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  • C07K16/10—RNA viruses
  • C07K16/11—Paramyxoviridae (F); Pneumoviridae (F), e.g. respiratory syncytial virus [RSV]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
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  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
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  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/55—Fab or Fab'
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  • C07K2317/622—Single chain antibody (scFv)
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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  • C12N2760/00011—Details
  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
  • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/18011—Paramyxoviridae
  • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
  • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the invention is directed to improved methods and materials for prophylaxis and treatment of respiratory syncytial virus (RSV) infection.
• RSV respiratory syncytial virus
• RSV is a negative- strand RNA virus with 10 genes encoding 11 proteins, which has resisted effective management for over 60 years in part because infection does not provide robust immunity (Broadbent, L. et al. Influenza Other Re spir Viruses (2015) 9: 169-78). To date, no vaccine has been approved despite several substantial attempts (Jorquera, P.A. and Tripp, R.A. Expert Rev Respir Med (2017) 11 (8):609-6l 5). Over 50% of infants in the US are infected during their first year, with nearly 5% requiring hospitalization (American Academy of Pediatrics Committee on Infectious Diseases, American Academy of Pediatrics Bronchiolitis Guidelines. Pediatrics (2014) l34:e620-38).
• Severe RSV disease in infancy is an established risk factor for childhood asthma-like symptoms (Gelfand, E.W. Curr Opin Immunol (2012) 24(6):7l3-9).
• Preterm infants ⁇ 29 weeks gestational age
• Preterm infants who are at particularly high risk have been the focus for prophylaxis with a humanized mouse monoclonal antibody or mAh (palivizumab, marketed by Medlmmune as Synagis®), an expensive treatment which reduces morbidity but not mortality (Meissner, H.C. and Kimberlin, D.W. Pediatrics (2013) 132:915-8).
• RSV has two major surface glycoproteins, F and G.
• palivizumab commercially available under the brand name Synagis®
• This mAh is broadly useful due to conservation of the F protein sequence among strains, although escape mutations are readily detected (Zhu, Q., et al. J Infect Dis (2011) 203:674-82).
• Efforts to develop a vaccine based on the RSV F protein have been described (Jorquera, P.A. and Tripp, R.A. Expert Rev Respir Med (2017) 11(8):609-615).
• antibody titers to the F protein produced in immunized subjects have reached levels that inhibit RSV propagation in cell culture, RSV disease has not been sufficiently prevented for any such vaccine to receive FDA approval.
• RSV G protein has been shown to exacerbate RSV infection by depressing the interferon response (Oshansky, C.M. et al. Viral Immunol (2009) 22(3): 147-161). Vaccines based on variants of RSV G protein but lacking the CX3CR1 interaction have been proposed.
• U.S. Pat. No. 8,173,131 discloses vaccines wherein the CX3C motif is modified or compositions that comprise antibodies or other moieties that prevent interaction of the motif with the receptor, thus preventing cell entry.
• U.S. Pat. No. 8,846,056 discloses immunogenic peptides from RSV G protein as vaccine components, specifically including residues 164-176 or 155-206.
• 9,321,830 discloses mAbs useful in treating or preventing RSV infections that bind to conserved linear sequences of the G protein of RSV in the region including residues 167-176; as these antibodies were derived from the natural human immune repertoire, they are expected to be minimally immunogenic when administered to a human subject.
• Such an attenuated live virus may be useful as a vaccine, although the high transmissibility of RSV poses a substantial risk of transfer to people at risk for severe disease despite the attenuation, including the very young, the elderly, and the immunocompromised.
• the secreted G protein poses an additional obstacle to an effective vaccine or treatment, because high affinity for antigen is needed for neutralizing soluble factors; otherwise the mAh simply provides a circulating reservoir for the antigen, extending its serum half-life and distributing it more efficiently around the body (Tabrizi, M., et al. AAPS J (2010) 12:33-43).
• mAbs with low picomolar (pM) affinity for the RSV G protein CCD such as those disclosed in US 9,321,830 are generally advantageous.
