Field of the invention

The current invention is in the field of viral infections, in particular viral infections of the respiratory tract. The invention is further in the field of neutralising antibodies for use in the treatment of viral infections of the respiratory tract, particularly in infants.

Background art

Infections of the respiratory tract are very common, especially in infants and small children. However, respiratory tract viral infections may have severe consequences. Viruses infecting the respiratory tract include respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PI V), adenovirus, rhinovirus (RhV), metapneumovirus and coronavirus.

Human respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in young infants (Smyth and Openshaw, 2006) and the second cause of death in the infant period after malaria worldwide. It is estimated that RSV was associated with 33,1 million cases of acute respiratory tract infection (ARTI) in 2015. Currently, there is no vaccine or treatment for RSV.

During the winter season RSV bronchiolitis is one the most common causes of hospitalization (Yorita KL et al, Infectious Disease Hospitalizations Among Infants in the United States. Pediatrics. 2008, 121 (2): 244-52). There are several recognized risk factors for severe RSV disease including prematurity (Hall CB et al, Respiratory syncytial virus-associated hospitalizations among children less than 24 months of age. Pediatrics. 2013,132(2):e341 -8), down syndrome (Bloemers BLP et al. Down Syndrome: A Novel Risk Factor for Respiratory Syncytial Virus Bronchiolitis A Prospective Birth-Cohort Study. Pediatrics. 2007, 120(4):e1076-81), immunodeficiency, and congenital heart disease. However, nearly 80% of children who become hospitalized with RSV infection are previously healthy and do not have any known risk factors for severe disease (Hall et al, supra).

There is no vaccine or evidence-based therapy for RSV bronchiolitis besides supportive care (Mazur Nl et al, Lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics. Lancet Respir Med. 2015, 3(1 1):888-900). Palivizumab, a humanized monoclonal antibody against the surface F protein of RSV, is the only approved preventive intervention, which is administered to high-risk infants. However, this intervention has two main limitations. To prevent one RSV hospitalization the current estimated cost for palivizumab is 100,000 euros. Furthermore, not only are costs high, but prophylaxis is now administered via monthly intramuscular injections, which is burdensome in infants and young children.

There is therefore a strong need in the art to reduce the costs for the prevention or treatment of a viral infection of the respiratory tract. In addition, there is a need in the art to make the administration of the antiviral antibodies less burdensome, especially for young infants.

Summary of the invention

The invention is summarized in the following numbered embodiments: Embodiment 1. A formulation comprising a neutralising antiviral antibody for use in the prevention, treatment or amelioration of a symptom of a viral infection of the respiratory tract, wherein the formulation is administered intranasally.

Embodiment 2. An antibody-comprising formulation for use according to embodiment 1 , wherein the viral infection is an infection caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), adenovirus, rhinovirus (RhV metapneumovirus and coronavirus, preferably the infection is caused by RSV.

Embodiment 3. An antibody-comprising formulation for use according to embodiment 1 or 2, wherein the antibody is a humanized serum antibody, preferably a humanized IgG type antibody. Embodiment 4. An antibody-comprising formulation for use according to embodiment 3, wherein the humanized IgG type antibody is palivizumab.

Embodiment 5. An antibody-comprising formulation for use according to any one of the preceding embodiments, wherein the concentration of the antibody in the formulation is at least about 0.1 , 0.2, 0.5 or 0.9 mg/ml, preferably about 0.1 - 64, 0.2 - 50, 0.5 - 20, 0.9 - 10 mg/ml, preferably about 1 mg/ml.

Embodiment 6. An antibody-comprising formulation for use according to any one of the preceding embodiments, wherein the formulation is administered intranasally at least once a week.

Embodiment 7. An antibody-comprising formulation for use according to any one of the preceding embodiments, wherein at least one drop of the formulation is administered per nostril, wherein preferably one drop comprises about 10-100 pi of the formulation.

Embodiment 8. An antibody-comprising formulation for use according to any one of the preceding embodiments, wherein the subject to be treated is an infant, preferably born at least 30 weeks gestational age, preferably a late preterm infant born 32-35 weeks gestational age. Embodiment 9. An antibody-comprising formulation for use according to any one of the preceding embodiments, wherein the age of the infant to be treated is between about 0-12 months at the start of the treatment. Embodiment 10. An antibody-comprising formulation for use according to any one of the preceding embodiments, wherein the duration of the treatment is at least 2 weeks, wherein preferably the duration of the treatment is about 1 - 6 months.

Embodiment 11. An antibody-comprising formulation comprising at least 0.1 mg/ml, preferably 0.5 mg/ml - 50 mg/ml of an antibody as defined in any one of embodiments 1-3 and a isotonic solution, wherein preferably the isotonic solution is 0.9% NaCI, wherein preferably the isotonic solution is a buffered isotonic solution.

Embodiment 12. An antibody-comprising formulation according to embodiment 11 , wherein the antibody-comprising formulation further comprises a preservative, wherein preferably the preservative is Benzalkoniumchloride.

Embodiment 13. A nasal dropper bottle comprising a container comprising the composition according to embodiment 11 or 12.

Embodiment 14. A nasal dropper bottle according to embodiment 13, wherein at least the container is made from glass, preferably tinted glass.

Embodiment 15. A nasal dropper bottle according to embodiment 13 of 14, further comprising a pipette made from glass.

Definitions

Various terms relating to the methods, compositions, formulations, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al.. Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al.. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego. “A,”“an,” and“the”: these singular form terms include plural referents unless the content clearly dictates otherwise. The indefinite article "a" or "an" thus usually means "at least one". Thus, for example, reference to“a cell” includes a combination of two or more cells, and the like.

“About” and“approximately”: these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

“And/or”: The term“and/or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

“Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

Exemplary": this terms means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein.

The terms“protein” or“polypeptide” are used interchangeably herein and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin. A“fragment” or“portion” of a protein may thus still be referred to as a“protein.” A protein as defined herein and as used in any method as defined herein may be an isolated protein. An“isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

The term "antibody" is used in the broadest sense and specifically covers, e.g. single antiviral monoclonal antibodies, including antagonist, neutralizing antibodies, full length or intact monoclonal antibodies, anti-viral antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, single chain anti-viral antibodies and fragments of anti-viral antibodies, including Fab, Fab’, F(ab’)2 and Fv fragments, diabodies, single domain antibodies (sdAbs), as long as they exhibit the desired biological and/or immunological activity.

The term "immunoglobulin" (Ig) is used interchangeable with antibody herein. An antibody can be human and/or humanized.

The term "anti-RSV antibody" specifically covers, e.g. single anti-RSV monoclonal antibodies, including antagonist, neutralizing antibodies, full length or intact monoclonal antibodies, anti-RSV antibody compositions with polyepitopic specificity, polyclonal antibodies, naked antibodies, multivalent antibodies, single chain anti-RSV antibodies and fragments of anti-RSV antibodies, including Fab, Fab’, F(ab’)2 and Fv fragments, diabodies, single domain antibodies (sdAbs), as long as they exhibit the desired biological and/or immunological activity.

The term "anti-RSV antibody" or "an antibody that binds to RSV" refers to an antibody that is capable of binding RSV with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting RSV. Preferably, the extent of binding of an anti-RSV antibody to an unrelated, non-RSV protein is less than about 10% of the binding of the antibody to RSV as measured, e.g. , by a radioimmunoassay (RIA) or ELISA. In certain embodiments, an antibody that binds to RSV has a dissociation constant (Kd) of < 1 mM, < 100 nM, < 10 nM, < 1 nM, or < 0.1 nM. In certain embodiments, anti-RSV antibody binds to an epitope that is conserved among RSV from different species.

Preferably, the antibody is a neutralizing antibody, i.e. the antibody blocks or neutralizes the biological effect of the virus, preferably of RSV.

