EP3974447A2_D0023.tif" /></entry><entry>76</entry><entry>20</entry><entry>8</entry></row><row><entry>ADI-18888</entry><entry>243</entry><entry>VH3-33</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0024.tif" /></entry><entry><img file="EP3974447A2_D0025.tif" /></entry><entry>24</entry><entry>8</entry><entry>10</entry></row><row><entry>ADI-18889</entry><entry>244</entry><entry>VH4-34</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0026.tif" /></entry><entry><img file="EP3974447A2_D0027.tif" /></entry><entry>3</entry><entry>25</entry><entry>12</entry></row><row><entry>ADI-18890</entry><entry>245</entry><entry>VH1-69</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0028.tif" /></entry><entry><img file="EP3974447A2_D0029.tif" /></entry><entry>21</entry><entry>27</entry><entry>10</entry></row><row><entry>ADI-18891</entry><entry>246</entry><entry>VH1-69</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0030.tif" /></entry><entry><img file="EP3974447A2_D0031.tif" /></entry><entry>21</entry><entry>22</entry><entry>10</entry></row><row><entry>ADI-18892</entry><entry>247</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0032.tif" /></entry><entry><img file="EP3974447A2_D0033.tif" /></entry><entry>39</entry><entry>7</entry><entry>1</entry></row><row><entry>ADI-18893</entry><entry>248</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0034.tif" /></entry><entry><img file="EP3974447A2_D0035.tif" /></entry><entry>61</entry><entry>8</entry><entry>3</entry></row><row><entry>ADI-18894</entry><entry>249</entry><entry>VH3-30</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0036.tif" /></entry><entry><img file="EP3974447A2_D0037.tif" /></entry><entry>54</entry><entry>11</entry><entry>5</entry></row><row><entry>ADI-18895</entry><entry>250</entry><entry>VH5-51</entry><entry>VK1-33</entry><entry>ASVMLRGIM</entry><entry><img file="EP3974447A2_D0038.tif" /></entry><entry>84</entry><entry>12</entry><entry>1</entry></row><row><entry>ADI-18896</entry><entry>251</entry><entry>VH3-30</entry><entry>VK3-15</entry><entry><img file="EP3974447A2_D0039.tif" /></entry><entry><img file="EP3974447A2_D0040.tif" /></entry><entry>16</entry><entry>10</entry><entry>6</entry></row><row><entry>ADI-18897</entry><entry>252</entry><entry>VH3-7</entry><entry>VK4-1</entry><entry><img file="EP3974447A2_D0041.tif" /></entry><entry><img file="EP3974447A2_D0042.tif" /></entry><entry>42</entry><entry>11</entry><entry>8</entry></row><row><entry>ADI-18898</entry><entry>253</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0043.tif" /></entry><entry><img file="EP3974447A2_D0044.tif" /></entry><entry>52</entry><entry>9</entry><entry>8</entry></row><row><entry>ADI-18899</entry><entry>254</entry><entry>VH3-30</entry><entry>VK3-15</entry><entry><img file="EP3974447A2_D0045.tif" /></entry><entry><img file="EP3974447A2_D0046.tif" /></entry><entry>16</entry><entry>10</entry><entry>6</entry></row><row><entry>ADI-18900</entry><entry>255</entry><entry>VH3-23</entry><entry>VK1-5</entry><entry><img file="EP3974447A2_D0047.tif" /></entry><entry><img file="EP3974447A2_D0048.tif" /></entry><entry>38</entry><entry>22</entry><entry>7</entry></row><row><entry>ADI-18901</entry><entry>256</entry><entry>VH1-2</entry><entry>VL1-51</entry><entry><img file="EP3974447A2_D0049.tif" /></entry><entry><img file="EP3974447A2_D0050.tif" /></entry><entry>72</entry><entry>18</entry><entry>8</entry></row><row><entry>ADI-18902</entry><entry>257</entry><entry>VH4-59</entry><entry>VL3-21</entry><entry><img file="EP3974447A2_D0051.tif" /></entry><entry><img file="EP3974447A2_D0052.tif" /></entry><entry>73</entry><entry>8</entry><entry>7</entry></row><row><entry>ADI-18903</entry><entry>258</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0053.tif" /></entry><entry><img file="EP3974447A2_D0054.tif" /></entry><entry>46</entry><entry>9</entry><entry>12</entry></row><row><entry>ADI-18904</entry><entry>259</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0055.tif" /></entry><entry><img file="EP3974447A2_D0056.tif" /></entry><entry>36</entry><entry>8</entry><entry>7</entry></row><row><entry>ADI-18905</entry><entry>260</entry><entry>VH4-39</entry><entry>VK1-5</entry><entry><img file="EP3974447A2_D0057.tif" /></entry><entry><img file="EP3974447A2_D0058.tif" /></entry><entry>1</entry><entry>29</entry><entry>2</entry></row><row><entry>ADI-18906</entry><entry>261</entry><entry>VH3-33</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0059.tif" /></entry><entry><img file="EP3974447A2_D0060.tif" /></entry><entry>24</entry><entry>8</entry><entry>10</entry></row><row><entry>ADI-18907</entry><entry>262</entry><entry>VH1-69</entry><entry>VK3-11</entry><entry><img file="EP3974447A2_D0061.tif" /></entry><entry><img file="EP3974447A2_D0062.tif" /></entry><entry>55</entry><entry>21</entry><entry>7</entry></row><row><entry>ADI-18908</entry><entry>263</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0063.tif" /></entry><entry><img file="EP3974447A2_D0064.tif" /></entry><entry>98</entry><entry>16</entry><entry>7</entry></row><row><entry>ADI-18909</entry><entry>264</entry><entry>VH4-304</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0065.tif" /></entry><entry><img file="EP3974447A2_D0066.tif" /></entry><entry>27</entry><entry>13</entry><entry>9</entry></row><row><entry>ADI-18910</entry><entry>265</entry><entry>VH1-69</entry><entry>VK2-28</entry><entry><img file="EP3974447A2_D0067.tif" /></entry><entry><img file="EP3974447A2_D0068.tif" /></entry><entry>74</entry><entry>8</entry><entry>3</entry></row><row><entry>ADI-18911</entry><entry>266</entry><entry>VH5-51</entry><entry>VK1-33</entry><entry><img file="EP3974447A2_D0069.tif" /></entry><entry><img file="EP3974447A2_D0070.tif" /></entry><entry>60</entry><entry>12</entry><entry>7</entry></row><row><entry>ADI-18912</entry><entry>267</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0071.tif" /></entry><entry><img file="EP3974447A2_D0072.tif" /></entry><entry>31</entry><entry>6</entry><entry>3</entry></row><row><entry>ADI-18913</entry><entry>268</entry><entry>VH5-51</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0073.tif" /></entry><entry><img file="EP3974447A2_D0074.tif" /></entry><entry>67</entry><entry>13</entry><entry>6</entry></row><row><entry>ADI-18915</entry><entry>269</entry><entry>VH3-15</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0075.tif" /></entry><entry><img file="EP3974447A2_D0076.tif" /></entry><entry>86</entry><entry>4</entry><entry>3</entry></row><row><entry>ADI-18916</entry><entry>270</entry><entry>VH5-51</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0077.tif" /></entry><entry><img file="EP3974447A2_D0078.tif" /></entry><entry>67</entry><entry>13</entry><entry>6</entry></row><row><entry>ADI-18917</entry><entry>271</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0079.tif" /></entry><entry><img file="EP3974447A2_D0080.tif" /></entry><entry>98</entry><entry>16</entry><entry>7</entry></row><row><entry>ADI-18918</entry><entry>272</entry><entry>VH5-51</entry><entry>VK3-15</entry><entry><img file="EP3974447A2_D0081.tif" /></entry><entry><img file="EP3974447A2_D0082.tif" /></entry><entry>62</entry><entry>8</entry><entry>13</entry></row><row><entry>ADI-18919</entry><entry>273</entry><entry>VH4-31</entry><entry>VK2-28</entry><entry><img file="EP3974447A2_D0083.tif" /></entry><entry><img file="EP3974447A2_D0084.tif" /></entry><entry>75</entry><entry>22</entry><entry>4</entry></row><row><entry>ADI-18920</entry><entry>274</entry><entry>VH3-30</entry><entry>VL1-51</entry><entry><img file="EP3974447A2_D0085.tif" /></entry><entry><img file="EP3974447A2_D0086.tif" /></entry><entry>12</entry><entry>10</entry><entry>14</entry></row><row><entry>ADI-18921</entry><entry>275</entry><entry>VH1-24</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0087.tif" /></entry><entry><img file="EP3974447A2_D0088.tif" /></entry><entry>88</entry><entry>15</entry><entry>2</entry></row><row><entry>ADI-18922</entry><entry>276</entry><entry>VH4-b</entry><entry>VK3-15</entry><entry><img file="EP3974447A2_D0089.tif" /></entry><entry><img file="EP3974447A2_D0090.tif" /></entry><entry>83</entry><entry>22</entry><entry>8</entry></row><row rowsep="0"><entry>ADI-18923</entry><entry>277</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0091.tif" /></entry><entry><img file="EP3974447A2_D0092.tif" /></entry><entry>48</entry><entry>0</entry><entry>0</entry></row><row><entry /><entry /><entry /><entry /><entry><img file="EP3974447A2_D0093.tif" /></entry><entry><img file="EP3974447A2_D0094.tif" /></entry><entry /><entry /><entry /></row><row><entry>ADI-18924</entry><entry>278</entry><entry>VH5-51</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0095.tif" /></entry><entry><img file="EP3974447A2_D0096.tif" /></entry><entry>18</entry><entry>9</entry><entry>11</entry></row><row><entry>ADI-18925</entry><entry>279</entry><entry>VH3-30</entry><entry>VK3-11</entry><entry><img file="EP3974447A2_D0097.tif" /></entry><entry><img file="EP3974447A2_D0098.tif" /></entry><entry>9</entry><entry>8</entry><entry>0</entry></row><row><entry>ADI-18926</entry><entry>280</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0099.tif" /></entry><entry><img file="EP3974447A2_D0100.tif" /></entry><entry>36</entry><entry>19</entry><entry>8</entry></row><row><entry>ADI-18927</entry><entry>281</entry><entry>VH1-2</entry><entry>VK1-13</entry><entry><img file="EP3974447A2_D0101.tif" /></entry><entry><img file="EP3974447A2_D0102.tif" /></entry><entry>82</entry><entry>21</entry><entry>21</entry></row><row><entry>ADI-18928</entry><entry>282</entry><entry>VH1-69</entry><entry>VL7-46</entry><entry><img file="EP3974447A2_D0103.tif" /></entry><entry><img file="EP3974447A2_D0104.tif" /></entry><entry>25</entry><entry>15</entry><entry>8</entry></row><row><entry>ADI-18929</entry><entry>283</entry><entry>VH3-11</entry><entry>VL1-44</entry><entry><img file="EP3974447A2_D0105.tif" /></entry><entry><img file="EP3974447A2_D0106.tif" /></entry><entry>19</entry><entry>9</entry><entry>6</entry></row><row><entry>ADI-18930</entry><entry>284</entry><entry>VH1-69</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0107.tif" /></entry><entry><img file="EP3974447A2_D0108.tif" /></entry><entry>21</entry><entry>14</entry><entry>8</entry></row><row><entry>ADI-18931</entry><entry>285</entry><entry>VH3-49</entry><entry>VK3-15</entry><entry><img file="EP3974447A2_D0109.tif" /></entry><entry><img file="EP3974447A2_D0110.tif" /></entry><entry>92</entry><entry>6</entry><entry>6</entry></row><row><entry>ADI-18932</entry><entry>286</entry><entry>VH4-304</entry><entry>VK3-11</entry><entry><img file="EP3974447A2_D0111.tif" /></entry><entry><img file="EP3974447A2_D0112.tif" /></entry><entry>29</entry><entry>25</entry><entry>10</entry></row><row><entry>ADI-18933</entry><entry>287</entry><entry>VH1-46</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0113.tif" /></entry><entry><img file="EP3974447A2_D0114.tif" /></entry><entry>69</entry><entry>26</entry><entry>10</entry></row><row><entry>ADI-18935</entry><entry>288</entry><entry>VH4-304</entry><entry>VK3-11</entry><entry><img file="EP3974447A2_D0115.tif" /></entry><entry><img file="EP3974447A2_D0116.tif" /></entry><entry>30</entry><entry>19</entry><entry>6</entry></row><row><entry>ADI-18936</entry><entry>289</entry><entry>VH1-8</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0117.tif" /></entry><entry><img file="EP3974447A2_D0118.tif" /></entry><entry>82</entry><entry>26</entry><entry>12</entry></row><row><entry>ADI-18937</entry><entry>290</entry><entry>VH4-304</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0119.tif" /></entry><entry><img file="EP3974447A2_D0120.tif" /></entry><entry>45</entry><entry>25</entry><entry>10</entry></row><row><entry>ADI-18938</entry><entry>291</entry><entry>VH3-74</entry><entry>VK1-5</entry><entry><img file="EP3974447A2_D0121.tif" /></entry><entry><img file="EP3974447A2_D0122.tif" /></entry><entry>32</entry><entry>15</entry><entry>10</entry></row><row><entry>ADI-18939</entry><entry>292</entry><entry>VH4-34</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0123.tif" /></entry><entry><img file="EP3974447A2_D0124.tif" /></entry><entry>4</entry><entry>3</entry><entry>7</entry></row><row><entry>ADI-18940</entry><entry>293</entry><entry>VH1-8</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0125.tif" /></entry><entry><img file="EP3974447A2_D0126.tif" /></entry><entry>82</entry><entry>33</entry><entry>14</entry></row><row><entry>ADI-18941</entry><entry>294</entry><entry>VH5-51</entry><entry>VK3-15</entry><entry><img file="EP3974447A2_D0127.tif" /></entry><entry><img file="EP3974447A2_D0128.tif" /></entry><entry>90</entry><entry>7</entry><entry>5</entry></row><row><entry>ADI-18942</entry><entry>295</entry><entry>VH4-304</entry><entry>VK3-11</entry><entry><img file="EP3974447A2_D0129.tif" /></entry><entry><img file="EP3974447A2_D0130.tif" /></entry><entry>28</entry><entry>19</entry><entry>6</entry></row><row><entry>ADI-18943</entry><entry>296</entry><entry>VH4-b</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0131.tif" /></entry><entry><img file="EP3974447A2_D0132.tif" /></entry><entry>2</entry><entry>38</entry><entry>7</entry></row><row><entry>ADI-18944</entry><entry>297</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0133.tif" /></entry><entry><img file="EP3974447A2_D0134.tif" /></entry><entry>34</entry><entry>20</entry><entry>8</entry></row><row><entry>ADI-18946</entry><entry>298</entry><entry>VH3-30</entry><entry>VL2-14</entry><entry><img file="EP3974447A2_D0135.tif" /></entry><entry><img file="EP3974447A2_D0136.tif" /></entry><entry>7</entry><entry>8</entry><entry>3</entry></row><row><entry>ADI-18947</entry><entry>299</entry><entry>VH3-30</entry><entry>VK3-15</entry><entry><img file="EP3974447A2_D0137.tif" /></entry><entry><img file="EP3974447A2_D0138.tif" /></entry><entry>16</entry><entry>16</entry><entry>7</entry></row><row><entry>ADI-18948</entry><entry>300</entry><entry>VH3-30</entry><entry>VK3-15</entry><entry><img file="EP3974447A2_D0139.tif" /></entry><entry><img