• a recombinant fusion protein comprising the central region (residues 131-230) of the G proteins of both RSV A and B subtypes was shown to be an effective immunogen in mice (Lee, J.Y. and Chang, J. PLoS One (2017) 12:e0175384).
• the present invention enables the conserved CCD of the RSV G protein to elicit neutralizing antibodies in a subject while eliminating the undesired interaction of the CX3C motif with its receptor. This is accomplished by mutating the G protein to prevent this interaction or by providing antibodies or other moieties that compete with the G protein for this receptor.
• an anti-G mAh is used to bind RSV G protein in a vaccine composition.
• the antibodies employed in the complexes of the present invention prevent the interaction of the RSV G protein with the host cell receptor CX3CR1, thereby preventing adverse events tied to that biological activity, without interfering with the ability of the G protein or the CCD portion thereof to induce neutralizing antibodies.
• Such a combination has been shown to block the activation of the CX3CR1 -receptor ( see Example 4 below), and such complexes are able to increase efficiency of immune response as explained below.
• the invention relates to an immunogen and vaccine compositions that contain it that comprise RSV G protein or a fragment comprising the CCD, or a homolog or stabilized forms thereof or peptidomimetic of either, complexed with a mAh (or fragments or derivative thereof) that binds to RSV G protein with high affinity and prevents interaction with CX3CR1.
• the immunogen may be administered along with a vaccine adjuvant.
• the CCD is that of residues 131-230 or 167-176 of a G protein.
• the mAh needs to have high enough affinity to support formation of stable immune complexes, such as an affinity equal to or better than that associated with a Kd of 100 pM or 50 pM or 25 pM or less.
• the invention includes methods to provide prophylaxis with respect to RSV infection in a subject which method comprises administering to a subject in need of such treatment a pharmaceutical or veterinary composition including these immunogens.
• the invention includes sequence modifications to a mAh against the CCD of the RSV G protein wherein the modifications result in extended serum half- life.
• Technology for achieving this goal has long been known, as described in U.S. Patent No. 8,318,907.
• Targeting the G protein in this fashion is a contribution to passive immunotherapy / prophylaxis against RSV, wherein the selection of antigen to target is important. That is, the vast majority of vaccine / antibody efforts on RSV have been focused on the F protein, with no prior suggestion that the long half-life approach currently being applied to the F protein should or could be applied to the G protein.
• Other techniques for extending the half-life include fusion to an albumin binding domain or modification of the Fc region to enhance binding to mucin.
• the invention includes expression in the cells of a subject of the mAh by Vectored Immunoprophylaxis (VIP), also referred to as Immunoprophylaxis by Gene Trasfer (IGT) or V ector-Mediated Antibody Gene transfer, which comprises introducing genes encoding a Ah into the subject’s cells which then secrete the Ah (Sanders, J.W. and Ponzio, T.A. Tropical Diseases, Travel Medicine and Vaccines (2017) 3:3).
• VIP Vectored Immunoprophylaxis
• ITT Immunoprophylaxis by Gene Trasfer
• V ector-Mediated Antibody Gene transfer which comprises introducing genes encoding a Ah into the subject’s cells which then secrete the Ah (Sanders, J.W. and Ponzio, T.A. Tropical Diseases, Travel Medicine and Vaccines (2017) 3:3).
• the invention includes formulation of a mAh against the CCD of RSV G protein that enables delivery by inhalation.
• Suitable technology to achieve this goal is described in U.S. Patent No. 9,718,875.
• a review of suitable nebulizers is: (Ari, A. and Fink, J.B. Curr Pharm Biotechnol. (2016) 17(14): 1268-1277).
• Protein “peptide” and“polypeptide” are used interchangeably and to refer to chains of naturally occurring amino acids coupled through amide bonds such that they can be synthesized by recombinant methods regardless of the length of the chain.“Homologs” are peptides or proteins with similar sequences, but alterations such that the homolog is 80% or 85% or 95% or 99% homologous to the referent.