A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. The“blocking” antibody or“antagonist” antibody can be a neutralizing antibody, preferably a neutralizing antibody as defined herein. A neutralizing antibody is thus an antibody that keeps an infectious agent, usually a virus, from infecting a cell (or a subject) by neutralizing or inhibiting its biological effect, for example by blocking the virus to interact with the receptors on the cell.

An antibody "which binds" an antigen of interest, e.g. a RSV protein, is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic or prophylactic agent, in particular for blocking the biological effects of RSV, when the antibody administered in an effective amount, and does not significantly cross-react with other proteins. The neutralizing antibody preferably binds with sufficient affinity to at least one at least one of the fusion glycoprotein (F protein), the attachment glycoprotein (G protein) and the small hydrophobic protein (SH protein) of RSV, preferably the A antigenic site of the F protein of RSV.

In an embodiment, the anti-RSV antibody is palivizumab (SYNAGIS®).

As used herein, the term "RSV epitope" refers to a portion of a RSV polypeptide or protein having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. An RSV epitope having immunogenic activity is a portion of a RSV polypeptide or protein that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a RSV polypeptide or protein to which an antibody immunospecifically binds as determined by any method well known in the art, for example, by the immunoassays. Antigenic epitopes need not necessarily be immunogenic.

Preferably, the epitope is an epitope present on at least one of the F protein, the G protein and the SH protein of RSV, preferably present on the G protein and/or F protein of RSV, preferably present on the F protein of RSV. Preferably, the epitope is in the A antigenic site of the F protein of RSV. In an embodiment, the epitope is an epitope bound by palivizumab, i.e. an epitope in the A antigenic site of the F protein of RSV.

The antibody, in particular the neutralizing antibody, can be a basic 4-chain antibody. Such basic 4-chain antibody unit is preferably a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains (an IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain).

In the case of IgGs, the 4-chain unit is generally about 150,000 Daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for m and e isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1 ). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated a, d, e, y, and m, respectively. The y and a classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: lgG1 , lgG2, lgG3, lgG4, IgAI, and lgA2.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 1 10-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" (HVRs) that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a b-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the b-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

An "intact" antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CH1 , CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

A "naked antibody" for the purposes herein is an antibody that is not conjugated to e.g. a cytotoxic moiety or radiolabel.

"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641 ,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen.

The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes). Monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581 -597 (1991), for example.

The monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851 -6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigenbinding sequences derived from a non-human primate (e.g. Old World Monkey, Ape etc.), and human constant region sequences.

"Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability. In some instances, a few framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol., 1 :105-1 15 (1998); Harris, Biochem. Soc. Transactions, 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech., 5:428-433 (1994).

The term "hypervariable region", "HVR", when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops that are responsible for antigen binding. Generally, antibodies comprise six hypervariable regions; three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The hypervariable regions generally comprise amino acid residues from a "complementarity determining region" or "CDR" (e.g., around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31 -35 (H1 ), 50- 65 (H2) and 95-102 (H3) in the VH when numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and/or those residues from a "hypervariable loop" (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (H1), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol. 196:901 -917 (1987)); and/or those residues from a "hypervariable loop'YCDR (e.g., residues 27-38 (L1), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (H1 ), 56-65 (H2) and 105-120 (H3) in the VH when numbered in accordance with the IMGT numbering system; Lefranc, M. P. et al. Nucl. Acids Res. 27:209-212 (1999), Ruiz, M. et al. Nucl. Acids Res. 28:219-221 (2000)). Optionally the antibody has symmetrical insertions at one or more of the following points 28, 36 (L1 ), 63, 74-75 (L2) and 123 (L3) in the VL, and 28, 36 (H1), 63, 74- 75 (H2) and 123 (H3) in the VH when numbered in accordance with Honneger, A. and Plunkthun, A. J. (Mol. Biol. 309:657-670 (2001)). The hypervariable regions/CDRs of the antibodies of the invention are preferably defined and numbered in accordance with the IMGT numbering system.

"Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues herein defined.

An "agonist antibody", as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.

"Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.

A "Kd" or "Kd value" can be measured by using surface plasmon resonance assays using a BIAcore™-2000 or a BIAcore™- 3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized antigen CM5 chips at ~10 - 50 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10mM sodium acetate, pH 4.8, into 5 pg/ml (-0.2 pM) before injection at a flow rate of 5pl/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of the antibody or Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25pl/min. Association rates (k ₀ n) and dissociation rates (k _0ff ) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneous fitting the association and dissociation sensorgram. The equilibrium dissociation constant (Kd) is calculated as the ratio kotf/kon. See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds 10 ⁶ M ^_1 S ^_1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

An "on-rate" or "rate of association" or "association rate" or "k ₀ n" according to this invention can also be determined with the same surface plasmon resonance technique described above using a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, NJ) as described above.

In an embodiment, the extent of binding of the antibody to a "non-target" protein will be less than about 10% of the binding of the antibody to its particular target protein as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With regard to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labelled target. In this case, specific binding is indicated if the binding of the labelled target to a probe is competitively inhibited by excess unlabelled target. The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target (which may be determined as described above) of at least about 10 ⁴ M, alternatively at least about 10 ^-5 M, alternatively at least about 10 ^-6 M, alternatively at least about 10 ⁷ M, alternatively at least about 10 ^-8 M, alternatively at least about 10 ^-9 M, alternatively at least about 10 ^_1 ° M, alternatively at least about 10 ^-11 M, alternatively at least about 10 ^-12 M, or greater. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

Preferably, the antibody is a neutralizing antibody, e.g. it blocks the interaction between molecules. However, the invention is not limited to neutralizing antibodies. In an embodiment, the antibody for use in the invention may have additional effector functions.

Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1 q binding, fixation of complement factors such as C4 and C3, and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

A "functional Fc region" possesses an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g. an antibody variable domain) and can be assessed using various assays as disclosed, for example, in definitions herein.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies "arm" the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991 ). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821 ,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. (USA) 95:652-656 (1998). WO 2000/42072 (Presta) describes antibody variants with improved or diminished binding to FcRs. See also, e.g., Shields et al. J. Biol. Chem. 9(2):6591 -6604 (2001).

"Human effector cells" include leukocytes which express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g., from blood.

"Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1 q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. Antibody variants with altered Fc region amino acid sequences (antibodies with a variant Fc region) and increased or decreased C1 q binding capability are described, e.g., in US Patent No. 6,194,551 B1 and WO 1999/51642. See also, e.g., Idusogie et al., J. Immunol. 164: 4178-4184 (2000). One such substitution that increases C1 q binding, and thereby an increases CDC activity, is the E333A substitution, which can advantageously be applied in the antibodies of the invention. The term "Fc region-comprising antibody" refers to an antibody that comprises an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during purification of the antibody or by recombinant engineering of the nucleic acid encoding the antibody. Accordingly, a composition comprising an antibody having an Fc region according to this invention can comprise an antibody with K447, with all K447 removed, or a mixture of antibodies with and without the K447 residue.

An "isolated antibody" is one which has been identified and separated and/or recovered from a component of its natural environment.

“Amino acid sequence”: This refers to the order of amino acid residues of, or within a protein. In other words, any order of amino acids in a protein may be referred to as amino acid sequence.

The terms“homology”,“sequence identity” and the like are used interchangeably herein. Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

The term “complementarity” is herein defined as the sequence identity of a nucleotide sequence to a fully complementary strand (e.g. the second, or reverse, strand). For example, a sequence that is 100% complementary (or fully complementary) is herein understood as having 100% sequence identity with the complementary strand and e.g. a sequence that is 80% complementary is herein understood as having 80% sequence identity to the (fully) complementary strand.

"Identity" and "similarity" can be readily calculated by known methods.“Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121 -3752 USA, or using open source software, such as the program“needle” (using the global Needleman Wunsch algorithm) or“water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for‘needle’ and for‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blosum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred.

Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as a“query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403— 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, word length = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

As used herein, the terms "prevent", "preventing", and "prevention" refer to the prevention or reduction of the recurrence, onset, development or progression of an viral infection, or the prevention or reduction of the severity and/or duration ofthe viral infection or one or more symptoms thereof.

As used herein, the terms "therapies" and "therapy" can refer to any protocol(s), method(s) and/or agent(s) that can be used in the prevention, treatment, management or amelioration of the viral infection or one or more symptoms thereof.

As used herein, the terms "treat", "treating" and "treatment" refer to the reduction or amelioration of the progression, severity, and/or duration of the viral infection and/or reduces or ameliorates one or more symptoms of the viral infection. In specific embodiments, such terms refer to the reduction or inhibition of the replication of the virus, the inhibition or reduction in the spread of the virus to other tissues or subjects, the inhibition or reduction of infection of a cell with the virus, or the amelioration of one or more symptoms associated with the viral infection.

As used herein, the term "effective amount" refers to the amount of a therapy, e.g., a prophylactic or therapeutic agent, preferably a (neutralizing) antibody, which is sufficient to reduce the severity, and/or duration of a viral infection, ameliorate one or more symptoms thereof, prevent the advancement of a viral infection, or cause regression of a viral infection, or which is sufficient to result in the prevention of the development, recurrence, onset, or progression of a viral infection or one or more symptoms thereof, or enhance or improve the prophylactic and/or therapeutic effect(s) of another therapy (e.g., another therapeutic agent). Preferably, the viral infection is an RSV infection.

Signs and symptoms of RSV infection are herein understood to include: fever, severe cough, wheezing, rapid breathing or difficulty breathing and cyanosis.

In an embodiment, an effective amount of a therapeutic or a prophylactic agent reduces one or more of the following steps of a RSV life cycle: the docking of the virus particle to a cell, the introduction of viral genetic information into a cell, the expression of viral proteins, the production of new virus particles and the release of virus particles from a cell by at least about 5%, preferably at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.

In another specific embodiment, a prophylactically effective amount of a prophylactic agent reduces the replication, multiplication or spread of a virus by at least about 5%, preferably at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.

Detailed description

Currently, prophylactic neutralizing anti-RSV antibodies are administered intramuscularly in a dosage regime of 15 mg/kg body weight once every 4 weeks during a period of usually 5 months (during the RSV season from October to March in The Netherlands). The inventors now discovered that instead of this high dosage and painful administration, similar effects can be achieved when a much lower amount of the same antibody (e.g. 100 pg) is administered intranasally. Hence this invention allows for a far larger number of subjects to be treated at the same costs, and the treatment is also significantly less burdensome for its users, the patients.

The skilled person understands that the invention is not limited to an antibody for use in the prevention or treatment of an RSV infection. Instead, it is contemplated herein that other viral infections of the respiratory tract can equally be prevented by intranasal administration of the respective neutralizing antibody.

In a first aspect, the invention therefore pertains to a formulation comprising an antiviral antibody for use in the prevention, treatment or amelioration of a symptom of a viral infection of the respiratory tract.

In an embodiment, the invention therefore pertains to a formulation comprising an antiviral antibody for use in the prevention a viral infection of the respiratory tract. In an embodiment, the invention therefore pertains to a formulation comprising an antiviral antibody for use in the treatment of a viral infection of the respiratory tract.

In an embodiment, the invention pertains to a formulation comprising an antiviral antibody for use in the amelioration of a symptom of a viral infection of the respiratory tract.

The medical use herein described is formulated as a compound as defined herein for use as a medicament for treatment or prophylaxis of the stated disease(s) by administration of an effective amount of an antibody as defined herein, but could equally be formulated as a method of treatment or prophylaxis of the stated disease(s) using an antibody as defined herein comprising a step of administering to a subject an effective amount of the antibody, an antibody as defined herein for use in the preparation of a medicament to treat or prevent the stated disease(s) wherein the antibody is to be administered in an effective amount and use of an antibody as defined herein for the treatment of the stated disease(s) by administering an effective amount. Such medical uses are all envisaged by the present invention.

Preferably, the antibody is a neutralizing antibody. Hence in an embodiment, the antibody neutralizes the biological effects of a virus infecting the respiratory tract. Preferably, the antibody neutralizes the biological effects of a virus selected from the group consisting of respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), adenovirus, rhinovirus (RhV), metapneumovirus and coronavirus. Preferably, the antibody neutralizes an RSV virus.

In a preferred embodiment, the antibody comprises an Fc region.

The antibody can be any isotype. Preferably, the antibody is at least one of IgA, IgD, IgE, IgG and IgM. Preferably the antibody is a serum antibody, preferably a humanized serum antibody. Hence, preferably, the antibody is not isotype IgA. Preferably, the serum antibody has an isotype selected from the group consisting of IgD, IgE, IgG and IgM. Preferably, the antibody has isotype IgG. In one embodiment, the antibody is an IgG antibody with extended half-life, preferably the half-life of the antibody is extended through a modified Fc-region. In one preferred embodiment, the antibody is MEDI8897 as for example described in (Griffin et al, 2017, DOI: 10.1 128/AAC.01714- 16).

In one embodiment, the antibody is a biosimilar with neutralizing capacity. Preferably, the neutralizing antibody for use according to the invention can be a biosimilar for palivizumab.

In a preferred embodiment, the antibody is a human or humanized antibody. Preferably, the antibody is a neutralizing humanized antibody. Preferably, the antibody for use in the current invention is a neutralizing, humanized serum antibody, preferably isotype IgG.

In an embodiment, the viral infection is at least one of an upper respiratory tract infection and a lower respiratory tract infection. The symptoms of an upper respiratory tract invention predominantly occur in the nose and throat, and includes, but is not limited to, the common cold and influenza. Upper respiratory tract infections may be caused by e.g. rhinoviruses, adenoviruses, coronaviruses, human metapneumoviruses and influenza viruses. The symptoms of a lower respiratory tract infection predominantly occur in the windpipe, airways and lungs, and includes, but is not limited to, croup, bronchiolitis and pneumonia. Lower respiratory tract infections may be caused by parainfluenza viruses, RSV, influenza viruses and rhinoviruses. In a preferred embodiment, the formulation as defined herein is for use in the prevention, treatment or amelioration of a symptom of a viral infection of the upper respiratory tract. Preferably, the formulation is administered intranasally.

In a preferred embodiment, the formulation as defined herein is for use in the prevention, treatment or amelioration of a symptom of a viral infection of the lower respiratory tract. Preferably, the formulation is administered intranasally.

In an embodiment, the viral infection of the respiratory tract is a lower respiratory tract infection. Preferably, the viral infection of the respiratory tract is bronchiolitis, i.e. an acute viral infection of the lower respiratory tract. The bronchiolitis may be caused by at least one of RSV, a rhinovirus and an (para)influenza virus. Preferably, the respiratory tract infection is caused by RSV.

In an embodiment, the invention concerns a formulation comprising an antiviral antibody for use in the prevention, treatment or amelioration of a symptom of a viral infection of the respiratory tract, wherein the viral infection is an infection caused by a virus selected from the group consisting of respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), adenovirus, rhinovirus (RhV), metapneumovirus and coronavirus. Preferably the infection is caused by RSV, more preferably human RSV.

A RSV virus is a pneumovirus in the family Paramyxoviridae. It has a single-stranded, negative-sense genome of about 15 kilobases with 10 gene start sites that encode 1 1 proteins. Three of those proteins are displayed on the viral envelope. The small hydrophobic (SH) protein is a pentameric ion channel; the attachment protein (G) is a heavily O-glycosylated mucin-like glycoprotein; and the fusion glycoprotein (F) is responsible for mediating viral entry through pH- independent membrane fusion. There are two major virus subtypes, A and B, largely defined by genetic variation in the G glycoprotein. Compared to other RNA viruses, RSV exhibits relatively little antigenic variation (Graham BS et al, Novel antigens for RSV vaccines, Curr Opin Immunol. (2015); 35: 30-8).