file="EP3974447A2_D0140.tif" /></entry><entry>16</entry><entry>13</entry><entry>7</entry></row><row><entry>ADI-18949</entry><entry>301</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0141.tif" /></entry><entry><img file="EP3974447A2_D0142.tif" /></entry><entry>34</entry><entry>20</entry><entry>8</entry></row><row><entry>ADI-18950</entry><entry>302</entry><entry>VH4-39</entry><entry>VK3-11</entry><entry>ATAWTFDH</entry><entry><img file="EP3974447A2_D0143.tif" /></entry><entry>85</entry><entry>13</entry><entry>2</entry></row><row><entry>ADI-18951</entry><entry>303</entry><entry>VH3-23</entry><entry>VL2-14</entry><entry><img file="EP3974447A2_D0144.tif" /></entry><entry><img file="EP3974447A2_D0145.tif" /></entry><entry>5</entry><entry>19</entry><entry>16</entry></row><row><entry>ADI-18952</entry><entry>304</entry><entry>VH3-23</entry><entry>VL2-14</entry><entry><img file="EP3974447A2_D0146.tif" /></entry><entry><img file="EP3974447A2_D0147.tif" /></entry><entry>5</entry><entry>18</entry><entry>11</entry></row><row><entry>ADI-18953</entry><entry>305</entry><entry>VH1-69</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0148.tif" /></entry><entry><img file="EP3974447A2_D0149.tif" /></entry><entry>21</entry><entry>12</entry><entry>11</entry></row><row><entry>ADI-18955</entry><entry>306</entry><entry>VH3-30</entry><entry>VK3-15</entry><entry><img file="EP3974447A2_D0150.tif" /></entry><entry><img file="EP3974447A2_D0151.tif" /></entry><entry>16</entry><entry>11</entry><entry>6</entry></row><row><entry>ADI-18956</entry><entry>307</entry><entry>VH1-69</entry><entry>VL2-11</entry><entry><img file="EP3974447A2_D0152.tif" /></entry><entry><img file="EP3974447A2_D0153.tif" /></entry><entry>89</entry><entry>23</entry><entry>10</entry></row><row><entry>ADI-18957</entry><entry>308</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0154.tif" /></entry><entry><img file="EP3974447A2_D0155.tif" /></entry><entry>78</entry><entry>12</entry><entry>3</entry></row><row><entry>ADI-18958</entry><entry>309</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0156.tif" /></entry><entry><img file="EP3974447A2_D0157.tif" /></entry><entry>51</entry><entry>18</entry><entry>3</entry></row><row><entry>ADI-18959</entry><entry>310</entry><entry>VH1-24</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0158.tif" /></entry><entry><img file="EP3974447A2_D0159.tif" /></entry><entry>88</entry><entry>18</entry><entry>4</entry></row><row><entry>ADI-18960</entry><entry>311</entry><entry>VH4-39</entry><entry>VL3-21</entry><entry><img file="EP3974447A2_D0160.tif" /></entry><entry><img file="EP3974447A2_D0161.tif" /></entry><entry>50</entry><entry>15</entry><entry>5</entry></row><row><entry>ADI-18962</entry><entry>312</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0162.tif" /></entry><entry><img file="EP3974447A2_D0163.tif" /></entry><entry>80</entry><entry>1</entry><entry>0</entry></row><row><entry>ADI-18965</entry><entry>313</entry><entry>VH1-3</entry><entry>VK4-1</entry><entry><img file="EP3974447A2_D0164.tif" /></entry><entry><img file="EP3974447A2_D0165.tif" /></entry><entry>91</entry><entry>14</entry><entry>9</entry></row><row><entry>ADI-18966</entry><entry>314</entry><entry>VH3-30</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0166.tif" /></entry><entry><img file="EP3974447A2_D0167.tif" /></entry><entry>35</entry><entry>13</entry><entry>3</entry></row><row><entry>ADI-18967</entry><entry>315</entry><entry>VH1-24</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0168.tif" /></entry><entry><img file="EP3974447A2_D0169.tif" /></entry><entry>88</entry><entry>23</entry><entry>12</entry></row><row><entry>ADI-18968</entry><entry>316</entry><entry>VH4-304</entry><entry>VK3-11</entry><entry><img file="EP3974447A2_D0170.tif" /></entry><entry><img file="EP3974447A2_D0171.tif" /></entry><entry>93</entry><entry>17</entry><entry>8</entry></row><row><entry>ADI-18969</entry><entry>317</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0172.tif" /></entry><entry><img file="EP3974447A2_D0173.tif" /></entry><entry>43</entry><entry>15</entry><entry>4</entry></row><row><entry>ADI-18970</entry><entry>318</entry><entry>VH4-304</entry><entry>VK3-11</entry><entry><img file="EP3974447A2_D0174.tif" /></entry><entry><img file="EP3974447A2_D0175.tif" /></entry><entry>26</entry><entry>6</entry><entry>9</entry></row><row><entry>ADI-18971</entry><entry>319</entry><entry>VH3-23</entry><entry>VL3-21</entry><entry><img file="EP3974447A2_D0176.tif" /></entry><entry><img file="EP3974447A2_D0177.tif" /></entry><entry>6</entry><entry>17</entry><entry>12</entry></row><row><entry>ADI-18972</entry><entry>320</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0178.tif" /></entry><entry><img file="EP3974447A2_D0179.tif" /></entry><entry>34</entry><entry>20</entry><entry>8</entry></row><row><entry>ADI-18973</entry><entry>321</entry><entry>VH2-70</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0180.tif" /></entry><entry><img file="EP3974447A2_D0181.tif" /></entry><entry>13</entry><entry>18</entry><entry>14</entry></row><row rowsep="0"><entry>ADI-18974</entry><entry>322</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0182.tif" /></entry><entry><img file="EP3974447A2_D0183.tif" /></entry><entry>41</entry><entry>22</entry><entry>5</entry></row><row><entry /><entry /><entry /><entry /><entry><img file="EP3974447A2_D0184.tif" /></entry><entry><img file="EP3974447A2_D0185.tif" /></entry><entry /><entry /><entry /></row><row><entry>ADI-18975</entry><entry>323</entry><entry>VH1-18</entry><entry>VK2-24</entry><entry><img file="EP3974447A2_D0186.tif" /></entry><entry><img file="EP3974447A2_D0187.tif" /></entry><entry>40</entry><entry>11</entry><entry>3</entry></row><row><entry>ADI-18976</entry><entry>324</entry><entry>VH4-31</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0188.tif" /></entry><entry><img file="EP3974447A2_D0189.tif" /></entry><entry>33</entry><entry>5</entry><entry>9</entry></row><row><entry>ADI-18977</entry><entry>325</entry><entry>VH1-3</entry><entry>VK1-33</entry><entry><img file="EP3974447A2_D0190.tif" /></entry><entry><img file="EP3974447A2_D0191.tif" /></entry><entry>68</entry><entry>17</entry><entry>6</entry></row><row><entry>ADI-18978</entry><entry>326</entry><entry>VH3-30</entry><entry>VK1-5</entry><entry><img file="EP3974447A2_D0192.tif" /></entry><entry><img file="EP3974447A2_D0193.tif" /></entry><entry>11</entry><entry>9</entry><entry>11</entry></row><row><entry>ADI-18979</entry><entry>327</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0194.tif" /></entry><entry><img file="EP3974447A2_D0195.tif" /></entry><entry>39</entry><entry>11</entry><entry>1</entry></row><row><entry>ADI-18980</entry><entry>328</entry><entry>VH5-51</entry><entry>VK3-11</entry><entry><img file="EP3974447A2_D0196.tif" /></entry><entry><img file="EP3974447A2_D0197.tif" /></entry><entry>10</entry><entry>8</entry><entry>9</entry></row><row><entry>ADI-18981</entry><entry>329</entry><entry>VH4-30</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0198.tif" /></entry><entry><img file="EP3974447A2_D0199.tif" /></entry><entry>17</entry><entry>27</entry><entry>9</entry></row><row><entry>ADI-18982</entry><entry>330</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0200.tif" /></entry><entry><img file="EP3974447A2_D0201.tif" /></entry><entry>39</entry><entry>7</entry><entry>1</entry></row><row><entry>ADI-18983</entry><entry>331</entry><entry>VH3-30</entry><entry>VK1-17</entry><entry><img file="EP3974447A2_D0202.tif" /></entry><entry><img file="EP3974447A2_D0203.tif" /></entry><entry>49</entry><entry>14</entry><entry>5</entry></row><row><entry>ADI-18984</entry><entry>332</entry><entry>VH4-61</entry><entry>VK1-12</entry><entry><img file="EP3974447A2_D0204.tif" /></entry><entry><img file="EP3974447A2_D0205.tif" /></entry><entry>56</entry><entry>29</entry><entry>5</entry></row><row><entry>ADI-18985</entry><entry>333</entry><entry>VH1-2</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0206.tif" /></entry><entry><img file="EP3974447A2_D0207.tif" /></entry><entry>95</entry><entry>22</entry><entry>16</entry></row><row><entry>ADI-18986</entry><entry>334</entry><entry>VH3-30</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0208.tif" /></entry><entry><img file="EP3974447A2_D0209.tif" /></entry><entry>54</entry><entry>12</entry><entry>5</entry></row><row><entry>ADI-18987</entry><entry>335</entry><entry>VH1-69</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0210.tif" /></entry><entry><img file="EP3974447A2_D0211.tif" /></entry><entry>20</entry><entry>12</entry><entry>8</entry></row><row><entry>ADI-18988</entry><entry>336</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0212.tif" /></entry><entry><img file="EP3974447A2_D0213.tif" /></entry><entry>97</entry><entry>22</entry><entry>6</entry></row><row><entry>ADI-18989</entry><entry>337</entry><entry>VH3-48</entry><entry>VK2-28</entry><entry><img file="EP3974447A2_D0214.tif" /></entry><entry><img file="EP3974447A2_D0215.tif" /></entry><entry>71</entry><entry>14</entry><entry>6</entry></row><row><entry>ADI-18990</entry><entry>338</entry><entry>VH4-304</entry><entry>VK3-11</entry><entry><img file="EP3974447A2_D0216.tif" /></entry><entry><img file="EP3974447A2_D0217.tif" /></entry><entry>28</entry><entry>19</entry><entry>6</entry></row><row><entry>ADI-18991</entry><entry>339</entry><entry>VH4-304</entry><entry>VL3-21</entry><entry><img file="EP3974447A2_D0218.tif" /></entry><entry><img file="EP3974447A2_D0219.tif" /></entry><entry>81</entry><entry>22</entry><entry>14</entry></row><row><entry>ADI-18992</entry><entry>340</entry><entry>VH1-46</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0220.tif" /></entry><entry><img file="EP3974447A2_D0221.tif" /></entry><entry>15</entry><entry>14</entry><entry>7</entry></row><row><entry>ADI-18993</entry><entry>341</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0222.tif" /></entry><entry><img file="EP3974447A2_D0223.tif" /></entry><entry>53</entry><entry>10</entry><entry>7</entry></row><row><entry>ADI-18994</entry><entry>342</entry><entry>VH1-18</entry><entry>VL2-23</entry><entry><img file="EP3974447A2_D0224.tif" /></entry><entry><img file="EP3974447A2_D0225.tif" /></entry><entry>58</entry><entry>13</entry><entry>7</entry></row><row><entry>ADI-18995</entry><entry>343</entry><entry>VH4-304</entry><entry>VL3-21</entry><entry><img file="EP3974447A2_D0226.tif" /></entry><entry><img file="EP3974447A2_D0227.tif" /></entry><entry>81</entry><entry>18</entry><entry>12</entry></row><row><entry>ADI-18996</entry><entry>344</entry><entry>VH1-2</entry><entry>VL1-51</entry><entry><img file="EP3974447A2_D0228.tif" /></entry><entry><img file="EP3974447A2_D0229.tif" /></entry><entry>72</entry><entry>17</entry><entry>10</entry></row><row><entry>ADI-18997</entry><entry>345</entry><entry>VH4-34</entry><entry>VK3-15</entry><entry><img file="EP3974447A2_D0230.tif" /></entry><entry><img file="EP3974447A2_D0231.tif" /></entry><entry>77</entry><entry>16</entry><entry>9</entry></row><row><entry>ADI-18998</entry><entry>346</entry><entry>VH4-304</entry><entry>VL3-21</entry><entry><img file="EP3974447A2_D0232.tif" /></entry><entry><img file="EP3974447A2_D0233.tif" /></entry><entry>81</entry><entry>15</entry><entry>15</entry></row><row><entry>ADI-18999</entry><entry>347</entry><entry>VH1-69</entry><entry>VL2-11</entry><entry><img file="EP3974447A2_D0234.tif" /></entry><entry><img file="EP3974447A2_D0235.tif" /></entry><entry>65</entry><entry>13</entry><entry>3</entry></row><row><entry>ADI-19000</entry><entry>348</entry><entry>VH1-8</entry><entry>VL1-36</entry><entry><img file="EP3974447A2_D0236.tif" /></entry><entry><img file="EP3974447A2_D0237.tif" /></entry><entry>96</entry><entry>10</entry><entry>6</entry></row><row><entry>ADI-19001</entry><entry>349</entry><entry>VH3-49</entry><entry>VL2-8</entry><entry><img file="EP3974447A2_D0238.tif" /></entry><entry><img file="EP3974447A2_D0239.tif" /></entry><entry>94</entry><entry>10</entry><entry>7</entry></row><row><entry>ADI-19002</entry><entry>350</entry><entry>VH4-59</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0240.tif" /></entry><entry><img file="EP3974447A2_D0241.tif" /></entry><entry>66</entry><entry>17</entry><entry>8</entry></row><row><entry>ADI-19003</entry><entry>351</entry><entry>VH4-39</entry><entry>VL3-21</entry><entry><img file="EP3974447A2_D0242.tif" /></entry><entry><img file="EP3974447A2_D0243.tif" /></entry><entry>50</entry><entry>15</entry><entry>4</entry></row><row><entry>ADI-19004</entry><entry>352</entry><entry>VH1-18</entry><entry>VK2-30</entry><entry><img file="EP3974447A2_D0244.tif" /></entry><entry><img file="EP3974447A2_D0245.tif" /></entry><entry>36</entry><entry>19</entry><entry>7</entry></row><row><entry>ADI-19005</entry><entry>353</entry><entry>VH1-18</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0246.tif" /></entry><entry><img file="EP3974447A2_D0247.tif" /></entry><entry>82</entry><entry>21</entry><entry>9</entry></row><row><entry>ADI-19006</entry><entry>354</entry><entry>VH3-11</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0248.tif" /></entry><entry><img file="EP3974447A2_D0249.tif" /></entry><entry>14</entry><entry>30</entry><entry>12</entry></row><row><entry>ADI-19007</entry><entry>355</entry><entry>VH3-23</entry><entry>VK4-1</entry><entry><img file="EP3974447A2_D0250.tif" /></entry><entry><img file="EP3974447A2_D0251.tif" /></entry><entry>8</entry><entry>18</entry><entry>5</entry></row><row><entry>ADI-19008</entry><entry>356</entry><entry>VH3-30</entry><entry>VK3-11</entry><entry><img file="EP3974447A2_D0252.tif" /></entry><entry><img file="EP3974447A2_D0253.tif" /></entry><entry>37</entry><entry>15</entry><entry>6</entry></row><row><entry>ADI-19009</entry><entry>357</entry><entry>VH1-69</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0254.tif" /></entry><entry><img file="EP3974447A2_D0255.tif" /></entry><entry>21</entry><entry>19</entry><entry>6</entry></row><row><entry>ADI-19010</entry><entry>358</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0256.tif" /></entry><entry><img file="EP3974447A2_D0257.tif" /></entry><entry>47</entry><entry>0</entry><entry>0</entry></row><row><entry>ADI-19011</entry><entry>359</entry><entry>VH3-30</entry><entry>VL4-60</entry><entry><img