• “Peptidomimetics” have similar homologies but include unnatural or synthetic amino acids, including D and L isomers and amino acid analogs linked by amide linkages or other bonds, e.g., ester, ether, etc.“Peptidomimetics” also include organic molecules not obviously analogous to peptides, including, for example, aptamers. As defined herein, based on previous studies, the CCD of the G protein is that portion of the protein represented by residues 169-198.
• subject refers to a human or non-human animal, including laboratory models for RSV such as rodents, or to livestock or pets.
• binding moiety includes antibodies and alternative non immunoglobulin binding moieties as set forth hereinbelow.
• Antibodies include immunoreactive fragments of traditional antibodies and their various fragmented forms that still retain immunospecificity such as Fab, F(ab')2, F v fragments, single-chain antibodies in which the variable regions of heavy and light chain are directly bound without some or all of the constant regions. Since light chains are often interchangeable without destroying specificity, antibodies composed of a heavy chain variable region that determines the specificity of the antibody may be combined with a heterologous light chain variable region. Chimeric antibodies with constant and variable regions derived, for example, from different species are also included. Also included are antibodies in which the Fc portion of the molecule has been mutated to enhance or reduce binding to Fc receptors (Kontermann, R.E. BioDrugs (2009) 23(2):93-l09).
• the critical amino acid sequences are the complementarity-determining region (CDR) sequences arranged on a framework, which framework can vary without necessarily affecting specificity or decreasing affinity to an unacceptable level. Definition of these CDR regions is accomplished by art- known methods. Specifically, the most commonly used method for identifying the relevant CDR regions is that of Rabat as disclosed in Wu, T. T., et al. , J. Exp. Med. (1970) 132:211-250 and in the book Rabat, E. A., et al. (1983) Sequence of Proteins of Immunological Interest, Bethesda National Institute of Flealth, 323 pages.
• CDR complementarity-determining region
• the mAbs of the invention may contain the three CDR regions of a heavy chain and optionally the three CDR’s of a light chain that matches it. Because binding affinity is also determined by the manner in which the CDR’s are arranged on a framework, the mAbs may contain complete variable regions of the heavy chain containing the three relevant CDR’s as well as, optionally, the complete light chain variable region comprising the three CDR’s associated with the light chain complementing the heavy chain in question.
• mAbs of the invention may include variable regions of the heavy and light chains of mAh 3G12, 3D3, 2B11, or 2D10, i.e., SEQ ID NO: 2 and 16; SEQ ID NO: 4 and 18; SEQ ID NO: 6 and 20; SEQ ID NO: 8 and 22, respectively.
• the invention also includes binding moieties that mimic the binding characteristics of m Ahs. Suitable mAh mimics include aptamers (Yu, Y., et al.
• An alternative to active immunization is to provide a mAh as passive immunotherapy, either prophylactically or therapeutically.
• a mAh for prophylaxis, it is useful to modify the Fc region of the mAh to extend serum half-life as has been described for an anti-F protein mAh now in clinical development (U.S. Patent No. 7,323,172).
• an inhaled formulation is useful, as has been described for m Ahs against influenza (U.S. Patent No. 9,718,875).
• any proteins or peptides of the invention including antibodies or antigen binding fragments thereof may be produced recombinantly using known techniques.
• the invention also includes nucleic acid molecules comprising nucleotide sequences encoding them, as well as vectors or expression systems that comprise these nucleotide sequences, cells containing expression systems or vectors for expression of these nucleotide sequences and methods to produce the peptides by culturing these cells and recovering the binding moieties produced.
• Any type of cell typically used in recombinant methods can be employed including prokaryotes, yeast, mammalian cells, insect cells and plant cells. Also included are human cells (e.g. , muscle cells or lymphocytes) transformed with one or more recombinant molecules that encode the relevant peptides.
• RSV G (298 residues) contains an approximately 40 amino acid central conserved domain (CCD) that is highly conserved, and devoid of glycosylation; this portion of the protein has been shown to play key roles in both virus infection and viral pathogenesis.