The F protein is the major target for antiviral drug development, and both G and F glycoproteins are the antigens targeted by neutralizing antibodies induced by infection. Hence, the neutralizing antibody for use in the invention preferably binds an epitope of at least one of the RSV G protein and the RSV F protein. In a preferred embodiment, the neutralizing antibody preferably binds to an epitope of the RSV F protein.

In an embodiment, the neutralizing antibody for use according to the invention binds to a conformational or a linear epitope, preferably a linear epitope, preferably a linear epitope of the RSV F protein.

In a preferred embodiment, the RSV F protein linear epitope is, or is close to, Antigenic site II, also called site A. Hence preferably, the neutralizing antibody binds, or binds close to, the A site of the RSV F protein. Site A is the target of palivizumab. Preferably, the neutralizing antibody binds, or binds close to, the same epitope that is bound by palivizumab. Preferably, the neutralizing antibody for use according to the invention is palivizumab, or a derivative thereof, or antigen-binding fragment thereof. In an embodiment, the epitope is, or is close to, RSV F protein antigenic site IV, also called site C. Preferably, the neutralizing antibody binds, or binds close to site C of the RSV F protein. Site C is the target of antibodies Mab19 and 101 F. Therefore preferably, the neutralizing antibody binds, or binds close to, the same epitope bound by at least one of Mab19 and 101 F (McLellan et al, Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F, J Virol. 2010 Dec; 84(23): 12236-12244).

In an embodiment, the epitope is, or is close to, RSV F protein antigenic site O. Preferably, the neutralizing antibody binds, or binds close to site O of the RSV F protein. Site O is the target of antibody nirsevimab (MEDI-8897). Preferably, the neutralizing antibody binds, or binds close to, the same epitope that is bound by nirsevimab. Preferably, the neutralizing antibody for use according to the invention is nirsevimab, or a derivative thereof, or antigen-binding fragment thereof. Preferably, the neutralizing antibody is selected from the group consisting of Palivizumab, nirsevimab (MEDI-8897), RSV-IVIG, Motavizumab, ALX-0171 and REGN2222 (Rezaee F, et al, Ongoing Developments in RSV Prophylaxis, Curr Opin Virol. 2017 Jun; 24: 70-78).

Preferably, the antibody is palivizumab (SYNAGIS®), or an antigen-binding fragment thereof. Palivizumab is known in the art and has been described e.g. in WO 1996/005229, which is incorporated herein by reference.

The antibody as defined herein is preferably present in a formulation suitable for intranasal administration, preferably suitable for nasal administration in a subject as defined herein below.

The antibody as defined herein is preferably present in the formulation in an effective amount. In an embodiment, the concentration of the antibody in the formulation is at least about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 or at least about 10.0 mg/ml. The concentration of the antibody in the formulation is preferably at least about 0.5 mg/ml of the antibody as defined herein.

Preferably, the concentration of the antibody in the formulation is in between about 0.1 - 100 mg/ml, 0.2 - 80 mg/ml, 0.5 - 75 mg/ml, 0.6 - 60 mg/ml, 0.7 - 50 mg/ml, 0.8 - 40 mg/ml, 0.9 - 30 mg/ml or between about 1 .0 - 20 mg/ml of the antibody as defined herein.

Preferably, the concentration of the antibody in the formulation is about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 or about 10.0 mg/ml. Preferably, the concentration of the antibody is about 1 mg/ml.

In an embodiment, the antibody-comprising formulation is preferably suitable for intranasal administration and the antibody-comprising formulation is preferably for use in the current invention

In an embodiment, the antibody as defined herein may be dispersed in a nasal drop solution. The nasal drop solution can be any conventional, e.g. commercial, nasal drop solution known in the art, such as but not limited to Fagron NL BV (article number 5086). The nasal drop solution preferably comprises about 0.9% sodium chloride.

In an embodiment, the antibody-comprising formulation comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier", as used herein, is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (see e.g.“Handbook of Pharmaceutical Excipients”, Rowe et al eds. 7 ^th edition, 2012, www.pharmpress.com). The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the antibody, use thereof in the compositions is contemplated.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In a preferred embodiment, the formulation comprises an antibody as defined herein and a pharmaceutically acceptable carrier, wherein the carrier is an isotonic solution. A preferred isotonic solution is an isotonic saline solution, preferably a solution comprising about 0.9% sodium chloride.

Preferably, the pharmaceutically acceptable carrier is a buffered solution, preferably a buffered isotonic solution. A preferred buffered isotonic solution may be selected from the group consisting of phosphate-buffered saline (PBS), TRIS-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES- buffered saline (HBS), Grey's balanced salt solution (GBSS), or normal saline (NaCI), hypotonic (saline) solutions with addition of e.g. glucose or dextrose. Preferably, the buffered solution is PBS.

In an embodiment, the formulation comprises a preservative. Preferably, the preservative is selected from the group consisting of benzethonium chloride, benzoic acid, sodium benzoate, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorbutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, sodium propionate, thimerosal, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, isobutyl paraben, benzyl paraben, sorbic acid, and potassium sorbate. Preferably, the preservative is benzalkonium chloride.

The concentration of the preservative is preferably present in an amount wherein the preservative is effective. In a preferred embodiment, the concentration of the preservative in the formulation is between about 0.005% - 0.5% (w/w), 0.008% - 0.1 % (w/w) or between about 0.01 % - 0.05% (w/w). Preferably, the concentration of the preservative present in in the formulation is about 0.01 % (w/w).

The formulation may further comprise a compound to increase the viscosity of the formulation, such as, but not limited to, any suitable water-soluble polymer. A preferred compound increasing the viscosity of the formulation is a water-soluble polymer derived from cellulose. A preferred compound is methocel™, preferably methocel K4M. However, the skilled person understands that other compounds can be equally suitable to increase to viscosity. Preferably, the viscosity is increased to a viscosity suitable for intranasal administration of the formulation.

In a preferred embodiment, the concentration of the viscosity-increasing compound is in between about 0.05 % - 5% (w/w), preferably in between about 0.25% - 2.5% (w/w), preferably about 0.5% (w/w).

In a particularly preferred embodiment, the antibody-comprising formulation further comprises at least sodium chloride and benzalkonium chloride. The formulation is preferably buffered. The formulation may further comprise a viscosity-increasing compound, such as, but not limited to, methocel K4M.

In addition, the antibody-comprising formulation may further comprise at least one of sodium dihydrogen phosphate dihydrate, disodium phosphate dodecahydrate and disodium edetate.

The concentration disodium phosphate dodecahydrate in the formulation is preferably in between about 0.01 % - 1.0% (w/w) or in between about 0.05 - 0.5% (w/w), preferably about 0.1 % (w/w).

The concentration sodium dihydrogen phosphate dehydrate in the formulation is preferably in between about 0.015% - 1.5% (w/w) or in between about 0.075 - 0.75% (w/w), preferably about 0.15% (w/w)

The concentration disodium edetate in the formulation is preferably in between about 0.01 %

- 1.0% (w/w) or in between about 0.05 - 0.5% (w/w), preferably about 0.1 % (w/w).

In a preferred embodiment, the formulation is comprises the following components as specified in Table 1. Table 1. Additional components antibody-comprising formulation

Supplementary active compounds can also be incorporated into the formulation of the invention. Hence in an embodiment, the formulation of the invention may contain an antibody as defined herein, in combination with an additional active compound, which is required for a particular indication being treated. Preferably, the antibody and the additional active compound have complementary activities that do not adversely affect each other. In a particularly preferred embodiment, the antibody-comprising formulation is administered intranasally. Nasal, or intranasal, administration is herein understood as a route of administration in which the formulation is insufflated through the nose. Preferably, the formulation is sprayed or dripped in at least one nostril, preferably into both nostrils.