file="EP3974447A2_D0258.tif" /></entry><entry><img file="EP3974447A2_D0259.tif" /></entry><entry>87</entry><entry>19</entry><entry>9</entry></row><row><entry>ADI-19012</entry><entry>360</entry><entry>VH4-4</entry><entry>VK1-39</entry><entry><img file="EP3974447A2_D0260.tif" /></entry><entry><img file="EP3974447A2_D0261.tif" /></entry><entry>63</entry><entry>12</entry><entry>10</entry></row><row><entry>ADI-19013</entry><entry>361</entry><entry>VH5-51</entry><entry>VK1-12</entry><entry><img file="EP3974447A2_D0262.tif" /></entry><entry><img file="EP3974447A2_D0263.tif" /></entry><entry>64</entry><entry>4</entry><entry>2</entry></row><row><entry>ADI-19014</entry><entry>362</entry><entry>VH4-304</entry><entry>VK3-20</entry><entry><img file="EP3974447A2_D0264.tif" /></entry><entry><img file="EP3974447A2_D0265.tif" /></entry><entry>70</entry><entry>21</entry><entry>10</entry></row><row><entry>ADI-19016</entry><entry>363</entry><entry>VH3-21</entry><entry>VL1-40</entry><entry><img file="EP3974447A2_D0266.tif" /></entry><entry><img file="EP3974447A2_D0267.tif" /></entry><entry>59</entry><entry>13</entry><entry>5</entry></row><row><entry>ADI-19017</entry><entry>364</entry><entry>VH1-18</entry><entry>VL3-25</entry><entry><img file="EP3974447A2_D0268.tif" /></entry><entry><img file="EP3974447A2_D0269.tif" /></entry><entry>82</entry><entry>22</entry><entry>0</entry></row></tbody></tgroup></table></tables><tables id="tabl0003" num="0003"><table frame="all"><title><b>Table 3: Affinity and Neutralization data for anti-RSV antibodies</b></title><tgroup cols="8"><colspec colnum="1" colname="col1" colwidth="15mm" align="center" /><colspec colnum="2" colname="col2" colwidth="20mm" align="center" /><colspec colnum="3" colname="col3" colwidth="25mm" align="center" /><colspec colnum="4" colname="col4" colwidth="23mm" align="center" /><colspec colnum="5" colname="col5" colwidth="25mm" align="center" /><colspec colnum="6" colname="col6" colwidth="23mm" align="center" /><colspec colnum="7" colname="col7" colwidth="20mm" align="center" /><colspec colnum="8" colname="col8" colwidth="20mm" align="center" /><thead valign="middle"><row><entry><b>Name</b></entry><entry><b>Antibody number (Ab #)</b></entry><entry><b>Prefusion subtype A K<sub>D</sub> (M)*</b></entry><entry><b>Postfusion subtype A K<sub>D</sub> (M)*</b></entry><entry><b>Prefusion subtype B K<sub>D</sub> (M)*</b></entry><entry><b>Postfusion subtype B K<sub>D</sub> (M)*</b></entry><entry><b>Neut IC<sub>50</sub> (µg/ml) subtype A*</b></entry><entry><b>Neut IC<sub>50</sub> (µg/ml) subtype B*</b></entry></row></thead><tbody><row><entry>ADI-18875</entry><entry>232</entry><entry>7.36E-10</entry><entry>NB</entry><entry>7.64E-10</entry><entry>NB</entry><entry>0.040</entry><entry>0.035</entry></row><row><entry>ADI-18876</entry><entry>233</entry><entry>7.07E-10</entry><entry>1.71E-09</entry><entry>3.16E-10</entry><entry>1.79E-10</entry><entry>0.037</entry><entry>0.179</entry></row><row><entry>ADI-18877</entry><entry>234</entry><entry>3.03E-10</entry><entry>NB</entry><entry>3.83E-10</entry><entry>NB</entry><entry>0.410</entry><entry>0.130</entry></row><row><entry>ADI-18878</entry><entry>235</entry><entry>4.53E-09</entry><entry>4.83E-10</entry><entry>5.82E-09</entry><entry>3.88E-10</entry><entry>>10</entry><entry>8.308</entry></row><row><entry>ADI-18879</entry><entry>236</entry><entry>3.12E-10</entry><entry>NB</entry><entry>3.58E-10</entry><entry>NB</entry><entry>0.041</entry><entry>0.103</entry></row><row><entry>ADI-18880</entry><entry>237</entry><entry>2.55E-10</entry><entry>NB</entry><entry>3.04E-10</entry><entry>NB</entry><entry>0.041</entry><entry>0.055</entry></row><row><entry>ADI-18882</entry><entry>238</entry><entry>4.27E-10</entry><entry>NB</entry><entry>4.76E-10</entry><entry>NB</entry><entry>0.041</entry><entry>0.057</entry></row><row><entry>ADI-18883</entry><entry>239</entry><entry>4.31E-10</entry><entry>NB</entry><entry>5.66E-10</entry><entry>NB</entry><entry>0.041</entry><entry>0.050</entry></row><row><entry>ADI-18884</entry><entry>240</entry><entry>3.38E-10</entry><entry>NB</entry><entry>2.04E-10</entry><entry>NB</entry><entry>0.073</entry><entry>0.239</entry></row><row><entry>ADI-18885</entry><entry>241</entry><entry>2.18E-09</entry><entry>3.84E-10</entry><entry>3.89E-09</entry><entry>3.07E-10</entry><entry>0.376</entry><entry>8.635</entry></row><row><entry>ADI-18887</entry><entry>242</entry><entry>NB</entry><entry>7.43E-10</entry><entry>1.22E-08</entry><entry>5.49E-10</entry><entry>1.110</entry><entry>>10</entry></row><row><entry>ADI-18888</entry><entry>243</entry><entry>NB</entry><entry>2.54E-08</entry><entry>NB</entry><entry>1.16E-09</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18889</entry><entry>244</entry><entry>5.54E-10</entry><entry>NB</entry><entry>5.87E-10</entry><entry>NB</entry><entry>0.040</entry><entry>0.019</entry></row><row><entry>ADI-18890</entry><entry>245</entry><entry>4.89E-10</entry><entry>NB</entry><entry>4.58E-10</entry><entry>NB</entry><entry>0.041</entry><entry>0.041</entry></row><row><entry>ADI-18891</entry><entry>246</entry><entry>5.34E-10</entry><entry>NB</entry><entry>5.13E-10</entry><entry>NB</entry><entry>0.012</entry><entry>0.026</entry></row><row><entry>ADI-18892</entry><entry>247</entry><entry>2.17E-10</entry><entry>NB</entry><entry>2.53E-10</entry><entry>NB</entry><entry>0.018</entry><entry>0.117</entry></row><row><entry>ADI-18893</entry><entry>248</entry><entry>2.45E-10</entry><entry>NB</entry><entry>2.78E-10</entry><entry>NB</entry><entry>0.123</entry><entry>0.182</entry></row><row><entry>ADI-18894</entry><entry>249</entry><entry>2.54E-09</entry><entry>NB</entry><entry>3.27E-10</entry><entry>NB</entry><entry>0.345</entry><entry>0.123</entry></row><row><entry>ADI-18895</entry><entry>250</entry><entry>NB</entry><entry>2.37E-09</entry><entry>NB</entry><entry>4.86E-10</entry><entry>2.303</entry><entry>>10</entry></row><row><entry>ADI-18896</entry><entry>251</entry><entry>2.27E-09</entry><entry>2.79E-10</entry><entry>1.81E-09</entry><entry>2.70E-10</entry><entry>1.100</entry><entry>4.722</entry></row><row><entry>ADI-18897</entry><entry>252</entry><entry>1.47E-09</entry><entry>2.19E-10</entry><entry>1.53E-09</entry><entry>1.85E-10</entry><entry>0.288</entry><entry>0.762</entry></row><row><entry>ADI-18898</entry><entry>253</entry><entry>3.05E-10</entry><entry>NB</entry><entry>3.25E-10</entry><entry>NB</entry><entry>0.030</entry><entry>0.097</entry></row><row><entry>ADI-18899</entry><entry>254</entry><entry>1.92E-09</entry><entry>2.66E-10</entry><entry>1.59E-09</entry><entry>2.51E-10</entry><entry>0.742</entry><entry>2.700</entry></row><row><entry>ADI-18900</entry><entry>255</entry><entry>1.19E-09</entry><entry>NB</entry><entry>3.31E-10</entry><entry>NB</entry><entry>0.035</entry><entry>0.059</entry></row><row><entry>ADI-18901</entry><entry>256</entry><entry>2.17E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>5.646</entry><entry>5.762</entry></row><row><entry>ADI-18902</entry><entry>257</entry><entry>1.07E-10</entry><entry>NB</entry><entry>1.01E-10</entry><entry>NB</entry><entry>0.024</entry><entry>0.150</entry></row><row><entry>ADI-18903</entry><entry>258</entry><entry>3.24E-10</entry><entry>NB</entry><entry>2.72E-10</entry><entry>NB</entry><entry>0.036</entry><entry>0.118</entry></row><row><entry>ADI-18904</entry><entry>259</entry><entry>2.51E-10</entry><entry>NB</entry><entry>2.37E-10</entry><entry>NB</entry><entry>0.018</entry><entry>0.089</entry></row><row><entry>ADI-18905</entry><entry>260</entry><entry>3.38E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>0.685</entry><entry>3.676</entry></row><row><entry>ADI-18906</entry><entry>261</entry><entry>NB</entry><entry>2.33E-08</entry><entry>NB</entry><entry>1.10E-09</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18907</entry><entry>262</entry><entry>1.74E-10</entry><entry>NB</entry><entry>2.04E-10</entry><entry>NB</entry><entry>3.300</entry><entry>>10</entry></row><row><entry>ADI-18908</entry><entry>263</entry><entry>3.02E-10</entry><entry>NB</entry><entry>3.52E-10</entry><entry>NB</entry><entry>0.018</entry><entry>0.095</entry></row><row><entry>ADI-18909</entry><entry>264</entry><entry>5.08E-10</entry><entry>5.81E-10</entry><entry>2.88E-10</entry><entry>2.25E-10</entry><entry>0.110</entry><entry>0.169</entry></row><row><entry>ADI-18910</entry><entry>265</entry><entry>4.67E-09</entry><entry>NB</entry><entry>1.06E-08</entry><entry>NB</entry><entry>>10</entry><entry>0.767</entry></row><row><entry>ADI-18911</entry><entry>266</entry><entry>NB</entry><entry>3.53E-10</entry><entry>NB</entry><entry>2.80E-10</entry><entry>0.301</entry><entry>4.853</entry></row><row><entry>ADI-18912</entry><entry>267</entry><entry>2.58E-10</entry><entry>NB</entry><entry>2.86E-10</entry><entry>NB</entry><entry>0.024</entry><entry>0.061</entry></row><row><entry>ADI-18913</entry><entry>268</entry><entry>5.68E-10</entry><entry>NB</entry><entry>4.71E-10</entry><entry>NB</entry><entry><0.01</entry><entry><0.01</entry></row><row><entry>ADI-18915</entry><entry>269</entry><entry>2.81E-08</entry><entry>NB</entry><entry>4.21E-10</entry><entry>NB</entry><entry>1.199</entry><entry>0.021</entry></row><row><entry>ADI-18916</entry><entry>270</entry><entry>5.85E-10</entry><entry>NB</entry><entry>4.65E-10</entry><entry>NB</entry><entry><0.01</entry><entry><0.01</entry></row><row><entry>ADI-18917</entry><entry>271</entry><entry>3.56E-10</entry><entry>NB</entry><entry>3.32E-10</entry><entry>NB</entry><entry>0.024</entry><entry>0.091</entry></row><row><entry>ADI-18918</entry><entry>272</entry><entry>NB</entry><entry>5.67E-10</entry><entry>NB</entry><entry>4.09E-10</entry><entry>0.377</entry><entry>4.590</entry></row><row><entry>ADI-18919</entry><entry>273</entry><entry>2.02E-10</entry><entry>NB</entry><entry>1.63E-10</entry><entry>NB</entry><entry>0.123</entry><entry>0.261</entry></row><row><entry>ADI-18920</entry><entry>274</entry><entry>6.78E-10</entry><entry>NB</entry><entry>9.77E-11</entry><entry>NB</entry><entry>0.041</entry><entry>0.049</entry></row><row><entry>ADI-18921</entry><entry>275</entry><entry>5.75E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>1.703</entry><entry>1.172</entry></row><row><entry>ADI-18922</entry><entry>276</entry><entry>3.47E-09</entry><entry>3.22E-10</entry><entry>5.27E-09</entry><entry>2.87E-10</entry><entry>>10</entry><entry>5.051</entry></row><row><entry>ADI-18923</entry><entry>277</entry><entry>5.17E-10</entry><entry>NB</entry><entry>1.79E-09</entry><entry>NB</entry><entry>0.078</entry><entry>0.147</entry></row><row><entry>ADI-18924</entry><entry>278</entry><entry>6.48E-09</entry><entry>4.11E-10</entry><entry>NB</entry><entry>3.21E-10</entry><entry>0.572</entry><entry>1.073</entry></row><row><entry>ADI-18925</entry><entry>279</entry><entry>4.99E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18926</entry><entry>280</entry><entry>2.52E-10</entry><entry>NB</entry><entry>2.50E-10</entry><entry>NB</entry><entry>0.023</entry><entry>0.092</entry></row><row><entry>ADI-18927</entry><entry>281</entry><entry>3.58E-09</entry><entry>NB</entry><entry>2.99E-09</entry><entry>NB</entry><entry>0.022</entry><entry>0.067</entry></row><row><entry>ADI-18928</entry><entry>282</entry><entry>4.49E-10</entry><entry>NB</entry><entry>5.15E-10</entry><entry>NB</entry><entry>0.034</entry><entry>0.062</entry></row><row><entry>ADI-18929</entry><entry>283</entry><entry>1.61E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>0.261</entry><entry>0.369</entry></row><row><entry>ADI-18930</entry><entry>284</entry><entry>3.87E-10</entry><entry>NB</entry><entry>3.72E-10</entry><entry>NB</entry><entry>0.013</entry><entry>0.051</entry></row><row><entry>ADI-18931</entry><entry>285</entry><entry>5.65E-10</entry><entry>NB</entry><entry>4.88E-10</entry><entry>NB</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18932</entry><entry>286</entry><entry>9.17E-10</entry><entry>NB</entry><entry>1.39E-09</entry><entry>NB</entry><entry>0.184</entry><entry>0.351</entry></row><row><entry>ADI-18933</entry><entry>287</entry><entry>NB</entry><entry>2.00E-08</entry><entry>NB</entry><entry>6.16E-10</entry><entry>0.075</entry><entry>0.137</entry></row><row><entry>ADI-18935</entry><entry>288</entry><entry>6.60E-10</entry><entry>NB</entry><entry>5.82E-10</entry><entry>NB</entry><entry>0.779</entry><entry>0.355</entry></row><row><entry>ADI-18936</entry><entry>289</entry><entry>3.03E-10</entry><entry>NB</entry><entry>2.98E-10</entry><entry>NB</entry><entry>0.032</entry><entry>0.035</entry></row><row><entry>ADI-18937</entry><entry>290</entry><entry>2.89E-10</entry><entry>NB</entry><entry>2.73E-10</entry><entry>NB</entry><entry>0.084</entry><entry>0.508</entry></row><row><entry>ADI-18938</entry><entry>291</entry><entry>1.65E-10</entry><entry>2.16E-10</entry><entry>1.50E-10</entry><entry>1.68E-10</entry><entry>0.837</entry><entry>4.255</entry></row><row><entry>ADI-18939</entry><entry>292</entry><entry>5.74E-10</entry><entry>NB</entry><entry>5.60E-10</entry><entry>NB</entry><entry>0.018</entry><entry>0.038</entry></row><row><entry>ADI-18940</entry><entry>293</entry><entry>1.12E-09</entry><entry>NB</entry><entry>1.56E-09</entry><entry>NB</entry><entry>0.023</entry><entry>0.063</entry></row><row><entry>ADI-18941</entry><entry>294</entry><entry>NB</entry><entry>1.91E-08</entry><entry>NB</entry><entry>6.45E-10</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18942</entry><entry>295</entry><entry>8.65E-10</entry><entry>2.81E-10</entry><entry>5.12E-10</entry><entry>2.58E-10</entry><entry>0.374</entry><entry>0.614</entry></row><row><entry>ADI-18943</entry><entry>296</entry><entry>6.46E-10</entry><entry>NB</entry><entry>7.25E-10</entry><entry>NB</entry><entry>0.027</entry><entry>0.043</entry></row><row><entry>ADI-18944</entry><entry>297</entry><entry>3.09E-10</entry><entry>NB</entry><entry>3.37E-10</entry><entry>NB</entry><entry>0.026</entry><entry>0.074</entry></row><row><entry>ADI-18946</entry><entry>298</entry><entry>1.58E-10</entry><entry>2.06E-10</entry><entry>1.57E-10</entry><entry>1.66E-10</entry><entry>0.093</entry><entry>0.227</entry></row