• CCD central conserved domain
• RSV G CCD contains a CX3C chemokine motif that facilitates binding to the human chemokine receptor CX3CR1 to promote RSV infection in human airway epithelial cells as well as modulating signaling that affects trafficking of CX3CRl + immune cells resulting in airway congestion (Tripp, R.A., et al. J Virol (2016) 92:e0l302-l7).
• the ability to activate or inhibit the CX3C receptor can be assessed using, for example, the chemotaxis assay set forth in Example 4 herein below or any other suitable method, such as a calcium flux assay that has shown good correlation with chemotaxis in a study of mutants of the endogenous ligand for CX3CR1 (fractalkine) (Dorgham, K. et al. J Leukoc Biol (2009) 86(4):903-l l).
• the pharmacological activity of the G protein is deleterious in the context of RSV infection and it is thus preferable to minimize that activity in the immunogen either by modification of the immunogen structure as previously described or by masking that site as disclosed herein.
• conformational stability achieved by stabilization of the structure using mutations chosen based on antibody-antigen high resolution structural data can result in higher titer more uniformly across immunized subjects than for the parental virus, as shown for the RSV F protein (McClellan, J.S., et al. Science (2013) 340(6136): 1113-1117).
• the natural G protein CCD is poorly immunogenic, and thus modifications to improve immunogenicity are important. Since the G protein CCD can be made either synthetically or recombinantly, methods well known in the art can be used to systematically mutate this peptide. To facilitate evaluation of a large number of such variants, in vitro assays are needed.
• Peptides incorporating non-natural motifs are often quite resistant to proteolytic degradation, which is an advantageous feature unrelated to the mimicry itself.
• a disadvantage of small molecules (“haptens”) is that they are often not immunogenic themselves; however, they can become effective immunogens when presented to the immune system embedded in virus like particles (Buonaguro, L., et al. Exp. Rev. Vaccines (2011) 10: 1569-1583).
• Still another aspect of the invention is providing a passive vaccine wherein an antibody directed to the RSV G protein is modified to provide an extended time period of effective lifetime in a subject or wherein the lifetime is extended by conjugation to albumin or wherein the antibody or antigen binding portion is expressed in the cells of the subject.
• RSV is a seasonal virus, with a typical season lasting from October to April in the US. Over that time period, among children younger than 5 years old, there are 2.1 million outpatient visits and 57,527 hospitalizations (Hall, C.B., et al. New Engl J Med. (2009) 360(6):588-98). For adults older than 65 years, there are 177,000 hospitalizations and 14,000 deaths (Falsey, A.R., et al. New Engl J Med. (2005) 352(17): 1749-59). The advantage of an extended half-life antibody for RSV to provide protection throughout the season is thus well recognized in the field.
• the Fc region of the mAh is modified to enhance the binding of the Fc region of IgGl to the neonatal Fc receptor (FcRn).
• FcRn neonatal Fc receptor
• An Fc modified mAh against the RSV F protein (MEDI8897) is in clinical development by Sanofi in partnership with AstraZeneca (Medimmune) (ClinicalTrials.gov Identifier: NCT02290340). This mAh has 100- fold higher potency in vitro as compared to the only approved mAh targeting RSV (palivizumab).
• the mAh has been engineered with a triple-amino-acid (M252Y/S254T/T256E [YTE]) substitution within its Fc region (Griffin, M.P., et al. Antimicrob Agents Chemother. (2017) 61(3): e01714-16).
• the YTE substitution enhances the binding of IgGl to the FcRn under the acidic conditions (pH 6.0) of the lysosome. This prevents degradation and increases recirculation to the surface of the cell, thereby prolonging the serum half-life of the antibody.
• the combination of higher potency and longer half-life enables a single dose to provide neutralizing activity for 3-4 months.
• the Fc region of the mAh is modified to bind to mucus components.
• mucus components are of particular utility for inhaled formulations (Wessler, T., et al. ACS Infect. Dis. (2016) 2(l):82-92).