In an embodiment, the antibody-comprising formulation is administered at least once a day, twice a day, 3, 4, 5, or 6 times a day. Preferably, the antibody-comprising formulation is administered at least once a week, twice a week, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or at least 14 times a week. Preferably, the antibody-comprising formulation is administered at most once or twice a day, preferably at most once a day.

In an embodiment, the antibody-comprising formulation is administered once a day, twice a day, 3, 4, 5, or 6 times a day. Preferably, the antibody-comprising formulation is administered once a week, twice a week, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or at least 14 times a week. Preferably, the antibody-comprising formulation is administered once or twice a day, preferably once a day.

Preferably, the antibody-comprising formulation is administered at least once a week, or at least once every two weeks. Preferably, the antibody-comprising formulation is administered intranasally at least once a week. Preferably, the antibody-comprising formulation is administered once a week. Preferably, the time in between two consecutive administrations is at least about 1 , 2, 3, 4, 5, 6, 7 or 14 days.

Preferably, the antibody-comprising formulation is administered once a week, or once every two weeks. Preferably, the antibody-comprising formulation is administered once a week. Preferably, the time in between two consecutive administrations is about 1 , 2, 3, 4, 5, 6, 7 or 14 days.

The administration is preferably in both nostrils. Preferably, the administration is in both nostrils at the same time or in one nostril and shortly thereafter in the second nostril. However, it is further understood herein that the administration encompasses the administration in only one nostril. As a non-limiting example, the first administration is in the left nostril and the second administration is in the right nostril, e.g. alternating between the left and right nostril. In an embodiment, the administration is predominantly, or only, in the right nostril or predominantly, or only, in the left nostril.

In a preferred embodiment, the antibody-comprising formulation is administered once a day in both nostrils.

The volume administered per nostril of the antibody-comprising formulation as defined herein is preferably at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least about 100 pi. The volume administered per nostril is preferably between about 10 - 100 pi, between about 20 -90 pi between about 30 - 80 pi between about 40 - 70 pi, 45 - 60 pi or between about 48 - 55 pi. Preferably, the volume administered per nostril is about 50 pi.

The total volume administered per administration is preferably at least about 20, 40, 60, 80, 100, 120, 140, 160, 180 or at least about 200 pi per administration. The volume administered in total per administration is preferably in between about 20 - 200 pi, between about 40 - 180 pi, between about 60 - 160 pi, between about 80 - 140 pi, between about 90 - 120 pi or between about 95 - 105 mI. Preferably, the total volume per nasal administration of the antibody-comprising formulation as defined herein is about 100 mI.

In an embodiment, the total amount of antibody per administration is at least about 0.1 pg, 0.2 pg, 0.3 pg, 0.4 pg, 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1 .0 pg, 2.0 pg, 3.0 pg, 4.0 pg, 5.0 pg, 6.0 pg, 7.0 pg, 8.0 pg, 9.0 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 1 10 pg, 120 pg, 130 pg, 140 pg, 150 pg, 200 pg, 250 pg, 300 pg, 350 pg, 400 pg, 450 pg or at least about 500 pg. The total amount of the antibody per administration is preferably between about 0.1-1000 pg, 1 -900 pg, 5-800 pg, 10-700 pg, 30-600 pg, 40-400 pg, 50-200 pg, 60-170 pg, 70- I SO pg, 80-120 pg or between about 90 - 1 10 pg. Preferably, the total amount of antibody that is administered per administration is about 100 pg of the antibody, preferably of the neutralizing antibody, as defined herein.

The total amount of antibody per administration may be divided over both nostrils. The total amount of antibody can be equally divided over both nostrils, however it is also contemplated within the invention that one nostril receives a larger portion (e.g. at least about 60%, 70%, 80% or at least 90%) of the antibody than the other nostril per administration.

Preferably, the amount administered is about one drop per nostril, preferably a drop from any conventional nose dropper known in the art. One drop from a nose dropper is about 50 pi, although this may vary among nose droppers. Preferably, the amount administered intranasally is one drop per nostril from a nose dropper as defined herein below.

The duration of the treatment as defined herein is preferably the duration of the RSV season, which usually begins in the fall and runs into spring— although year-round RSV activity has been reported in warmer climates. The duration of the treatment per year is preferably at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks or more. The duration of the treatment per year is preferably at least about 3, 4, 5, 6, 7, 8, 9, or 10 weeks or more. Preferably, the duration of the treatment is at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 months. Preferably, the duration of the treatment per year is less than about 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or less than about 1 month. Preferably, the duration of the treatment per year is between about 1 - 10 months, 2 - 9 months, about 3-8 months, about 4-7 months, or about 4-6 months. Preferably, the duration of the treatment is about 5 months.

The subject may be treated during only one RSV season, e.g. is treated during only one year. However, the treatment may be repeated yearly, e.g. during consecutive RSV seasons. Hence, a subject may be treated for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more.

The subject to be treated according to the invention is preferably a subject that is susceptible for RSV infection. The terms "subject" and "patient" can be used interchangeably herein. As used herein, the terms "subject" and "subjects" refer to an animal, preferably a mammal including a nonprimate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a chimpanzee, a monkey such as a cynomolgous monkey, and a human), and more preferably a human.

In one embodiment, the formulations as defined herein are administered, preferably intranasally, to a mammal, preferably a human, to prevent, treat, manage or ameliorate a RSV infection or one or more symptoms thereof. In another embodiment, the formulations as defined herein are administered, preferably administered intranasally, to a human with cystic fibrosis, bronchopulmonary dysplasia, congenital heart disease, congenital immunodeficiency or acquired immunodeficiency, or to a human who has had a bone marrow transplant to prevent, treat, manage or ameliorate a RSV infection or one or more symptoms thereof.

In another embodiment, the formulations as defined herein are administered, preferably are administered intranasally, to a human infant, preferably a human infant born prematurely or a human infant at risk of hospitalization for a RSV infection to prevent, treat, manage or ameliorate a RSV infection or one or more symptoms thereof.

In another embodiment, the formulations as defined herein are administered, preferably are administered intranasally, to an elderly person to prevent, treat, manage or ameliorate a RSV infection or one or more symptoms thereof.

In another embodiment, the formulations as defined herein are administered, preferably administered intranasally, to a subject in an institution or group home (e.g., a nursing home or orphanage).

In a preferred embodiment, the formulation as defined herein is administered, preferably is administered intranasally, to an infant. Preferably, the infant is at least about 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , or 42 weeks gestational age. Preferably, the infant is at least about 36 weeks gestational age. Preferably, the infant is a pre-term, early term, full term or late term infant. Preferably, the infant is a pre-term or early term infant. Preferably, the infant is a pre-term infant, preferably a late pre-term infant.

In a preferred embodiment, the infant is born 30-37 weeks gestational age, preferably born 32-35 weeks gestational age.

In an embodiment, the subject to be treated is preferably a neonate (first 28 days after birth), an infant (0-1 year), a toddler (1 -3 years), a pre-schooler (3-5 years), a school-aged child (5-12 years) or an adolescent (13-18 years). Preferably, the subject is a neonate, an infant or a toddler.

In an embodiment, the subject to be treated can start the treatment directly after birth. Alternatively or in addition, the subject to be treated is at the start of the treatment at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 days old. Alternatively or in addition, the subject to be treated is at the start of the treatment at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks old. Alternatively or in addition, the subject to be treated is at the start of the treatment at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 1 1 months old. Alternatively or in addition, the subject to be treated is at the start of the treatment at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 years old.