><row><entry>ADI-18947</entry><entry>299</entry><entry>2.45E-09</entry><entry>2.96E-10</entry><entry>2.20E-09</entry><entry>2.82E-10</entry><entry>1.299</entry><entry>3.602</entry></row><row><entry>ADI-18948</entry><entry>300</entry><entry>4.55E-09</entry><entry>2.57E-10</entry><entry>2.10E-09</entry><entry>2.47E-10</entry><entry>1.123</entry><entry>4.346</entry></row><row><entry>ADI-18949</entry><entry>301</entry><entry>3.07E-10</entry><entry>NB</entry><entry>3.08E-10</entry><entry>NB</entry><entry>0.040</entry><entry>0.076</entry></row><row><entry>ADI-18950</entry><entry>302</entry><entry>1.13E-09</entry><entry>3.93E-10</entry><entry>4.18E-09</entry><entry>3.85E-10</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18951</entry><entry>303</entry><entry>7.52E-10</entry><entry>1.29E-09</entry><entry>5.35E-09</entry><entry>1.13E-09</entry><entry>3.398</entry><entry>>10</entry></row><row><entry>ADI-18952</entry><entry>304</entry><entry>7.15E-10</entry><entry>7.29E-10</entry><entry>1.01E-09</entry><entry>6.33E-10</entry><entry>1.589</entry><entry>2.745</entry></row><row><entry>ADI-18953</entry><entry>305</entry><entry>5.13E-10</entry><entry>NB</entry><entry>4.21E-10</entry><entry>NB</entry><entry>0.034</entry><entry>0.022</entry></row><row><entry>ADI-18955</entry><entry>306</entry><entry>5.99E-10</entry><entry>2.56E-10</entry><entry>2.37E-09</entry><entry>2.50E-10</entry><entry>1.933</entry><entry>3.116</entry></row><row><entry>ADI-18956</entry><entry>307</entry><entry>1.82E-10</entry><entry>NB</entry><entry>2.24E-10</entry><entry>NB</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18957</entry><entry>308</entry><entry>4.69E-10</entry><entry>NB</entry><entry>3.24E-10</entry><entry>NB</entry><entry>1.339</entry><entry>6.084</entry></row><row><entry>ADI-18958</entry><entry>309</entry><entry>2.86E-10</entry><entry>NB</entry><entry>3.02E-10</entry><entry>NB</entry><entry>0.587</entry><entry>3.364</entry></row><row><entry>ADI-18959</entry><entry>310</entry><entry>4.68E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>7.214</entry><entry>2.258</entry></row><row><entry>ADI-18960</entry><entry>311</entry><entry>1.78E-10</entry><entry>NB</entry><entry>1.83E-10</entry><entry>NB</entry><entry>0.034</entry><entry>0.107</entry></row><row><entry>ADI-18962</entry><entry>312</entry><entry>8.83E-09</entry><entry>NB</entry><entry>2.28E-08</entry><entry>NB</entry><entry>4.439</entry><entry>>10</entry></row><row><entry>ADI-18965</entry><entry>313</entry><entry /><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>3.023</entry><entry>6.892</entry></row><row><entry>ADI-18966</entry><entry>314</entry><entry>5.78E-10</entry><entry>NB</entry><entry>5.62E-10</entry><entry>NB</entry><entry>0.044</entry><entry>0.130</entry></row><row><entry>ADI-18967</entry><entry>315</entry><entry>8.09E-10</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>6.737</entry><entry>3.651</entry></row><row><entry>ADI-18968</entry><entry>316</entry><entry>1.98E-09</entry><entry>4.38E-10</entry><entry>6.02E-10</entry><entry>3.05E-10</entry><entry>0.909</entry><entry>0.541</entry></row><row><entry>ADI-18969</entry><entry>317</entry><entry>3.03E-10</entry><entry>NB</entry><entry>2.97E-10</entry><entry>NB</entry><entry>0.035</entry><entry>0.187</entry></row><row><entry>ADI-18970</entry><entry>318</entry><entry>1.04E-08</entry><entry>6.84E-09</entry><entry>4.45E-10</entry><entry>4.12E-10</entry><entry>>10</entry><entry>0.333</entry></row><row><entry>ADI-18971</entry><entry>319</entry><entry>1.57E-10</entry><entry>NB</entry><entry>1.68E-10</entry><entry>NB</entry><entry>0.039</entry><entry>0.114</entry></row><row><entry>ADI-18972</entry><entry>320</entry><entry>2.98E-10</entry><entry>NB</entry><entry>3.68E-10</entry><entry>NB</entry><entry>0.016</entry><entry>0.107</entry></row><row><entry>ADI-18973</entry><entry>321</entry><entry>3.78E-09</entry><entry>4.95E-10</entry><entry>2.63E-09</entry><entry>3.94E-10</entry><entry>9.605</entry><entry>6.273</entry></row><row><entry>ADI-18974</entry><entry>322</entry><entry>2.53E-10</entry><entry>NB</entry><entry>2.90E-10</entry><entry>NB</entry><entry>0.030</entry><entry>0.105</entry></row><row><entry>ADI-18975</entry><entry>323</entry><entry>2.67E-10</entry><entry>NB</entry><entry>2.98E-10</entry><entry>NB</entry><entry>0.037</entry><entry>0.174</entry></row><row><entry>ADI-18976</entry><entry>324</entry><entry>4.03E-09</entry><entry>2.36E-09</entry><entry>1.24E-09</entry><entry>2.09E-10</entry><entry>6.290</entry><entry>10.600</entry></row><row><entry>ADI-18977</entry><entry>325</entry><entry>7.86E-10</entry><entry>NB</entry><entry>9.66E-10</entry><entry>NB</entry><entry>0.108</entry><entry>0.117</entry></row><row><entry>ADI-18978</entry><entry>326</entry><entry>3.00E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18979</entry><entry>327</entry><entry>1.89E-10</entry><entry>NB</entry><entry>1.84E-10</entry><entry>NB</entry><entry>0.012</entry><entry>0.031</entry></row><row><entry>ADI-18980</entry><entry>328</entry><entry>NB</entry><entry>5.33E-10</entry><entry>NB</entry><entry>3.50E-10</entry><entry>3.599</entry><entry>>10</entry></row><row><entry>ADI-18981</entry><entry>329</entry><entry>1.53E-09</entry><entry>3.53E-10</entry><entry>1.15E-09</entry><entry>2.80E-10</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18982</entry><entry>330</entry><entry>1.92E-10</entry><entry>7.65E-10</entry><entry>1.95E-10</entry><entry>7.47E-10</entry><entry>0.018</entry><entry>0.053</entry></row><row><entry>ADI-18983</entry><entry>331</entry><entry>1.71E-09</entry><entry>NB</entry><entry>5.81E-10</entry><entry>NB</entry><entry>0.028</entry><entry>0.075</entry></row><row><entry>ADI-18984</entry><entry>332</entry><entry>1.29E-08</entry><entry>8.03E-10</entry><entry>6.08E-09</entry><entry>6.59E-10</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18985</entry><entry>333</entry><entry>5.66E-10</entry><entry>NB</entry><entry>1.70E-09</entry><entry>NB</entry><entry>0.034</entry><entry>0.090</entry></row><row><entry>ADI-18986</entry><entry>334</entry><entry>2.68E-09</entry><entry>NB</entry><entry>2.38E-10</entry><entry>NB</entry><entry>0.464</entry><entry>0.123</entry></row><row><entry>ADI-18987</entry><entry>335</entry><entry>4.49E-10</entry><entry>NB</entry><entry>5.24E-10</entry><entry>NB</entry><entry>0.015</entry><entry>0.027</entry></row><row><entry>ADI-18988</entry><entry>336</entry><entry>2.93E-10</entry><entry>NB</entry><entry>3.70E-10</entry><entry>NB</entry><entry>0.089</entry><entry>0.370</entry></row><row><entry>ADI-18989</entry><entry>337</entry><entry>3.51E-09</entry><entry>3.56E-10</entry><entry>3.92E-09</entry><entry>3.77E-10</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18990</entry><entry>338</entry><entry>8.90E-10</entry><entry>2.94E-10</entry><entry>4.91E-10</entry><entry>2.52E-10</entry><entry>0.580</entry><entry>0.845</entry></row><row><entry>ADI-18991</entry><entry>339</entry><entry>1.35E-10</entry><entry /><entry>1.52E-10</entry><entry /><entry>0.028</entry><entry>0.228</entry></row><row><entry>ADI-18992</entry><entry>340</entry><entry>7.66E-10</entry><entry>1.53E-09</entry><entry>9.69E-10</entry><entry>9.07E-10</entry><entry>2.546</entry><entry>5.692</entry></row><row><entry>ADI-18993</entry><entry>341</entry><entry>2.55E-10</entry><entry>NB</entry><entry>2.77E-10</entry><entry>NB</entry><entry>0.078</entry><entry>0.128</entry></row><row><entry>ADI-18994</entry><entry>342</entry><entry>3.10E-10</entry><entry>NB</entry><entry>3.31E-10</entry><entry>NB</entry><entry>0.047</entry><entry>0.108</entry></row><row><entry>ADI-18995</entry><entry>343</entry><entry>1.20E-10</entry><entry>1.23E-08</entry><entry>1.27E-10</entry><entry /><entry>0.043</entry><entry>0.125</entry></row><row><entry>ADI-18996</entry><entry>344</entry><entry>2.52E-09</entry><entry>NB</entry><entry>3.60E-09</entry><entry>NB</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18997</entry><entry>345</entry><entry>5.01E-09</entry><entry>NB</entry><entry>5.32E-09</entry><entry>NB</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-18998</entry><entry>346</entry><entry>1.57E-10</entry><entry>1.24E-08</entry><entry>1.72E-10</entry><entry>NB</entry><entry>0.055</entry><entry>0.458</entry></row><row><entry>ADI-18999</entry><entry>347</entry><entry>5.92E-10</entry><entry>1.67E-10</entry><entry>1.02E-09</entry><entry>1.41E-10</entry><entry>1.805</entry><entry>6.465</entry></row><row><entry>ADI-19000</entry><entry>348</entry><entry>1.10E-10</entry><entry>1.75E-10</entry><entry>1.04E-10</entry><entry>1.28E-10</entry><entry>0.037</entry><entry>0.129</entry></row><row><entry>ADI-19001</entry><entry>349</entry><entry>1.07E-09</entry><entry>1.93E-10</entry><entry>1.06E-09</entry><entry>1.49E-10</entry><entry>>10</entry><entry>3.259</entry></row><row><entry>ADI-19002</entry><entry>350</entry><entry>1.63E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>2.886</entry><entry>4.507</entry></row><row><entry>ADI-19003</entry><entry>351</entry><entry>1.61E-10</entry><entry>NB</entry><entry>1.68E-10</entry><entry>NB</entry><entry>0.047</entry><entry>0.125</entry></row><row><entry>ADI-19004</entry><entry>352</entry><entry>2.28E-10</entry><entry>NB</entry><entry>2.73E-10</entry><entry>NB</entry><entry>0.020</entry><entry>0.128</entry></row><row><entry>ADI-19005</entry><entry>353</entry><entry>9.63E-10</entry><entry>NB</entry><entry>9.64E-10</entry><entry>NB</entry><entry>0.041</entry><entry>0.110</entry></row><row><entry>ADI-19006</entry><entry>354</entry><entry>1.75E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>4.891</entry><entry>5.059</entry></row><row><entry>ADI-19007</entry><entry>355</entry><entry>6.18E-10</entry><entry>9.69E-10</entry><entry>6.08E-10</entry><entry>4.57E-10</entry><entry>0.208</entry><entry>0.370</entry></row><row><entry>ADI-19008</entry><entry>356</entry><entry>3.63E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>8.293</entry><entry>>10</entry></row><row><entry>ADI-19009</entry><entry>357</entry><entry>4.42E-10</entry><entry>NB</entry><entry>4.66E-10</entry><entry>NB</entry><entry>0.062</entry><entry>0.066</entry></row><row><entry>ADI-19010</entry><entry>358</entry><entry>6.04E-09</entry><entry>NB</entry><entry>2.84E-09</entry><entry>NB</entry><entry>>10</entry><entry>0.650</entry></row><row><entry>ADI-19011</entry><entry>359</entry><entry>2.15E-09</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>>10</entry><entry>6.237</entry></row><row><entry>ADI-19012</entry><entry>360</entry><entry>2.89E-09</entry><entry>3.04E-10</entry><entry>1.14E-09</entry><entry>3.14E-10</entry><entry>>10</entry><entry>>10</entry></row><row><entry>ADI-19013</entry><entry>361</entry><entry>NB</entry><entry>1.61E-08</entry><entry>NB</entry><entry>5.83E-10</entry><entry>9.504</entry><entry>>10</entry></row><row><entry>ADI-19014</entry><entry>362</entry><entry>2.82E-09</entry><entry>3.59E-10</entry><entry>2.21E-09</entry><entry>2.77E-10</entry><entry>1.745</entry><entry>>10</entry></row><row><entry>ADI-19016</entry><entry>363</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>NB</entry><entry>0.052</entry><entry>0.092</entry></row><row><entry>ADI-19017</entry><entry>364</entry><entry>1.10E-08</entry><entry>NB</entry><entry>7.20E-09</entry><entry>NB</entry><entry>1.562</entry><entry>0.795</entry></row></tbody></tgroup><tgroup cols="8" rowsep="0"><colspec colnum="1" colname="col1" colwidth="15mm" align="justify" /><colspec colnum="2" colname="col2" colwidth="20mm" /><colspec colnum="3" colname="col3" colwidth="25mm" /><colspec colnum="4" colname="col4" colwidth="23mm" /><colspec colnum="5" colname="col5" colwidth="25mm" /><colspec colnum="6" colname="col6" colwidth="23mm" /><colspec colnum="7" colname="col7" colwidth="20mm" /><colspec colnum="8" colname="col8" colwidth="20mm" /><tbody><row><entry namest="col1" nameend="col8">*NN; non-neutralizing, NB; non-binding, ND; not determined. IgG KDs were calculated for antibodies with BLI binding responses >0.1 nm. Antibodies with BLI binding responses <0.05 nm were designated as NB</entry></row></tbody></tgroup></table></tables><tables id="tabl0004" num="0004"><table frame="all"><title><b>Table 4: Bin, patch, and antigenic site assignments for anti-RSV antibodies</b></title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="21mm" align="center" /><colspec colnum="2" colname="col2" colwidth="40mm" align="center" /><colspec colnum="3" colname="col3" colwidth="30mm" align="center" /><colspec colnum="4" colname="col4" colwidth="32mm" align="center" /><colspec colnum="5" colname="col5" colwidth="45mm" align="center" /><thead valign="top"><row><entry><b>Name</b></entry><entry><b>Antibody number (Ab #)</b></entry><entry><b>Bin Assignment</b></entry><entry><b>Patch Assignment</b></entry><entry><b>Antigenic Site Assignment</b></entry></row></thead><tbody><row><entry>ADI-18875</entry><entry>232</entry><entry>D25</entry><entry>1,2</entry><entry>Ø</entry></row><row><entry>ADI-18876</entry><entry>233</entry><entry>Mota</entry><entry>5</entry><entry>II</entry></row><row><entry>ADI-18877</entry><entry>234</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18878</entry><entry>235</entry><entry>101F/13390</entry><entry /><entry /></row><row><entry>ADI-18879</entry><entry>236</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18880</entry><entry>237</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18882</entry><entry>238</entry><entry>D25</entry><entry>1,2</entry><entry>Ø</entry></row><row><entry>ADI-18883</entry><entry>239</entry><entry>D25</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18884</entry><entry>240</entry><entry>14469</entry><entry /><entry>I</entry></row><row><entry>ADI-18885</entry><entry>241</entry><entry>101F/13390</entry><entry /><entry /></row><row><entry>ADI-18887</entry><entry>242</entry><entry>Mota/13390</entry><entry /><entry /></row><row><entry>ADI-18888</entry><entry>243</entry><entry>Mota/101F/13390</entry><entry /><entry /></row><row><entry>ADI-18889</entry><entry>244</entry><entry>D25</entry><entry>1,2</entry><entry>Ø</entry></row><row><entry>ADI-18890</entry><entry>245</entry><entry>D25</entry><entry>2</entry><entry>Ø</entry></row><row><entry>ADI-18891</entry><entry>246</entry><entry>D25</entry><entry>2,1</entry><entry>Ø</entry></row><row><entry>ADI-18892</entry><entry>247</entry><entry>Mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18893</entry><entry>248</entry><entry>Mota/MPE8</entry><entry /><entry>III</entry></row><row><entry>ADI-18894</entry><entry>249</entry><entry>D25</entry><entry /><entry /></row><row><entry>ADI-18895</entry><entry>250</entry><entry>Unknown</entry><entry /><entry /></row><row><entry>ADI-18896</entry><entry>251</entry><entry>101F/13390</entry><entry /><entry /></row><row><entry>ADI-18897</entry><entry>252</entry><entry>Mota/101F/13390</entry><entry /><entry>III</entry></row><row><entry>ADI-18898</entry><entry>253</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18899</entry><entry>254</entry><entry>101F/13390</entry><entry /><entry>I</entry></row><row><entry>ADI-18900</entry><entry>255</entry><entry>D25</entry><entry>1</entry><entry>Ø</entry></row><row><entry>ADI-18901</entry><entry>256</entry><entry>Unknown</entry><entry /><entry /></row><row><entry>ADI-18902</entry><entry>257</entry><entry>14443</entry><entry>9</entry><entry>IV</entry></row><row><entry>ADI-18903</entry><entry>258</entry><entry>D25/mota/MPE8</entry><entry>4,3</entry><entry>V</entry></row><row><entry>ADI-18904</entry><entry>259</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18905</entry><entry>260</entry><entry>MPE8</entry><entry /><entry /></row><row><entry>ADI-18906</entry><entry>261</entry><entry>Mota/101F/13390</entry><entry /><entry /></row><row><entry>ADI-18907</entry><entry>262</entry><entry /><entry /><entry>UK</entry></row><row><entry>ADI-18908</entry><entry>263</entry><entry>Mota/MPE8</entry><entry /><entry>III</entry></row><row><entry>ADI-18909</entry><entry>264</entry><entry>Mota</entry><entry>5</entry><entry>II</entry></row><row><entry>ADI-18910</entry><entry>265</entry><entry>Unknown</entry><entry /><entry /></row><row><entry>ADI-18911</entry><entry>266</entry><entry>Mota</entry><entry /><entry /></row><row><entry>ADI-18912</entry><entry>267</entry><entry>Mota/MPE8</entry><entry /><entry>III</entry></row><row><entry>ADI-18913</entry><entry>268</entry><entry>D25</entry><entry>1</entry><entry>Ø</entry></row><row><entry>ADI-18915</entry><entry>269</entry><entry>D25/mota</entry><entry /><entry /></row><row><entry>ADI-18916</entry><entry>270</entry><entry>D25</entry><entry>1</entry><entry>Ø</entry></row><row><entry>ADI-18917</entry><entry>271</entry><entry>Mota/MPE8</entry><entry /><entry>III</entry></row><row><entry>ADI-18918</entry><entry>272</entry><entry>Mota</entry><entry /><entry /></row><row><entry>ADI-18919</entry><entry>273</entry><entry /><entry /><entry>UK</entry></row><row><entry>ADI-18920</entry><entry>274</entry><entry>101F</entry><entry>9</entry><entry>IV</entry></row><row><entry>ADI-18921</entry><entry>275</entry><entry>101F</entry><entry /><entry /></row><row><entry>ADI-18922</entry><entry>276</entry><entry>Mota</entry><entry /><entry /></row><row><entry>ADI-18923</entry><entry>277</entry><entry>Mota/MPE8/101F</entry><entry /><entry>III</entry></row><row><entry>ADI-18924</entry><entry>278</entry><entry>Unknown</entry><entry /><entry /></row><row><entry>ADI-18925</entry><entry>279</entry><entry>101F</entry><entry /><entry /></row><row><entry>ADI-18926</entry><entry>280</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18927</entry><entry>281</entry><entry>D25/mota</entry><entry /><entry /></row><row><entry>ADI-18928</entry><entry>282</entry><entry>D25</entry><entry>1,2</entry><entry>Ø</entry></row><row><entry>ADI-18929</entry><entry>283</entry><entry>101F</entry><entry>1</entry><entry>UK</entry></row><row><entry>ADI-18930</entry><entry>284</entry><entry>D25</entry><entry>1,2</entry><entry>Ø</entry></row><row><entry>ADI-18931</entry><entry>285</entry><entry>101F</entry><entry /><entry>IV</entry></row><row><entry>ADI-18932</entry><entry>286</entry><entry>Mota</entry><entry>5</entry><entry>II</entry></row><row><entry>ADI-18933</entry><entry>287</entry><entry>Unknown</entry><entry /><entry /></row><row><entry>ADI-18935</entry><entry>288</entry><entry>Mota</entry><entry>6,5</entry><entry>III</entry></row><row><entry>ADI-18936</entry><entry>289</entry><entry>D25/mota</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18937</entry><entry>290</entry><entry>Mota</entry><entry>5</entry><entry>II</entry></row><row><entry>ADI-18938</entry><entry>291</entry><entry>Mota/101F</entry><entry /><entry>III</entry></row><row><entry>ADI-18939</entry><entry>292</entry><entry>D25</entry><entry>9</entry><entry>Ø</entry></row><row><entry>ADI-18940</entry><entry>293</entry><entry>D25/mota</entry><entry>1,2</entry><entry>V</entry></row><row><entry>ADI-18941</entry><entry>294</entry><entry>Mota</entry><entry /><entry /></row><row><entry>ADI-18942</entry><entry>295</entry><entry>Mota</entry><entry /><entry>II</entry></row><row><entry>ADI-18943</entry><entry>296</entry><entry>D25</entry><entry>5</entry><entry>UK</entry></row><row><entry>ADI-18944</entry><entry>297</entry><entry>D25/mota/MPE8</entry><entry>1,2</entry><entry>V</entry></row><row><entry>ADI-18946</entry><entry>298</entry><entry>101F</entry><entry>4</entry><entry>IV</entry></row><row><entry>ADI-18947</entry><entry>299</entry><entry>101F/13390</entry><entry /><entry /></row><row><entry>ADI-18948</entry><entry>300</entry><entry>101F/13390</entry><entry /><entry /></row><row><entry>ADI-18949</entry><entry>301</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18950</entry><entry>302</entry><entry>13390</entry><entry /><entry>I</entry></row><row><entry>ADI-18951</entry><entry>303</entry><entry>Mota/13390</entry><entry /><entry>III</entry></row><row><entry>ADI-18952</entry><entry>304</entry><entry>Mota/13390</entry><entry /><entry>III</entry></row><row><entry>ADI-18953</entry><entry>305</entry><entry>D25</entry><entry>2</entry><entry>Ø</entry></row><row><entry>ADI-18955</entry><entry>306</entry><entry>101F/13390</entry><entry /><entry>I</entry></row><row><entry>ADI-18956</entry><entry>307</entry><entry>14469</entry><entry /><entry>I</entry></row><row><entry>ADI-18957</entry><entry>308</entry><entry>Mota/MPE8</entry><entry /><entry>III</entry></row><row><entry>ADI-18958</entry><entry>309</entry><entry>Mota</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18959</entry><entry>310</entry><entry>14443</entry><entry /><entry /></row><row><entry>ADI-18960</entry><entry>311</entry><entry>14469</entry><entry>9</entry><entry>IV</entry></row><row><entry>ADI-18962</entry><entry>312</entry><entry>Mota/MPE8</entry><entry /><entry /></row><row><entry>ADI-18965</entry><entry>313</entry><entry>Unknown</entry><entry /><entry /></row><row><entry>ADI-18966</entry><entry>314</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18967</entry><entry>315</entry><entry>101F</entry><entry>9</entry><entry>IV</entry></row><row><entry>ADI-18968</entry><entry>316</entry><entry>Mota</entry><entry>5</entry><entry>II</entry></row><row><entry>ADI-18969</entry><entry>317</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18970</entry><entry>318</entry><entry>Mota/MPE8</entry><entry /><entry /></row><row><entry>ADI-18971</entry><entry>319</entry><entry>14469</entry><entry>9</entry><entry>IV</entry></row><row><entry>ADI-18972</entry><entry>320</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18973</entry><entry>321</entry><entry>13390</entry><entry /><entry /></row><row><entry>ADI-18974</entry><entry>322</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18975</entry><entry>323</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18976</entry><entry>324</entry><entry>13390</entry><entry /><entry /></row><row><entry>ADI-18977</entry><entry>325</entry><entry>D25/mota</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18978</entry><entry>326</entry><entry>14469</entry><entry /><entry /></row><row><entry>ADI-18979</entry><entry>327</entry><entry>Mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18980</entry><entry>328</entry><entry>Mota</entry><entry /><entry /></row><row><entry>ADI-18981</entry><entry>329</entry><entry>101F/13390</entry><entry /><entry>I</entry></row><row><entry>ADI-18982</entry><entry>330</entry><entry>Mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18983</entry><entry>331</entry><entry>101F</entry><entry>3,9</entry><entry>Q</entry></row><row><entry>ADI-18984</entry><entry>332</entry><entry>13390</entry><entry /><entry /></row><row><entry>ADI-18985</entry><entry>333</entry><entry>D25/mota</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-18986</entry><entry>334</entry><entry>D25</entry><entry /><entry /></row><row><entry>ADI-18987</entry><entry>335</entry><entry>D25</entry><entry /><entry>Ø</entry></row><row><entry>ADI-18988</entry><entry>336</entry><entry>Mota/MPE8</entry><entry /><entry>III</entry></row><row><entry>ADI-18989</entry><entry>337</entry><entry>101F/13390</entry><entry /><entry /></row><row><entry>ADI-18990</entry><entry>338</entry><entry>Mota</entry><entry>5</entry><entry>II</entry></row><row><entry>ADI-18991</entry><entry>339</entry><entry>14443</entry><entry>9</entry><entry>IV</entry></row><row><entry>ADI-18992</entry><entry>340</entry><entry>101F</entry><entry /><entry>IV</entry></row><row><entry>ADI-18993</entry><entry>341</entry><entry>Mota/MPE8</entry><entry /><entry>III</entry></row><row><entry>ADI-18994</entry><entry>342</entry><entry>Mota/MPE8</entry><entry /><entry>III</entry></row><row><entry>ADI-18995</entry><entry>343</entry><entry>14443</entry><entry /><entry>IV</entry></row><row><entry>ADI-18996</entry><entry>344</entry><entry>Unknown</entry><entry /><entry /></row><row><entry>ADI-18997</entry><entry>345</entry><entry>D25/mota</entry><entry /><entry /></row><row><entry>ADI-18998</entry><entry>346</entry><entry>14443</entry><entry>9</entry><entry>IV</entry></row><row><entry>ADI-18999</entry><entry>347</entry><entry>101F</entry><entry>9</entry><entry>IV</entry></row><row><entry>ADI-19000</entry><entry>348</entry><entry>14443</entry><entry>9</entry><entry>IV</entry></row><row><entry>ADI-19001</entry><entry>349</entry><entry>101F/13390</entry><entry /><entry>I</entry></row><row><entry>ADI-19002</entry><entry>350</entry><entry>Unknown</entry><entry /><entry>UK</entry></row><row><entry>ADI-19003</entry><entry>351</entry><entry>14469</entry><entry>9</entry><entry>IV</entry></row><row><entry>ADI-19004</entry><entry>352</entry><entry>D25/mota/MPE8</entry><entry>4</entry><entry>V</entry></row><row><entry>ADI-19005</entry><entry>353</entry><entry>D25/mota</entry><entry /><entry>V</entry></row><row><entry>ADI-19006</entry><entry>354</entry><entry>Unknown</entry><entry /><entry>UK</entry></row><row><entry>ADI-19007</entry><entry>355</entry><entry>Mota/MPE8</entry><entry>5</entry><entry>II</entry></row><row><entry>ADI-19008</entry><entry>356</entry><entry>Unknown</entry><entry /><entry /></row><row><entry>ADI-19009</entry><entry>357</entry><entry>D25</entry><entry>1</entry><entry>Ø</entry></row><row><entry>ADI-19010</entry><entry>358</entry><entry>Mota/MPE8</entry><entry /><entry /></row><row><entry>ADI-19011</entry><entry>359</entry><entry>Unknown</entry><entry /><entry /></row><row><entry>ADI-19012</entry><entry>360</entry><entry>13390</entry><entry /><entry /></row><row><entry>ADI-19013</entry><entry>361</entry><entry>Unknown</entry><entry /><entry /></row><row><entry>ADI-19014</entry><entry>362</entry><entry>101F/13390</entry><entry /><entry /></row><row><entry>ADI-19016</entry><entry>363</entry><entry>Mota/MPE8</entry><entry /><entry /></row><row><entry>ADI-19017</entry><entry>364</entry><entry>D25/mota</entry><entry /><entry /></row></tbody></tgroup></table></tables><tables id="tabl0005" num="0005"><table frame="all"><title><b>Table 5: A subset of anti-RSV F antibodies cross-neutralize human metapneumovirus.</b></title><tgroup cols="7"><colspec colnum="1" colname="col1" colwidth="16mm" align="center" /><colspec colnum="2" colname="col2" colwidth="28mm" align="center" /><colspec colnum="3" colname="col3" colwidth="22mm" align="center" /><colspec colnum="4" colname="col4" colwidth="25mm" align="center" /><colspec colnum="5" colname="col5" colwidth="28mm" align="center" /><colspec colnum="6" colname="col6" colwidth="28mm" align="center" /><colspec colnum="7" colname="col7" colwidth="22mm" align="center" /><thead valign="middle"><row><entry>Name</entry><entry>Antibody number (Ab #)</entry><entry>HMPV-A1 IC<sub>50</sub> (µg/ml)</entry><entry>RSV-A2 IC<sub>50</sub> (µg/ml)</entry><entry>Prefusion RSV F <i>K</i><sub>D</sub> (M)</entry><entry>Postfusion RSV F <i>K</i><sub>D</sub> (M)</entry><entry>RSV F Binding Site</entry></row></thead><tbody valign="middle"><row><entry>ADI-18992</entry><entry /><entry>6.1</entry><entry>2.5</entry><entry>7.6 x 10<sup>-10</sup></entry><entry>1.5 x 10<sup>-9</sup></entry><entry>IV*</entry></row><row><entry>MPE8 Control</entry><entry>N/A</entry><entry>0.07</entry><entry>0.04</entry><entry>-</entry><entry>-</entry><entry>-</entry></row></tbody></tgroup><tgroup cols="7" rowsep="0"><colspec colnum="1" colname="col1" colwidth="16mm" align="justify" /><colspec colnum="2" colname="col2" colwidth="28mm" /><colspec colnum="3" colname="col3" colwidth="22mm" /><colspec colnum="4" colname="col4" colwidth="25mm" /><colspec colnum="5" colname="col5" colwidth="28mm" /><colspec colnum="6" colname="col6" colwidth="28mm" /><colspec colnum="7" colname="col7" colwidth="22mm" /><tbody><row><entry namest="col1" nameend="col7">N/A, not applicable *Binding site assignment based on competition only.</entry></row></tbody></tgroup></table></tables>
Materials and Methods
Study design
To profile the antibody response to RSV F, peripheral blood mononuclear cells were obtained from a adult donor approximately between 20-35 years of age, and monoclonal antibodies from RSV F-reactive B cells were isolated thereform. The antibodies were characterized by sequencing, binding, epitope mapping, and neutralization assays. All samples for this study were collected with informed consent of volunteers. This study was unblinded and not randomized. At least two independent experiments were performed for each assay.