• Another option for extending the half-life of a mAh or fragment thereof is to fuse or conjugate the sequence to an albumin binding domain (Malm, M., et al. Biotechnol J. (2014) 9(9): 1215-22).
• the mAh or antigen binding portion is expressed in the cells of the subject by Vectored Immunoprophylaxis, a process in which genes encoding previously characterized neutralizing antibodies are vectored into the subject’s cells which then secrete the monoclonal antibodies encoded by those genes.
• Vectored Immunoprophylaxis a process in which genes encoding previously characterized neutralizing antibodies are vectored into the subject’s cells which then secrete the monoclonal antibodies encoded by those genes.
• the technology has been proven effective in animals and is under consideration for providing extended protection in people against HIV (Sanders, J.W. and Ponzio, T.A. Tropical Diseases, Travel Medicine and Vaccines (2017) 3:3).
• a vector comprising self-complementary adeno- associated virus (scAAV) of serotype 8 supported long- lived expression of full-length human antibodies driven from CMV promoters after administration through a single injection of the gastrocnemius muscle.
• scAAV self-complementary adeno- associated virus
• a plasmid encoding the genes for the heavy and light chain of an antibody was introduced by electroporation into mouse muscle pre-treated with hyaluronidase to improve plasmid access to the cells (Yamazaki, T., et al. Vaccines (2016) 6(3):35). Plasmid vectors are considered to be safer than AAV, are easy to prepare and stable during storage. Moreover, plasmid DNA does not induce an immune response against itself. An antibody against the HA protein of influenza induced by this means in mice produced >10 pg/mL of the antibodies in serum for at least 70 days following antibody gene transfer, significantly higher than the level of HA-specific IgG antibody induced from vaccination (1-3 pg/mL).
• the device typically delivers 9 times more aerosol dose than a standard small volume nebuliser such as a pressurized metered dose inhaler (as is typical for asthma treatment). Aerosol particle size is measured using a cascade impactor (In- Tox Products, Moriarity, NM). Using this system, we have produced aerosols of an antibody with a 2.17 pm mass median aerodynamic diameter (MMAD) and a 2.24 pm geometric standard deviation (GSD).
• MMAD mass median aerodynamic diameter
• GSD geometric standard deviation
• a second generation Aerogen product is specifically designed to treat patients requiring a ventilator.
• This Photo Defined Aperture Plate (PDAP) device provides significant improvements in ease of use as well as further dose reduction over the first generation product by achieving tighter control over the mean particle size. This is important because aerosol droplets that are too large do not penetrate deeply into the lungs, but if the droplets are too small, they don’t settle out and are simply exhaled.
• the PDAP device also allows the nebulizer to be synchronized with the patient’s breathing. Only generating the aerosol during the inspiration phase of the breathing cycle substantially improves the fraction of drug that is deposited in the lungs.
• the invention is also directed to pharmaceutical and veterinary compositions which comprise as active ingredients the binding moieties, mutants or other peptides or peptidomimetics of the invention.
• the compositions contain suitable physiologically compatible excipients such as buffers and other simple excipients.
• the compositions may include additional active ingredients as well, in particular in the case of immunogens immune system stimulants as vaccine adjuvants.
• the pharmaceutical or veterinary compositions may also contain other formulation excipients, including formulations for intra-nasal or inhaled delivery of m Ahs as described in US Patent No. 9,718,875.
• the immunogens are employed in a method to generate an immune response to RSV, comprising administering formulations containing them to a subject, including a human subject, such as a pregnant woman, an infant, an elderly human, or an immunocompromised subject. Infections related to RSV in other animal species may also be treated prophylactically by the immunogens or binding moieties of the invention.
• Recombinant m Ahs 3D3 and 2D 10 were produced by transient-transfection in CHO cells and purification by immobilized protein A.
• the Fab fragment of 3D3 was generated from recombinantly produced 3D3 by incubation with immobilized papain, followed by removal of the Fc fragment with immobilized protein A. Fab 3D3 was then purified by Superdex 200 size-exclusion chromatography in 10 mM Tris-HCl pH 8.0 and 150 mM NaCl.