In an embodiment, the subject to be treated is at the start of the treatment between about 0- 10 years, 0-8 years, 0-6 years, 0-5 years, 0-4 years, 0-3 years, 0-2 years, or between about 0- 1 years old. Preferably, the subject to be treated is at the start of the treatment between about 0- 36 months, 0-34 months, 0-32 months, 0-30 months, 0-28 months, 0-24 months, 0-22 months, 0-20 months, 0-18 months, 0-16 months, 0-14 months, 0-12 months, 0-10 months, 0-8 months, 0-6 months, 0-4 months, or between about 0-2 months old. Preferably, the subject to be treated is at the start of the treatment between about 0-12 months, 1-10 months, 2-9 months, 3-8 months, or between about 4-7 months old. Preferably, the subject to be treated is at the start of the treatment between about 0-6 months old.

In another embodiment, the subject to be treated is preferably and elderly individual. RSV infection is a significant problem in elderly adults due to a diminished immune system in that population For purposes of this disclosure, a human is defined as "elderly" if they are at least 65 years of age. In an embodiment, therefore, the treatment discussed herein is intended for use with a human of 65 years of age or more. In an alternative embodiment, the subject to be treated is at the start of the treatment is at least 65, 70, 75 or more years old.

In a second aspect, the invention pertains to an antibody-comprising formulation as defined herein above. The antibody-comprising composition is preferably suitable for the use specified above. The antibody-comprising formulation preferably comprises the antibody at a concentration as defined herein above.

The antibody-comprising formulation may be a conventional, e.g. a commercially available, nose (nasal) drop formulation further comprising the antibody as defined in herein.

In an embodiment, the antibody-comprising formulation further comprises an isotonic solution, preferably as defined herein above. Preferably, the isotonic solution is a buffered isotonic solution as defined herein above.

In an embodiment, the antibody comprising formulation comprises a preservative, preferably a preservative as defined herein above. Preferably, the preservative is Benzalkonium chloride.

In an embodiment, the antibody-comprising formulation comprises at least one of Sodium Chloride and a preservative. The formulation is preferably buffered.

The antibody-comprising formulation may comprise a viscosity-increasing compound, such as, but not limited to, methocel K4M.

In addition or alternatively, the antibody-comprising formulation may comprise at least one of sodium dihydrogen phosphate dihydrate, disodium phosphate dodecahydrate and disodium edetate.

The concentration of at least one of the preservative, the viscosity-enhancing compound, sodium dihydrogen phosphate dihydrate, disodium phosphate dodecahydrate and disodium edetate is a concentration as specified herein above.

Preferably, at least one of these compounds are present in the formulation at a concentration as defined herein above in the first aspect of the invention.

In a third aspect, the invention concerns a method for preparing an antibody-comprising formulation as defined herein. Preferably, the method comprises a step of dispensing the antibody as defined herein in a suitable formulation. The antibody is preferably dispensed in a conventional nose (nasal) drop formulation. As a non-limiting example, commercially available Synagis® (Abbvie BV), comprising 100 mg/ml_ palivizumab (MEDI-493) can be diluted 100 times in a commercially available nose drop solution, such as but not limited to Fagron (article number 5086, Fagron NL BV).

In an embodiment, the method comprises a step of dispensing the antibody as defined herein in a formulation as defined herein. In a further aspect, the invention relates to a kit of parts. The kit of parts preferably comprises a first vial comprising a solution comprising the antibody as defined herein and a second vial comprising a solution suitable for intranasal administration. Preferably, the solution for intranasal administration is at least one of a conventional, e.g. commercially available nose drop and a solution as defined herein above in the first aspect of the invention.

In a further aspect, it is provided for a nasal dropper bottle comprising a container comprising the composition of the invention. The nasal dropper bottle can be any nasal dropper bottle described in the art. Typically, the nasal dropper bottle will comprise a pipette (open at both ends), a compressible bulb, a container for containing liquids and a bottle cap. One end of the pipette is placed into the container and the compressible bulb is mounted on the other end of the pipette. When the free end of the pipette is held into the container comprising liquid and the compressible bulb is compressed, the air inside the bulb will be expelled from into the container. When the pressure on the compressible bulb is subsequently released, The elasticity of the bulb allows it to return to its initial volume, creating a vacuum in the bulb which allows the pipet to be filled with the liquid. Compressing the bulb anew will release the liquid in drops from the pipet. The pipette is usually affixed to the bottle cap, usually mounted in the center of the cap in a sealed relationship. The bottle cap comprising the pipette can be screwed onto the container, thereby creating an air tight liquid container that will prevent spilling of the liquid.

In a further embodiment, the container of the nasal dropper bottle of the invention is made from glass. Preferably, the container is made from glass. In a further embodiment, the container is made from glass that protects the liquids inside the bottle from exposure to light. Preferably, the container is made from tinted glass, such as amber coloured or brown glass.

In a further embodiment, the pipette nasal dropper bottle according to the invention comprises a pipette made from glass. The pipette can be made from“clear” glass, allowing the user to view the level of liquid present in the pipette.

In yet another aspect, the invention provides for a nasal spray comprising a bottle or equivalent receptacle comprising the composition of the invention. The nasal spray can be any nasal spray described in the prior art. Typically, the nasal spray consists of a bottle containing a liquid solution. The bottle is further provided with a part for dispensing the preparation into the nostril. The solution can then be squirted into the nostril by any suitable means, for instance by means of a pump, by deformation of the bottle or by using a suitable propellant. In one embodiment, the bottle or equivalent receptacle is made from glass. Preferably, the bottle or equivalent receptacle is made from glass. In a further embodiment, the bottle or equivalent receptacle is made from glass that protects the liquids inside the bottle from exposure to light. Preferably, the bottle or equivalent receptacle is made from tinted glass, such as amber colored or brown glass. Figures legend

Figure 1 : schematic representation of the experimental set-up for stability testing. Dilutions, all from the same stock palivizumab were prepared and placed at three different temperatures (4°C, room- temperature (RT), and 37°C) for up to 52 weeks.

Figure 2: Set-up of the safety studies. The study is divided into two arms, the first arm will receive 1 mg/ml_ palivizumab (narsyn) daily for 7 days and the second arm will receive a placebo daily for 7 days. This intervention period is followed by a 14-day wash out period. After the wash out period, individuals from the first arm will be switched to the second arm and vice versa.

Figure 3: Kinetics of palivizumab in bronchoalveolar lavage (BAL). The palivizumab concentration was measured 1 (D-1), 3 (D-3), 4 (D-4), 5 (D-5) or 7 (D-7) days after administration.

Figure 4: Kinetics of palivizumab in nasal lavage (NAL). The palivizumab concentration was measured 1 (D-1), 3 (D-3), 4 (D-4), 5 (D-5) or 7 (D-7) days after administration.

Figure 5: Kinetics of palivizumab in bronchoalveolar lavage (BAL) after RSV challenge. RSV infection was 1 (D-1 ), 3 (D-3), 4 (D-4), 5 (D-5) or 7 (D-7) days after administration of palivizumab. The palivizumab concentration was measured one day after the RSV infection.

Figure 6: Kinetics of palivizumab in nasal lavage (NAL) after RSV challenge. RSV infection was 1 (D-1), 3 (D-3), 4 (D-4), 5 (D-5) or 7 (D-7) days after administration of palivizumab. The palivizumab concentration was measured one day after the RSV infection.

Examples

Example 1

Despite being fully protected against RSV infection for at least one week (Jacobino J Allerg Clinical Immunol, 2016 Nov; 138 (5): 1477-1480), we showed that monoclonal RSV-neutralizing antibodies (0.5 mg/kg palivizumab) administered into the lungs of naive wild-type BALB/c mice are detected in low levels or not at all detected on the mucosal level 7 days after administration in the nasal airway or lungs. Naive wild-type BALB/c mice were treated with palivizumab prophylaxis one (d-1), three (d-3), four (d-4), five (d-5) or seven (d-7) days before bronchoalveolar lavage (BAL) and nasal lavage (NAL) were performed (DO) to measure palivizumab concentrations in the mucosa.