Generation of RSV F sorting probes
The soluble prefusion and postfusion probes were based on the RSV F ΔFP and DS-Cav1 constructs that we previously crystallized and determined to be in the pre- and postfusion conformations, respectively (<i>11, 15</i>)<i>.</i> To increase the avidity of the probes and to uniformly orient the RSV F proteins, the trimeric RSV F proteins were coupled to tetrameric streptavidin through biotinylation of a C-terminal AviTag. For each probe, both a C-terminal His-Avi tagged version and a C-terminal StrepTagII version were co-transfected into FreeStyle 293-F cells. The secreted proteins were purified first over Ni-NTA resin to remove trimers lacking the His-Avi tag. The elution from the Ni-NTA purification was then purified over <i>Strep</i>-Tactin resin. Due to the low avidity of a single StrepTagII for the <i>Strep</i>-Tactin resin, additional washing steps were able to remove singly StrepTagged trimers. This resulted in the purification of trimers containing two StrepTagII tagged monomers and therefore only one His-Avi tagged monomer. This purification scheme results in a single AviTag per trimer which greatly reduces the aggregation or 'daisy-chaining' that occurs when trimeric proteins containing three AviTags are incubated with tetrameric streptavidin. RSV F trimers were biotinylated using biotin ligase BirA according to the manufacturer's instructions (Avidity, LLC). Biotinylated proteins were separated from excess biotin by size-exclusion chromotography on a Superdex 200 column (GE Healthcare). Quantification of the number of biotin moieties per RSV F trimer was performed using the Quant*Tag Biotin Kit per the manufacturer's instructions (Vector Laboratories).
Single B-cell sorting
Peripheral blood mononuclear cells were stained using anti-human IgG (BV605), IgA (FITC), CD27 (BV421), CD8 (PerCP-Cy5.5), CD14 (PerCP-Cy5.5), CD19 (PECy7), CD20 (PECy7) and a mixture of dual-labeled DS-Cavl and F ΔFP tetramers (50 nM each). Dual-labeled RSV F tetramers were generated by incubating the individual AviTagged RSV F proteins with premium-grade phycoerythrin-labeled streptavidin (Molecular Probes) or premium-grade allophycocyanin-labeled streptavidin for at least 20 minutes on ice at a molar ratio of 4:1. Tetramers were prepared fresh for each experiment. Single cells were sorted on a BD fluorescence-activated cell sorter Aria II into 96-well PCR plates (BioRad) containing 20 µL/well of lysis buffer [5 µL of 5X first strand cDNA buffer (Invitrogen), 0.25 µL RNaseOUT (Invitrogen), 1.25 µL dithiothreitol (Invitrogen), 0.625 µL NP-40 (New England Biolabs), and 12.6 µL dH<sub>2</sub>O]. Plates were immediately frozen on dry ice before storage at -80°C.
Amplification and cloning of antibody variable genes
Single B cell PCR was performed as described previously (22). Briefly, IgH, Igλ and Igκ variable genes were amplified by RT-PCR and nested PCR reactions using cocktails of IgG and IgA-specific primers (22). The primers used in the second round of PCR contained 40 base pairs of 5' and 3' homology to the cut expression vectors to allow for cloning by homologous recombination into <i>Saccharomyces cerevisiae</i> (40). PCR products were cloned into <i>S. cerevisiae</i> using the lithium acetate method for chemical transformation (41). Each transformation reaction contained 20 µL of unpurified heavy chain and light chain PCR product and 200 ng of cut heavy and light chain plasmids. Following transformation, individual yeast colonies were picked for sequencing and characterization.
Expression and purification of IgGs and Fab fragments
Anti-RSV F IgGs were expressed in <i>S. cerevisiae</i> cultures grown in 24-well plates, as described previously (23). Fab fragments used for competition assays were generated by digesting the IgGs with papain for 2 h at 30 °C. The digestion was terminated by the addition of iodoacetamide, and the Fab and Fc mixtures were passed over Protein A agarose to remove Fc fragments and undigested IgG. The flowthrough of the Protein A resin was then passed over CaptureSelect<sup>™</sup> IgG-CH1 affinity resin (ThermoFischer Scientific), and eluted with 200 mM acetic acid / 50 mM NaCl pH 3.5 into 1/8th volume 2M Hepes pH 8.0. Fab fragments then were buffer-exchanged into PBS pH 7.0.
Biolayer interferometry binding analysis
IgG binding to DS-Cavl and FΔ FP was determined by BLI measurements using a FortéBio Octet HTX instrument (Pall Life Sciences). For high-throughput <i>K</i><sub>D</sub> screening, IgGs were immobilized on AHQ sensors (Pall Life Sciences) and exposed to 100 nM antigen in PBS containing 0.1% BSA (PBSF) for an association step, followed by a dissociation step in PBSF buffer. Data was analyzed using the FortéBio Data Analysis Software 7. The data was fit to a 1:1 binding model to calculate an association and dissociation rate, and <i>K</i><sub>D</sub> was calculated using the ratio <i>k</i><sub>d</sub><i>lk</i><sub>a</sub>.
Antibody competition assays
Antibody competition assays were performed as previously described (23). Antibody competition was measured by the ability of a control anti-RSV F Fab to inhibit binding of yeast surface-expressed anti-RSV F IgGs to either DS-Cavl or FΔ FP. 50 nM biotinylated DS-Cavl or FΔ FP was pre-incubated with 1 µM competitor Fab for 30 min at room temperature and then added to a suspension of yeast expressing anti-RSV F IgG. Unbound antigen was removed by washing with PBS containing 0.1% BSA (PBSF). After washing, bound antigen was detected using streptavidin Alexa Fluor 633 at a 1:500 dilution (Life Technologies) and analyzed by flow cytometry using a FACSCanto II (BD Biosciences). The level of competition was assessed by measuring the fold reduction in antigen binding in the presence of competitor Fab relative to an antigen-only control. Antibodies were considered competitors when a greater than five-fold reduction was observed in the presence of control Fab relative to an antigen-only control.
Expression, purification and biotinylation of preF patch variants
A panel of 9 patches of 2-4 mutations uniformly covering the surface of the preF molecule was designed based on the structure of prefusion RSV F <i>(10).</i> For known antigenic sites, including those recognized by motavizumab, 101F, D25, AM14 and MPE8, patches incorporated residues associated with viral escape or known to be critical for antibody binding. Residues with high conservation across 184 subtype A, subtype B and bovine RSV F sequences were avoided where possible to minimize the likelihood of disrupting protein structure. The mutations present in each patch variant are shown in <figref idref="f0010">Figure 7A</figref>. Mutations for each patch variant were cloned into the prefusion stabilized RSV F (DS-Cav1) construct with a C-terminal AviTag for site specific biotinylation. Proteins were secreted from FreeStyle 293-F cells, purified over Ni-NTA resin and biotinylated using biotin ligase BirA according to the manufacturer's instructions (Avidity, LLC). Biotinylated proteins were separated from excess biotin by size-exclusion chromotography on a Superdex 200 column (GE Healthcare). A deglycosylated version of DS-Cavl was produced by expressing DS-Cavl in the presence of 1 µM kifunensine and digesting with 10% (wt/wt) EndoH before biotinylation.
Luminex assay for patch variant binding
Binding of isolated antibodies to the patch variants was determined using a high-throughput Luminex assay. Each biotinylated variant and a DS-Cavl control were coupled to avidin coated MagPlex beads (Bio-Rad), each with a bead identification number reflecting a unique ratio of red and infrared dyes embedded within the bead. The coupled beads were then mixed with a six-fold serial dilution of each antibody, ranging from 400 nM to 1.4 pM, in a 384-well plate. Beads were washed using a magnetic microplate washer (BioTek) before incubation with a PE conjugated mouse anti-human IgGFc secondary antibody (Southern Biotech). Beads were classified and binding of PE was measured using a FLEXMAP 3D flow cytometer (Luminex).
RSV neutralization assays
Viral stocks were prepared and maintained as previously described <i>(61).</i> Recombinant mKate-RSV expressing prototypic subtype A (strain A2) and subtype B (18537) F genes and the Katushka fluorescent protein were constructed as reported by Hotard <i>et al. (62).</i> HEp-2 cells were maintained in Eagle's minimal essential medium containing 10% fetal bovine serum supplemented with glutamine, penicillin and streptomycin. Antibody neutralization was measured by a fluorescence plate reader neutralization assay <i>(15).</i> A 30 µL solution of culture media containing 2.4 x 10<sup>4</sup> HEp-2 cells was seeded in 384-well black optical bottom plate (Nunc, Thermo Scientific). IgG samples were serially diluted four-fold from 1:10 to 1:163840 and an equal volume of recombinant mKate-RSV A2 was added. Samples were mixed and incubated at 37 °C for one hour. After incubation, 50 µL mixture of sample and virus was added to cells in 384-well plate, and incubated at 37 °C for 22-24 hours. The assay plate was then measured for fluorescence intensity in a microplate reader at Ex 588 nm and Em 635 nm (SpectraMax Paradigm, molecular devices). IC<sub>50</sub> of neutralization for each sample was calculated by curve fitting using Prism (GraphPad Software Inc.).
Human metapneuomovirus neutralization assays
Predetermined amounts of GFP-expressing hMPV recombinant virus (NL/1/00, A1 sublineage, a kind gift of Bernadette van den Hoogen and Ron Fouchier, Rotterdam, the Netherlands) were mixed with serial dilutions of monoclonal antibodies before being added to cultures of Vero-118 cells growing in 96-well plates with Dulbecco's Modified Eagle's medium supplemented with 10% fetal calf serum. Thirty-six hours later, the medium was removed, PBS was added and the amount of GFP per well was measured with a Tecan microplate reader M200. Fluorescence values were represented as percent of a virus control without antibody.
Polyreactivity assay
Antibody polyreactivity was assessed using a previously described high-throughput assay that measures binding to solubilized CHO cell membrane preparations (SMPs) (43). Briefly, two million IgG-presenting yeast were transferred into a 96-well assay plate and pelleted to remove the supernatant. The pellet was resuspended in 50 µL of 1:10 diluted stock b-SMPs and incubated on ice for 20 minutes. Cells were then washed twice with ice-cold PBSF and the cell pellet was re-suspended in 50 µL of secondary labeling mix (Extravidin-R-PE, anti-human LCFITC, and propidium iodide). The mix was incubated on ice for 20 minutes followed by two washes with ice-cold PBSF. Cells were then re-suspended in 100 µL of ice-cold PBSF, and the plate was run on a FACSCanto II (BD Biosciences) using a HTS sample injector. Flow cytometry data was analyzed for mean fluorescence intensity in the R-PE channel and normalized to proper controls in order to assess non-specific binding.