• scFv 2D 10 For recombinant scFv 2D 10, a synthetic gene codon-optimized for Drosophila melanogaster encoding 2D10 heavy chain variable region, a (GGGGS)3GGG linker, and 2D10 light chain variable region, was cloned into pMT-puro in-frame with an N-terminal BiP signal sequence and a C-terminal thrombin cleavage site followed by a Twin-Strep purification tag. The resulting scFv 2D 10 expression plasmid was used to obtain stably-transfected Schneider 2 (S2) insect cells.
• S2 stably-transfected Schneider 2
• Secreted scFv 2D 10 was affinity purified on a StrepTrap column, digested with thrombin protease to remove the purification tag, and then purified by Superdex 200 size- exclusion chromatography in 10 mM Tris-HCl pH 8.0 and 150 mM NaCl.
• G[ecto] RSV G ectodomain
• P03423 A synthetic gene encoding RSV G ectodomain (G[ecto]) P03423) was cloned into pCF in-frame with an N-terminal TPA signal sequence and C-terminal tandem 6-histidine and Twin-Strep purification tags.
• G[ecto] was produced by transient-transfection in CHO cells and secreted G[ecto] was affinity purified on a StrepTrap column.
• a synthetic gene codon-optimized for Escherichia coli encoding RSV G residues 161 to 197 (G [161-197]) with a C-terminal 6-histidine purification tag was cloned into pET52b. The peptide was expressed overnight in E. coli BL2l(DE3) at l8°C. The cells were then were lysed by ultrasonication in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, and 25 mM imidazole (Buffer A) containing 2 mM MgCk, benzonase, and protease inhibitors.
• RSV G[ 161 - 197] was purified from soluble lysates by HisTrap FF affinity chromatography and eluted with a gradient into Buffer B (Buffer A containing 500 mM imidazole). Analogous methods were used to produce related peptides (G [162-172] and G [169-198]).
• Efficacy can be evaluated by injecting mice with a vaccine comprising RSV G or fragments thereof, an anti-G mAh that blocks the interaction of the G protein with CX3CR1, along with standard vaccine adjuvants; alternatively, administration may be by intra-nasal route (Kruijsen, D., et al. J Virol (2013) 87(l3):7550-57). After adequate time for immunization, the mice are challenged with a virulent strain of RSV and observed for morbidity and mortality. The composition of the present invention results in protection of the subject mice. The immunogens of the invention are evaluated in a murine model using the following criteria. Table 1: Immunogen Efficacy Criteria
• the assay was performed using a transwell insert plate with an 8 pm pore size. Approximately 2 million log-phase THP-l cells (a human leukemia monocytic cell line) washed twice and suspended in serum-free RPMI 1640 media were added to the upper chamber of the insert plate. Negative control was serum-free media alone to which serum-free media containing 25 nM mAh was added to the lower chamber. As a positive control, media containing 10% FBS was added to the lower chamber. RSV G[ecto] or RSV G[ 161 -197] samples were added to the lower chamber at a final concentration of 5 nM in serum-free media.
• RSV G[l 61 - 197] and mAbs RSV G[l6l-l97] was pre-incubated with 5-fold excess mAh (on a molar basis) for 20 min at room temperature, and then added to serum-free media in the lower chamber, for a final concentration of 5 nM RSV G[ 161 -197] and 25 nM mAh.
• anti-CX3CRl antibody 2 pL 1 mg/mL anti-CX3CRl rabbit polyclonal antibody (ThermoFisher Scientific Cat# PA5-19910) was incubated with TF1P-1 cells for 30 minutes in the upper chamber before being placed into the well.
• the assembled plates were incubated in a C02 incubator at 37°C for 5 h. Cells migrated to the lower chamber were counted, and the chemotactic indices were determined by comparing the fold-increase in cell migration toward the chemoattractant to cell migration toward serum-free media alone. Experiments were performed in at least four biological replicates.

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