Uninfected Mice: We treated 3 mice per group with 50ul palivizumab on either day -1 , day - 3, day -4, day -5 or day -7 and collected 1 mL BAL and NAL on day 0. PBS prophylaxis was used as a negative control, administered on d-1 . One week after antibody administration we detected minimal concentrations of palivizumab in BAL [Fig 3] (mean 83.1 ng/ml) and NAL [Fig 4] (mean 1 .6 ng/ml) as compared to BAL [Fig 3] (mean 1718.2 ng/ml) and NAL [Fig 4] (mean 12.2 ng/ml) collected one day after antibody administration.

Infected Mice: Similarly, naive wild-type BALB/c mice were treated with palivizumab prophylaxis one (d-1), three (d-3), five (d-5) or seven (d-7) days before infection with RSV on day 0. 24 hours later (day 1), bronchoalveolar lavage (BAL) and nasal lavage (NAL) were performed (DO) to measure palivizumab concentrations in the mucosa. We treated 3 mice per group with 50ul palivizumab on either one (d-1), three (d-3), five (d-5) or seven (d-7) days before RSV infection on day 0 with 3x 10 ⁶ PFU RSV-A2 in a volume of 50ul PBS and collected 1 mL BAL and NAL 24 hours afterwards (day 1). PBS prophylaxis was used as a negative control and trastuzumab (HerC) was used as an isotype control lgG1 antibody because it has no RSV binding activity. In mice treated with antibody one week before infection we detected minimal concentrations of palivizumab 24 hours afterwards in BAL [Fig 5] (mean 0.7 ng/ml) and NAL [Fig 6] (no antibody detected) as compared to BAL [Fig 5] (mean 3.8 ng/ml) and NAL [Fig 6] (mean 77.9 ng/ml) of mice treated with antibody one day before infection.

These data indicate that intranasal antibody prophylaxis in vivo can protect fully against infection up to seven days after palivizumab administration (Jacobino J Allerg Clinical Immunol, 2016 Nov; 138 (5): 1477-1480), even though minimal or no antibody is detected in the mucosa of the nose and/or lungs after prophylaxis in infected and uninfected mice.

Example 2

Methods

HPSEC- stability measurement

HPSEC was used to determine stability over time. Decomposition chemistry was measured, specifically to indicate:

o chemical degradation or aggregate formation

o relative concentration

HP-SEC criteria were defined for acceptable stability based on Drug Stability Guidelines developed by the FDA (Guidance for Industry, 2008). In this guideline strength can be measured through concentration or potency. Consensus by the scientific community indicates that 90% of labeled potency is generally recognized as the minimum acceptable potency level. Therefore, our criterion for stability was 90% minimum potency level (a maximum decrease of 10% concentration). Furthermore, the appearance of new protein peaks indicating products of degradation (retention time > 7.17 minutes) and aggregate formation (retention time < 7.12 minutes) was considered chemical decomposition.

In summary, the following three criteria were used to measure stability:

1 . Concentration above 90% compared to concentration at t=0 weeks (potency) 2. No new protein peaks with retention time < 7.12 minutes (chemical degradation)

3. No new protein peaks with retention time > 7.17 minutes (chemical degradation)

HPSEC was performed with a Yarra 3um SEC 2000 column (Phenomenex; Part No: 00H-4512-E0) on Shimadzu HPLC system LC20-AT and as the mobile phase (12uL) in a mobile phase 100mM Sodium Phosphate (pH 6.8) and 150mM NaCI. Samples of equal injection volumes (12uL) were measured at a flow rate of 0.350ml_/min at room temperature and detection occurred at 280 nm. All samples were diluted to 50% in mobile phase buffer to reduce viscosity. To prepare for HPSEC samples were spun at 10000 ref for 5 minutes at 4°C using a 0.22um filter (Millipore cat no: UFC30GVOS). 60ul of filtrate was transferred to a HPLC sample injection vial (1 ,5mL; 548-0386; VWR) with silanized micro-insert (0.1 mL; 548-031 1 ; VWR) for small volumes and screw cap PP (548-0390; VWR). Analysis was performed using LabSolutions Version 5.72 (Shimadzu). Area under the curve was used to calculate concentration relative to concentration at t=0 weeks.

Experimental Set-up

Dilutions of 1 mg/mL and 0.1 mg/mL palivizumab in commercial 0.9% NaCI nasal drops were prepared in brown glass nose drop bottles (Spruyt Hillen; Ijsselstein, The Netherlands) and placed at three different temperature conditions (4°C, RT, and 37°C) according to Fig. 1 . All dilutions were prepared from the same stock of palivizumab. Aliquots were removed at different time points from each condition and stored at -80°C until testing.

Results

Stability was tested for the following storage temperature conditions:

• 4°C

• Room temperature (RT)

• 37°C

For proof-of-principle that lack of stability could be measured, the following stress conditions was tested:

• 10-fold lower concentration (0.1 mg/mL)

• 70°C

The stability data for the drug substance show that even at these low concentrations, narsyn is intrinsically very stable. No degradation and loss of integrity was observed for the intended use formulation when kept for 1 year at various temperature conditions (4°C, room temperature) and for 6 months at other temperature conditions (37°C).

We note that it could not have been predicted on forehand that these low concentrations of palivizumab would remain stable for a sufficient period of time to use the composition for the prevention or treatment of an RSV infection. 4°C

The concentration of 1 mg/ml palivizumab stored at 4°C remains above the FDA acceptable level of 90% after 52 weeks as can be seen in table 2 below and no degradation or aggregate formation was observed (data not shown).

Table 2: Observed concentrations of 1 mg/ml palivizumab at 4°C measured by HPSEC after different storage times

Temperature Time stored Concentration

(°C) (weeks) (mg/ml_)

0 1 ,00

1 1 ,03

4 0,98

4 8 1 ,00

12 1 ,00

26 1 ,04

52 1 ,05

37°C

There was no decrease in concentration below the FDA threshold of 90% after 26 weeks when 1 mg/mL is stored at 37°C (table 3). When 1 mg/ml_ palivizumab was stored at 37°C the appearance of very small protein peaks (retention time 8.8 minutes) was observed which increased with increasing time stored at 37°C (table 4).

Table 3: Observed concentrations of 1 mg/ml palivizumab at 37°C measured by HPSEC after different storage times

Temperature

Time stored Concentration

(°C)

(weeks) (mg/mL)

0 TOO

1 1 .02

4 0,95

37

8 0,92

12 0,90

26 1 ,03

52 0,89 Table 4. Observed concentrations of degradation product at 37°C measured by HPSEC after different storage times

Time stored at 37°C Retention time Area

(weeks) (minutes) (%)

0 No peak detected 0

1 8,796 0,06

4 8,821 0,18

8 8,822 0,27

12 8,845 0,61

26 8,895 1 ,36

52 8,895 2,69

Freshly prepared No peak detected 0 Room-temperature (RT)

When 1 mg/mL is stored at RT there is no decrease in concentration below the 90% FDA threshold after 52 weeks (Table 5). No chemical degradation is observed for at least 4 weeks. After 8 weeks a very small protein peak appeared with a retention time of 8.8 minutes (data not shown).

Table 5: Observed concentrations of 1 mg/ml palivizumab at RT measured by HPSEC after different storage times

Temperature Time stored Concentration

(weeks) (mg/mL)

0 1 ,00

1 1.03

4 0,97

RT 8 1 .02

12 0,95

26 1 ,03

52 1.04

Stress conditions (control)

The ability to measure both loss of potency and chemical degradation was demonstrated with 0.1 mg/mL palivizumab stored at 4°C a condition at which instability upon storage is expected.