References and Notes:
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An informal sequence listing is provided in Talbe 6, below. The informal sequence listing provides the following sixteen (16) sequence elements contained in each of the 133 antibodies, identified as described above and designated as Antibody Numbers (Ab #) 232 through 364, in the following order: <ul id="ul0002" list-style="bullet" compact="compact"><li>Heavy chain variable region ("HC") nucleic acid sequence</li><li>Heavy chain variable region ("HC") amino acid sequence</li><li>Heavy chain variable region CDR H1 ("H1") amino acid sequence</li><li>Heavy chain variable region CDR H1 ("H1") nucleic acid sequence</li><li>Heavy chain variable region CDR H2 ("H2") amino acid sequence</li><li>Heavy chain variable region CDR H2 ("H2") nucleic acid sequence</li><li>Heavy chain variable region CDR H3 ("H3") amino acid sequence</li><li>Heavy chain variable region CDR H3 ("H3") nucleic acid sequence</li><li>Light chain variable region("LC") nucleic acid sequence</li><li>Light chain variable region ("LC") amino acid sequence</li><li>Light chain variable region CDR L1 ("L1") amino acid sequence</li><li>Light chain variable region CDR L1 ("L1") nucleic acid sequence</li><li>Light chain variable region CDR L2 ("L2") amino acid sequence</li><li>Light chain variable region CDR L2 ("L2") nucleic acid sequence</li><li>Light chain variable region CDR L3 ("L3") amino acid sequence</li><li>Light chain variable region CDR L3 ("L3") nucleic acid sequence</li></ul>
The informal sequence listing for antibodies 365 - 372 provides the following ten (10) sequence elements contained in each of the 8 antibodies, identified as described above and designated as Antibody Numbers (Ab #) 365 through 372, in the following order: <ul id="ul0003" list-style="bullet" compact="compact"><li>Heavy chain variable region ("HC") nucleic acid sequence</li><li>Heavy chain variable region ("HC") amino acid sequence</li><li>Heavy chain variable region CDR H1 ("H1") amino acid sequence</li><li>Heavy chain variable region CDR H2 ("H2") amino acid sequence</li><li>Heavy chain variable region CDR H3 ("H3") amino acid sequence</li><li>Light chain variable region("LC") nucleic acid sequence</li><li>Light chain variable region ("LC") amino acid sequence</li><li>Light chain variable region CDR L1 ("L1") amino acid sequence</li><li>Light chain variable region CDR L2 ("L2") amino acid sequence</li><li>Light chain variable region CDR L3 ("L3") amino acid sequence</li></ul><tables id="tabl0006" num="0006"><img file="EP3974447A2_D0270.tif" /></tables><tables id="tabl0007" num="0007"><img file="EP3974447A2_D0271.tif" /></tables><tables id="tabl0008" num="0008"><img file="EP3974447A2_D0272.tif" /></tables><tables id="tabl0009" num="0009"><img file="EP3974447A2_D0273.tif" /></tables><tables id="tabl0010" num="0010"><img file="EP3974447A2_D0274.tif" /></tables><tables id="tabl0011" num="0011"><img file="EP3974447A2_D0275.tif" /></tables><tables id="tabl0012" num="0012"><img file="EP3974447A2_D0276.tif" /></tables><tables id="tabl0013" num="0013"><img file="EP3974447A2_D0277.tif" /></tables><tables id="tabl0014" num="0014"><img file="EP3974447A2_D0278.tif" /></tables><tables id="tabl0015" num="0015"><img file="EP3974447A2_D0279.tif" /></tables><tables id="tabl0016" num="0016"><img file="EP3974447A2_D0280.tif" /></tables><tables id="tabl0017" num="0017"><img file="EP3974447A2_D0281.tif" /></tables><tables id="tabl0018" num="0018"><img file="EP3974447A2_D0282.tif" /></tables><tables id="tabl0019" num="0019"><img file="EP3974447A2_D0283.tif" /></tables><tables id="tabl0020" num="0020"><img file="EP3974447A2_D0284.tif" /></tables><tables id="tabl0021" num="0021"><img file="EP3974447A2_D0285.tif" /></tables><tables id="tabl0022" num="0022"><img file="EP3974447A2_D0286.tif" /></tables><tables id="tabl0023" num="0023"><img file="EP3974447A2_D0287.tif" /></tables><tables id="tabl0024" num="0024"><img file="EP3974447A2_D0288.tif" /></tables><tables id="tabl0025" num="0025"><img file="EP3974447A2_D0289.tif" /></tables><tables id="tabl0026" num="0026"><img file="EP3974447A2_D0290.tif" /></tables><tables id="tabl0027" num="0027"><img file="EP3974447A2_D0291.tif" /></tables><tables id="tabl0028" num="0028"><img file="EP3974447A2_D0292.tif" /></tables><tables id="tabl0029" num="0029"><img file="EP3974447A2_D0293.tif" /></tables><tables id="tabl0030" num="0030"><img file="EP3974447A2_D0294.tif" /></tables><tables id="tabl0031" num="0031"><img 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file="EP3974447A2_D0346.tif" /></tables><tables id="tabl0083" num="0083"><img file="EP3974447A2_D0347.tif" /></tables><tables id="tabl0084" num="0084"><img file="EP3974447A2_D0348.tif" /></tables><tables id="tabl0085" num="0085"><img file="EP3974447A2_D0349.tif" /></tables><tables id="tabl0086" num="0086"><img file="EP3974447A2_D0350.tif" /></tables><tables id="tabl0087" num="0087"><img file="EP3974447A2_D0351.tif" /></tables><tables id="tabl0088" num="0088"><img file="EP3974447A2_D0352.tif" /></tables><tables id="tabl0089" num="0089"><img file="EP3974447A2_D0353.tif" /></tables><tables id="tabl0090" num="0090"><img file="EP3974447A2_D0354.tif" /></tables><tables id="tabl0091" num="0091"><img file="EP3974447A2_D0355.tif" /></tables><tables id="tabl0092" num="0092"><img file="EP3974447A2_D0356.tif" /></tables><tables id="tabl0093" num="0093"><img file="EP3974447A2_D0357.tif" /></tables><tables id="tabl0094" num="0094"><img file="EP3974447A2_D0358.tif" /></tables><tables id="tabl0095" num="0095"><img file="EP3974447A2_D0359.tif" /></tables><tables id="tabl0096" num="0096"><img file="EP3974447A2_D0360.tif" /></tables><tables id="tabl0097" num="0097"><img file="EP3974447A2_D0361.tif" /></tables><tables id="tabl0098" num="0098"><img file="EP3974447A2_D0362.tif" /></tables><tables id="tabl0099" num="0099"><img file="EP3974447A2_D0363.tif" /></tables><tables id="tabl0100" num="0100"><img file="EP3974447A2_D0364.tif" /></tables><tables id="tabl0101" num="0101"><img file="EP3974447A2_D0365.tif" /></tables><tables id="tabl0102" num="0102"><img file="EP3974447A2_D0366.tif" /></tables><tables id="tabl0103" num="0103"><img file="EP3974447A2_D0367.tif" /></tables><tables id="tabl0104" num="0104"><img file="EP3974447A2_D0368.tif" /></tables><tables id="tabl0105" num="0105"><img file="EP3974447A2_D0369.tif" /></tables><tables id="tabl0106" num="0106"><img file="EP3974447A2_D0370.tif" /></tables><tables id="tabl0107" num="0107"><img file="EP3974447A2_D0371.tif" /></tables><tables 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file="EP3974447A2_D0397.tif" /></tables><tables id="tabl0134" num="0134"><img file="EP3974447A2_D0398.tif" /></tables><tables id="tabl0135" num="0135"><img file="EP3974447A2_D0399.tif" /></tables><tables id="tabl0136" num="0136"><img file="EP3974447A2_D0400.tif" /></tables><tables id="tabl0137" num="0137"><img file="EP3974447A2_D0401.tif" /></tables><tables id="tabl0138" num="0138"><img file="EP3974447A2_D0402.tif" /></tables><tables id="tabl0139" num="0139"><img file="EP3974447A2_D0403.tif" /></tables><tables id="tabl0140" num="0140"><img file="EP3974447A2_D0404.tif" /></tables><tables id="tabl0141" num="0141"><img file="EP3974447A2_D0405.tif" /></tables><tables id="tabl0142" num="0142"><img file="EP3974447A2_D0406.tif" /></tables><tables id="tabl0143" num="0143"><img file="EP3974447A2_D0407.tif" /></tables><tables id="tabl0144" num="0144"><img file="EP3974447A2_D0408.tif" /></tables><tables id="tabl0145" num="0145"><img file="EP3974447A2_D0409.tif" /></tables><tables id="tabl0146" num="0146"><img file="EP3974447A2_D0410.tif" /></tables><tables id="tabl0147" num="0147"><img file="EP3974447A2_D0411.tif" /></tables><tables id="tabl0148" num="0148"><img file="EP3974447A2_D0412.tif" /></tables><tables id="tabl0149" num="0149"><img file="EP3974447A2_D0413.tif" /></tables><tables id="tabl0150" num="0150"><img file="EP3974447A2_D0414.tif" /></tables><tables id="tabl0151" num="0151"><img file="EP3974447A2_D0415.tif" /></tables><tables id="tabl0152" num="0152"><img file="EP3974447A2_D0416.tif" /></tables><tables id="tabl0153" num="0153"><img file="EP3974447A2_D0417.tif" /></tables><tables id="tabl0154" num="0154"><img file="EP3974447A2_D0418.tif" /></tables><tables id="tabl0155" num="0155"><img file="EP3974447A2_D0419.tif" /></tables><tables id="tabl0156" num="0156"><img file="EP3974447A2_D0420.tif" /></tables><tables id="tabl0157" num="0157"><img file="EP3974447A2_D0421.tif" /></tables><tables id="tabl0158" num="0158"><img file="EP3974447A2_D0422.tif" /></tables><tables id="tabl0159" num="0159"><img file="EP3974447A2_D0423.tif" /></tables><tables id="tabl0160" num="0160"><img file="EP3974447A2_D0424.tif" /></tables><tables id="tabl0161" num="0161"><img file="EP3974447A2_D0425.tif" /></tables><tables id="tabl0162" num="0162"><img file="EP3974447A2_D0426.tif" /></tables><tables id="tabl0163" num="0163"><img file="EP3974447A2_D0427.tif" /></tables><tables id="tabl0164" num="0164"><img file="EP3974447A2_D0428.tif" /></tables><tables id="tabl0165" num="0165"><img file="EP3974447A2_D0429.tif" /></tables><tables id="tabl0166" num="0166"><img file="EP3974447A2_D0430.tif" /></tables><tables id="tabl0167" num="0167"><img file="EP3974447A2_D0431.tif" /></tables><tables id="tabl0168" num="0168"><img file="EP3974447A2_D0432.tif" /></tables><tables id="tabl0169" num="0169"><img file="EP3974447A2_D0433.tif" /></tables><tables id="tabl0170" num="0170"><img file="EP3974447A2_D0434.tif" /></tables><tables id="tabl0171" num="0171"><img file="EP3974447A2_D0435.tif" /></tables><tables id="tabl0172" num="0172"><img file="EP3974447A2_D0436.tif" /></tables><tables id="tabl0173" num="0173"><img file="EP3974447A2_D0437.tif" /></tables>
Additional Embodiments:
Embodiment 1. An isolated antibody or an antigen-binding fragment there or that specifically binds to Respiratory Syncytial Virus (RSV) F protein (F), wherein at least one of the CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and CDRL3 amino acid sequence of the antibody or the antigen-binding fragment thereof is at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99%; and/or all percentages of identity in between; to at least one the CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and/or a CDRL3 amino acid sequences as disclosed in Table 6 of an antibody selected from Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; and wherein said antibody or the antigen-binding fragment thereof also has one or more of the following characteristics: <ol id="ol0002" compact="compact"><li>a) the antibody or antigen-binding fragment thereof cross-competes with said antibody ar antigen-binding fragment thereof for binding to RSV-F;</li><li>b) the antibody or antigen-binding fragment thereof displays better binding affinity for the PreF form of RSV-F relative to the PostF form;</li><li>c) the antibody or antigen-binding fragment thereof displays a clean or low polyreactivity profile;</li><li>d) the antibody or antigen-binding fragment thereof displays neutralization activity towoard RSV suptype A and RSV subtype B in vitro;</li><li>e) the antibody or antigen-binding fragment thereof displays antigenic site specificity for RSV-F at Site Ø, Site I, Site II, Site III, Site IV, or Site V;</li><li>f) the antibody or antigen-binding fragment thereof displays antigenic site specificity for RSV-F Site Ø, Site V, or Site III relative to RSV-F Site I, Site II, or Site IV;</li><li>g) at least a portion of the epitope with which the antibody or antigen-binding fragment thereof interacts comprises the α3 helix and β3/β4 hairpin of PreF;</li><li>h) the antibody or antigen-binding fragment thereof displays an in vitro neutralization potency (IC<sub>50</sub>) of between about 0.5 microgram/milliliter (ug/ml) to about 5 ug/ml; between about 0.05 ug/ml to about 0.5 ug/ml; or less than about 0.05 mg/ml;</li><li>i) the binding affinity and/or epitopic specificity of the antibody or antigen-binding fragment thereof for any one of the RSV-F variants designated as 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG in <figref idref="f0010">Figure 7A</figref> is reduced or eliminated relative to the binding affinity and/or epitopic specificity of said antibody or antigen-binding fragment thereof for the RSV-F or RSV-F DS-Cavl;</li><li>j) the antibody or antigen-binding fragment thereof of displays a cross-neutalization potency (IC<sub>50</sub>) against human metapneumovirus (HMPV);</li><li>k) the antibody or antigen-binding fragment thereof does not complete with D25, MPE8, palivisumab, motavizumab, or AM-14; or</li><li>l) the the antibody or antigen-binding fragment thereof displays at least about 2-fold; at least about 3-fold; at least about 4-fold; at least about 5-fold; at least about 6-fold; at least about 7-fold; at least about 8-fold; at least about 9-fold; at least about 10-fold; at least about 15-fold; at least about 20-fold; at least about 25-fold; at least about 30-fold; at least about 35-fold; at least about 40-fold; at least about 50-fold; at least about 55-fold; at least about 60-fold; at least about 70-fold; at least about 80-fold; at least about 90-fold; at least about 100-fold; greater than about 100-fold; and folds in between any of the foregoing; greater neutralization potency (IC50) than D25 and/or palivizumab.</li></ol>
Embodiment 2. The isolated antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antibody or antigen-binding fragment thereof comprises: at least two; at least three; at least 4; at least 5; at least 6; at least 7; at least 8; at least 9; at least 10; at least 11; or at least 12; of characteristics a) through 1).
Embodiment 3. The isolated antibody or antigen-binding fragment thereof of Embodiment 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: <ol id="ol0003" compact="compact"><li>a) the CDRH3 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6;</li><li>b) the CDRH2 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6;</li><li>c) the CDRH1 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6;</li><li>d) the CDRL3 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6;</li><li>e) the CDRL2 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6;</li><li>f) the CDRL1 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; or</li><li>g) any combination of two or more of a), b), c), d), e), and f).</li></ol>
Embodiment 4. The isolated antibody or antigen-binding fragment thereof of any one of Embodiments 1 through 3, wherein the antibody or antigen-binding fragment thereof comprises: <ol id="ol0004" compact="compact"><li>a) a heavy chain (HC) amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; and/or</li><li>b) a light chain (LC) amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.</li></ol>
Embodiment 5. The isolated antibody or antigen-binding fragment thereof of any one of Embodiments 1 through 4, wherein the antibody is selected from the group consisting antibodies that are at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99%; and/or all percentages of identity in between; to any one of the antibodies designated as Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.
Embodiment 6. The isolated antibody or antigen-binding fragment thereof of any one of Embodiments 1 through 5, wherein the antibody is selected from the group consisting of the antibodies designated as Antibody 232 through Antibody Number 372 as disclosed in Table 6.
Embodiment 7. An isolated nucleic acid sequence encoding an antibody or antigen-binding fragment thereof according to any one of Embodiments 1 through 6.
Embodiment 8. An expression vector comprising the isolated nucleic acid sequence according to Embodiment 7.
Embodiment 9. A host cell transfected, transformed, or transduced with the nucleic acid sequence according to Embodiment 7 or the expression vector according to Embodiment 8.
Embodiment 10. A pharmaceutical composition comprising: one or more of the isolated antibodies or antigen-binding fragments thereof according to any one of Embodiments 1 through 6; and a pharmaceutically acceptable carrier and/or excipient.
Embodiment 11. A pharmaceutical composition comprising: one or more nucleic acid sequences according to Embodiment 7; or one or more the expression vectors according to Embodiment 8; and a pharmaceutically acceptable carrier and/or excipient.
Embodiment 12. A transgenic organism comprising the nucleic acid sequence according to Embodiment 7; or the expression vector according to Embodiment 8.
Embodiment 13. A method of treating or preventing a Respiratory Syncytial Virus (RSV) infection, ar at least one symptom associated with RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof: <ol id="ol0005" compact="compact"><li>a) one or more antibodies or antigen-binding fragments thereof according to any of Embodiments 1 thorugh 6;</li><li>b) a nucleic acid sequences according to Embodiment 7;</li><li>c) an expression vector according to Embodiment 8;</li><li>d) a host cell according to Embodiment 9; or</li><li>e) a pharmaceutical composition according Embodiment 10 or Embodiment 11; such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity.</li></ol>
Embodiment 14. A method of treating or preventing either a Respiratory Syncytial Virus (RSV) infection or a human metapneumovirus (HMPV) infection, ar at least one symptom associated with said RSV infection or said HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof: <ol id="ol0006" compact="compact"><li>a) one or more antibodies or antigen-binding fragments thereof according to any of Embodiments 1 through 6;</li><li>b) a nucleic acid sequences according to Embodiment 7;</li><li>c) an expression vector according to Embodiment 8;</li><li>d) a host cell according to Embodiment 9; or</li><li>e) a pharmaceutical composition according Embodiment 10 or Embodiment 11; such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity.</li></ol>
Embodiment 15. The method according to Embodiment 14, wherein the one or more antibodies or antigen-binding fragments thereof comprises Antibody Number 340.
Embodiment 16. The method according to any one of Embodiments 13 through 15, wherein the method further comprises administering to the patient a second therapeutic agent.
Embodiment 17. The method according to Embodiment 16, wherein the second therapeutic agent is selected group consisting of: an antiviral agent; a vaccine specific for RSV, a vaccine specific for influenza virus, or a vaccine specific 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.
Embodiment 18. A pharmaceutical composition comprising any one or more of the isolated antibodies or antigen-binding fragments thereof of any one of Embodiments 1 through 7 and a pharmaceutically acceptable carrier and/oror excipient.
Embodiment 19. The pharmaceutical composition according to Embodiment 18 for use in preventing a respiratory syncytial virus (RSV) infection in a patient in need thereof or suspected of being in need thereof, or for treating a patient suffering from an RSVinfection, 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 prevented, ameliorated, or lessened in severity and/or duration as a result of such use.
Embodiment 20. The pharmaceutical composition according to Embodiment 18 for us in treating or preventing either a Respiratory Syncytial Virus (RSV) infection or a human metapneumovirus (HMPV) infection, or at least one symptom associated with said RSV infection or said HMPV infection, in a patient in need thereof or suspected of being in need thereof, wherein the infection is either prevented, or at least one symptom or complication associated with the infection is prevented, ameliorated, or lessened in severity and/or duration as a result of such use.
Embodiment 21. Use of the pharmaceutical composition of Embodiment 18 in the manufacture of a medicament for 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 prevented, ameliorated, or lessened in severity and/or duration.
Embodiment 22. Use of the pharmaceutical composition of Embodiment 18 in the manufacture of a medicament for preventing either a Respiratory Syncytial Virus (RSV) infection or a human metapneumovirus (HMPV) infection, or at least one symptom associated with said RSV infection or said HMPV infection, in a patient in need thereof or suspected of being in need thereof, wherein the infection is either prevented, or at least one symptom or complication associated with the infection is prevented, ameliorated, or lessened in severity and/or duration as a result of such use. In view of the above, it will be appreciated that the invention also encompasses the following items: <ol id="ol0007"><li>1. An isolated antibody or an antigen-binding fragment there or that specifically binds to Respiratory Syncytial Virus (RSV) F protein (F), wherein at least one of the CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and CDRL3 amino acid sequence of the antibody or the antigen-binding fragment thereof is at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99%; and/or all percentages of identity in between; to at least one the CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and/or a CDRL3 amino acid sequences as disclosed in Table 6 of an antibody selected from Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; and wherein said antibody or the antigen-binding fragment thereof also has one or more of the following characteristics: <ol id="ol0008" compact="compact"><li>a) the antibody or antigen-binding fragment thereof cross-competes with said antibody ar antigen-binding fragment thereof for binding to RSV-F;</li><li>b) the antibody or antigen-binding fragment thereof displays better binding affinity for the PreF form of RSV-F relative to the PostF form;</li><li>c) the antibody or antigen-binding fragment thereof displays a clean or low polyreactivity profile;</li><li>d) the antibody or antigen-binding fragment thereof displays neutralization activity towoard RSV suptype A and RSV subtype B in vitro;</li><li>e) the antibody or antigen-binding fragment thereof displays antigenic site specificity for RSV-F at Site Ø, Site I, Site II, Site III, Site IV, or Site V;</li><li>f) the antibody or antigen-binding fragment thereof displays antigenic site specificity for RSV-F Site Ø, Site V, or Site III relative to RSV-F Site I, Site II, or Site IV;</li><li>g) at least a portion of the epitope with which the antibody or antigen-binding fragment thereof interacts comprises the α3 helix and β3/β4 hairpin of PreF;</li><li>h) the antibody or antigen-binding fragment thereof displays an in vitro neutralization potency (IC<sub>50</sub>) of between about 0.5 microgram/milliliter (ug/ml) to about 5 ug/ml; between about 0.05 ug/ml to about 0.5 ug/ml; or less than about 0.05 mg/ml;</li><li>i) the binding affinity and/or epitopic specificity of the antibody or antigen-binding fragment thereof for any one of the RSV-F variants designated as 1, 2, 3, 4, 5, 6, 7, 8, 9, and DG in <figref idref="f0010">Figure 7A</figref> is reduced or eliminated relative to the binding affinity and/or epitopic specificity of said antibody or antigen-binding fragment thereof for the RSV-F or RSV-F DS-Cavl;</li><li>j) the antibody or antigen-binding fragment thereof of displays a cross-neutalization potency (IC<sub>50</sub>) against human metapneumovirus (HMPV);</li><li>k) the antibody or antigen-binding fragment thereof does not complete with D25, MPE8, palivisumab, motavizumab, or AM-14; or</li><li>l) the the antibody or antigen-binding fragment thereof displays at least about 2-fold; at least about 3-fold; at least about 4-fold; at least about 5-fold; at least about 6-fold; at least about 7-fold; at least about 8-fold; at least about 9-fold; at least about 10-fold; at least about 15-fold; at least about 20-fold; at least about 25-fold; at least about 30-fold; at least about 35-fold; at least about 40-fold; at least about 50-fold; at least about 55-fold; at least about 60-fold; at least about 70-fold; at least about 80-fold; at least about 90-fold; at least about 100-fold; greater than about 100-fold; and folds in between any of the foregoing; greater neutralization potency (IC50) than D25 and/or palivizumab.</li></ol></li><li>2. The isolated antibody or antigen-binding fragment thereof of item 1, wherein the antibody or antigen-binding fragment thereof comprises: at least two; at least three; at least 4; at least 5; at least 6; at least 7; at least 8; at least 9; at least 10; at least 11; or at least 12; of characteristics a) through 1).</li><li>3. The isolated antibody or antigen-binding fragment thereof of item 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: <ol id="ol0009" compact="compact"><li>a) the CDRH3 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6;</li><li>b) the CDRH2 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6;</li><li>c) the CDRH1 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6;</li><li>d) the CDRL3 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6;</li><li>e) the CDRL2 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6;</li><li>f) the CDRL1 amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; or</li><li>g) any combination of two or more of a), b), c), d), e), and f).</li></ol></li><li>4. The isolated antibody or antigen-binding fragment thereof of any one of items 1 through 3, wherein the antibody or antigen-binding fragment thereof comprises: <ol id="ol0010" compact="compact"><li>a) a heavy chain (HC) amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6; and/or</li><li>b) a light chain (LC) amino acid sequence of any one of the antibodies designated Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.</li></ol></li><li>5. The isolated antibody or antigen-binding fragment thereof of any one of items 1 through 4, wherein the antibody is selected from the group consisting antibodies that are at least 70% identical; at least 75% identical; 80% identical; at least 85% identical; at least 90% identical; at least 95% identical; at least 96% identical; at least 97% identical; at least 98% identical; at least 99%; and/or all percentages of identity in between; to any one of the antibodies designated as Antibody Number 232 through Antibody Number 372 as disclosed in Table 6.</li><li>6. The isolated antibody or antigen-binding fragment thereof of any one of items 1 through 5, wherein the antibody is selected from the group consisting of the antibodies designated as Antibody 232 through Antibody Number 372 as disclosed in Table 6.</li><li>7. An isolated nucleic acid sequence encoding an antibody or antigen-binding fragment thereof according to any one of items 1 through 6.</li><li>8. An expression vector comprising the isolated nucleic acid sequence according to item 7.</li><li>9. A host cell transfected, transformed, or transduced with the nucleic acid sequence according to item 7 or the expression vector according to item 8.</li><li>10. A pharmaceutical composition comprising: one or more of the isolated antibodies or antigen-binding fragments thereof according to any one of items 1 through 6; and a pharmaceutically acceptable carrier and/or excipient.</li><li>11. A pharmaceutical composition comprising: one or more nucleic acid sequences according to claim 7; or one or more the expression vectors according to item 8; and a pharmaceutically acceptable carrier and/or excipient.</li><li>12. A transgenic organism comprising the nucleic acid sequence according to item 7; or the expression vector according to item 8.</li><li>13. A method of treating or preventing a Respiratory Syncytial Virus (RSV) infection, ar at least one symptom associated with RSV infection, comprising administering to a patient in need thereof or suspected of being in need thereof: <ol id="ol0011" compact="compact"><li>a) one or more antibodies or antigen-binding fragments thereof according to any of items 1 thorugh 6;</li><li>b) a nucleic acid sequences according to item 7;</li><li>c) an expression vector according to item 8;</li><li>d) a host cell according to item 9; or</li><li>e) a pharmaceutical composition according item 10 or item 11;</li></ol> such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity.</li><li>14. A method of treating or preventing either a Respiratory Syncytial Virus (RSV) infection or a human metapneumovirus (HMPV) infection, ar at least one symptom associated with said RSV infection or said HMPV infection, comprising administering to a patient in need thereof or suspected of being in need thereof: <ol id="ol0012" compact="compact"><li>a) one or more antibodies or antigen-binding fragments thereof according to any of items 1 through 6;</li><li>b) a nucleic acid sequences according to item 7;</li><li>c) an expression vector according to item 8;</li><li>d) a host cell according to item 9; or</li><li>e) a pharmaceutical composition according item 10 or item 11;</li></ol> such that the RSV infection is treated or prevented, or the at least on symptom associated with RSV infection is treated, alleviated, or reduced in severity.</li><li>15. The method according to item 14, wherein the one or more antibodies or antigen-binding fragments thereof comprises Antibody Number 340.</li><li>16. The method according to any one of items 13 through 15, wherein the method further comprises administering to the patient a second therapeutic agent.</li><li>17. The method according to item 16, wherein the second therapeutic agent is selected group consisting of: an antiviral agent; a vaccine specific for RSV, a vaccine specific for influenza virus, or a vaccine specific 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.</li><li>18. A pharmacueitcal composition comprising any one or more of the isolated antibodies or antigen-binding fragments thereof of any one of items 1 through 7 and a pharmaceutically acceptable carrier and/oror excipient.</li><li>19. The pharmaceutical composition according to item 18 for use in preventing a respiratory syncytial virus (RSV) infection in a patient in need thereof or suspected of being in need thereof, or for treating a patient suffering from an RSVinfection, 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 prevented, ameliorated, or lessened in severity and/or duration as a result of such use.</li><li>20. The pharmaceutical composition according to item 18 for us in treating or preventing either a Respiratory Syncytial Virus (RSV) infection or a human metapneumovirus (HMPV) infection, ar at least one symptom associated with said RSV infection or said HMPV infection, in a patient in need thereof or suspected of being in need thereof, wherein the infection is either prevented, or at least one symptom or complication associated with the infection is prevented, ameliorated, or lessened in severity and/or duration as a result of such use.</li><li>21. Use of the pharmaceutical composition of item 18 in the manufacture of a medicament for 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 prevented, ameliorated, or lessened in severity and/or duration.</li><li>22. Use of the pharmaceutical composition of item 18 in the manufacture of a medicament for preventing either a Respiratory Syncytial Virus (RSV) infection or a human metapneumovirus (HMPV) infection, ar at least one symptom associated with said RSV infection or said HMPV infection, in a patient in need thereof or suspected of being in need thereof, wherein the infection is either prevented, or at least one symptom or complication associated with the infection is prevented, ameliorated, or lessened in severity and/or duration as a result of such use.</li></ol>
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16 members in 6 offices
Priority claims12
| Document | Office | Kind | Date |
|---|---|---|---|
| 201662411508 | United States of America | P | |
| 201662411508 | United States of America | P | |
| 201662411508P | United States of America | – | |
| 17794177 | European Patent Office (EPO) | A | |
| 17794177 | European Patent Office (EPO) | A | |
| 2017057737 | United States of America | W | |
| 2017057737 | United States of America | W | |
| 177941770 | – | – | – |
| 201662411508P | – | – | – |
| EP20170794177 | – | – | – |
| US201662411508P | – | – | – |
| WO2017US57737 | – | – | – |
Members16
| Document | Office | Kind | |
|---|---|---|---|
| CA3040893A1 | Canada | A1 | |
| WO2018075974A2 | World Intellectual Property Organization (WIPO) | A2 | |
| WO2018075974A3 | World Intellectual Property Organization (WIPO) | A3 | |
| AU2017345786A1 | Australia | A1 | |
| EP3529270A2 | European Patent Office (EPO) | A2 | |
| JP2020503843A | Japan | A | |
| US2020223906A1 | United States of America | A1 | |
| EP3974447A2This record | European Patent Office (EPO) | A2 | |
| JP2022065140A | Japan | A | |
| EP3974447A3 | European Patent Office (EPO) | A3 | |
| US11485775B2 | United States of America | B2 | |
| US2023192822A1 | United States of America | A1 | |
| AU2017345786B2 | Australia | B2 | |
| JP7644040B2 | Japan | B2 | |
| US12281156B2 | United States of America | B2 | |
| US2025388653A1 | United States of America | A1 |
18 legal events, as 2 offices reported them to INPADOC
Over the term
Point at a mark for the eventEvents
| Event | Code | Office | |
|---|---|---|---|
| First examination report despatched17Q | 17Q | EP | |
| Information on the status of an ep patent application or granted ep patentGrantedSTATUS: EXAMINATION IS IN PROGRESSSTAA | STAA | EP | |
| Request for examination filed17P | 17P | EP | |
| Designated contracting states (corrected)RBV | RBV | EP | |
| Information on the status of an ep patent application or granted ep patentGrantedSTATUS: REQUEST FOR EXAMINATION WAS MADESTAA | STAA | EP | |
| Requests to designate patent in hong kongDE | DE | HK | |
| Designated contracting statesAK | AK | EP | |
| Information provided on ipc code assigned before grantRIC1 | RIC1 | EP | |
| Information provided on ipc code assigned before grantRIC1 | RIC1 | EP | |
| Information provided on ipc code assigned before grantRIC1 | RIC1 | EP | |
| Search report despatchedORIGINAL CODE: 0009013PUAL | PUAL | EP | |
| Information provided on ipc code assigned before grantRIC1 | RIC1 | EP | |
| Information provided on ipc code assigned before grantRIC1 | RIC1 | EP | |
| Information provided on ipc code assigned before grantRIC1 | RIC1 | EP | |
| Divisional application: reference to earlier applicationAC | AC | EP | |
| Designated contracting statesAK | AK | EP | |
| Public reference made under article 153(3) epc to a published international application that has entered the european phaseORIGINAL CODE: 0009012PUAI | PUAI | EP | |
| Information on the status of an ep patent application or granted ep patentGrantedSTATUS: THE APPLICATION HAS BEEN PUBLISHEDSTAA | STAA | EP |
Numbers
- Publication
- 3974447
- Publication, DOCDB
- 3974447
- Publication, EPODOC
- EP3974447
- Application
- 211688189
- Application, DOCDB
- 21168818
- Application, EPODOC
- EP20210168818
Titles3
- German
- ANTIKÖRPER GEGEN DAS RESPIRATORISCHE SYNZYTIALVIRUS UND VERFAHREN ZU DEREN ERZEUGUNG UND VERWENDUNG
- English
- ANTI-RESPIRATORY SYNCYTIAL VIRUS ANTIBODIES, AND METHODS OF THEIR GENERATION AND USE
- French
- ANTICORPS ANTI-VIRUS RESPIRATOIRE SYNCYTIAL, ET LEURS PROCÉDÉS DE GÉNÉRATION ET D'UTILISATION
Classification
- CPC, 10
- C07K16/11
- A61K2039/505
- C07K2317/21
- C07K2317/33
- C07K2317/34
- C07K2317/565
- C07K2317/92
- C07K2317/76
- A61P31/14
- A61P43/00
- IPC, 3
- C07K16 10
- A61K39 00
- A61P31 14
Designated states1
- Contracting states, 1
- Türkiye