When stored at a ten-fold lower concentration (0.1 mg/mL) a decrease in potency below 90% is observed after 1 week as can be seen in table 6 below. Table 6: Observed concentrations of 0,1 mg/ml palivizumab at 4°C measured by HPSEC after different storage times

Temperature Time stored Concentration

(weeks) (mg/mL)

0 0,100

1 0,078

4 0,067

4 8 0,076

12 0,053

26 0,074

52 0,074

Table 7: Observed concentrations of 1 mg/ml palivizumab at 70°C measured by HPSEC after different storage times

Temperature Time stored Concentration

(°C) (hours) (mg/mL)

0 TOO

1 0,67

70 2 0,68

4 0,65

Freshly prepared 0,96

After 1 hour of storage at 70°C, there was aggregate formation with a new protein peak visible with a retention time of 6.0 minutes which increased with time stored at 70°C. Loss of potency <90% was observed after 1 hour of storage at 70°C (table 7).

Example 3

Stability testing was performed independently by Apotheek A15. HPSEC was used to determine stability over time of palivizumab nose drops. Dilutions of 1 mg/mL palivizumab in commercial 0.9% NaCI nasal drops were prepared and placed at three different temperature conditions (4°C, room temperature (RT), and 40°C).

HP-SEC criteria were defined for acceptable stability as 90% of labeled concentration (a maximum decrease of 10% concentration). Criteria for color was B9 (Ph. Eur.) and purity as OS1 (Ph. Eur.) and lightly viscous, colorless liquid upon visual inspection. Criteria for pH was between 5, 0-8, 3.

4°C

The concentration of palivizumab stored at 4°C remains above the FDA acceptable level of 90% after 12 months as can be seen in table 8 below. Color, purity and pH also met predefined stability criteria after 12 months. Even when product is removed from the storage condition for 2,5 months at room temperature, it still meets stability criteria after a period of 14 months.

Table 8. Observed color, purity, pH, and concentration of palivizumab at 4°C after different storage times

TIME Color Purity Color pH Concentration

STORED (Ph Eur) (Ph Eur) (Visual) (%)

(months)

*: The color was not determined at t=0 according to Ph. Eur. as this was not yet standard practice at A15 Pharmacy. **: Comparator for 3-month timepoint, which was performed from a separate batch. Rise in concentration at 3 months was accepted because it was less than 5% higher than t=0 comparator (**). *** Comparator for 14-month timepoint, which was performed from a separate batch. The stability for the 14-month timepoint was measured including a period of 2,5 months at room temperature. RT

The concentration of palivizumab stored at RT remains above the FDA acceptable level of 90% after 6 months as can be seen in table 9 below. Color, purity and pH met predefined stability criteria after 12 months.

Table 9. Observed color, purity, pH and concentration of palivizumab at RT after different storage times

TIME Color Purity Color pH Concentration

STORED (Ph Eur) (Ph Eur) (Visual) (%)

(months)

*: The color was not determined at t=0 according to Ph. Eur as this was not yet standard practice at A15 pharmacy. **: Comparator for 3-month timepoint, which was performed from a separate batch.

40 °C

The concentration of palivizumab stored at 40°C falls below the FDA acceptable level of 90% after 1 month as can be seen in table 10 below. Color upon visual inspection met predefined stability criteria after 1 month. Table 10. Observed color and concentration of palivizumab at 40°C after different storage times TIME Color pH Concentration

STORED (Visual) (%)

(months)

*: Comparator for 3-month timepoint, which was performed from a separate batch. In conclusion, the low concentrations of palivizumab remained surprisingly stable, indicating that such compositions can be used for the prevention or treatment of an RSV infection.

Example 4

Methods

The safety of the intranasal administration of palivizumab is tested first in healthy adults in a phase I safety study. A phase I double blind randomized placebo-controlled crossover trial was performed in healthy adult volunteers 18 - 60 years of age. Two arms of 10 individuals and randomize the individuals were enrolled to administer 1 mg/mL palivizumab (narsyn) or placebo daily for 7 days to the right nostril. The 7 days administration period was followed by a 14 day wash-out period before the group of individuals crosses over to the other arm of the study (see Fig 3.). Patients scored symptoms on the FDA scorecard scale from 0 to 3. The overall incidence of solicited local (pain, redness, stuffy nose, sneezing, runny nose, nasal congestion, sore throat) or systemic (fever >38C, headache, myaligia) adverse events (AE) was calculated for the vaccine and placebo group for each nostril separately.

Results

No toxicity was measured upon intranasal administration for this monoclonal antibody that has a non-human target (RSV F protein) and has been used clinically for over 20 years. Example 5

Safety of narsyn was determined through a phase I randomized-controlled trial. Crossover safety study in healthy adult volunteers. After favorable DSMB evaluation, study B will start. The objective of this trial was to measure the safety of intranasal administration of palivizumab in healthy adults. The study population consisted of healthy adult men and women 18-60 years of age. One nose drop in the right nostril once daily of 1 mg/mL palivizumab (narsyn) or placebo for 7 days; 14 day washout period, then crossover to other arm for 7 days. The main study outcome is self-reported symptoms according to the FDA scorecard and SAE’s. The phase lib was initiated based on the overall safety profile. The study will proceed to study B if no serious adverse events or other AE are considered to be treatment-related by the investigators and the DSMB. As there were no treatment- related adverse events and no severe adverse events, the DSMB found the safety profile to be acceptable and gave a positive advice to continue on to the phase MB trial.

Table 11. Airway patency 10 minutes after nasal drop during arm 1 of phase I crossover safety trial in trial participants who received Narsyn or placebo. There was one exclusion in the placebo group (9/10 participants).

Table 12. Local and general symptoms per participant in the placebo and narsyn trial arms. Symptoms were scored daily during treatment according to a symptom scorecard from 0 to 3. 0: no symptoms; 1 : mild, does not hinder daily activities; 2: moderate, hinders daily activities; 3: severe, not able to perform daily activities. If there were no reported symptoms (“0” in diary) then 0 was not reported in this table. D: day (days were numbered from 1 to 7 in each trial arm.

Table 13. Composite severe adverse events in either trial arm. *As determined by DSMB. SAE: severe adverse event. AE: adverse event, DSMB: data safety monitoring board

Table 14. Overview of adverse events from both trial arms including conclusion about whether or not adverse event was treatment-related. NA: not applicable.

Example 6

Methods

The effect of local administration of palivizumab on prevention of RSV infection in the target population is tested in a double-blind placebo controlled proof-of-concept trial (phase 2b clinical trial). The study population consists of late preterm infants 32-35 weeks gestational age who are less than 6 months of age at the onset of the RSV season, with at least one sibling will be tested. The infants are given 1 nose drop per nostril once daily of 1 mg/ml_ palivizumab (narsyn) or placebo for a duration of 2 - 5 months during the RSV season. These infants are traced to see if they develop RSV which can be used to determine the efficacy of this daily nasal administration.

Inclusion criteria of the study population

Healthy preterm infants with gestational age between 32 and 35 weeks and less than 6 months of age at the onset of the RSV season are eligible. Children must have a least one sibling as this increases their risk of RSV infection. The first five children are selected based on an expected duration of hospitalization for preterm birth for at least seven days to allow for clinical observation of AE after start of treatment for at least 3 days. Exclusion criteria

Children with a known cardiac anomaly, Down syndrome or other serious congenital disorders and children who received surfactant treatment are excluded from the study. Simultaneous use of other nose drops or nasal spray is an exclusion criterion except normal saline drops.

Results

The results of the placebo controlled phase 2b trial in non-high-risk infants will demonstrate the efficacy of local administration of narsyn in at least one of the prevention of any RSV infection, medically-attended RSV infection, RSV-associated hospitalization and infant wheeze during the first year of life.

In addition, using the same or similar methods as outlined above, the efficacy of local administration of narsyn will also be demonstrated in high risk children, High risk children include severe and mild preterm infants, children with congenital heart disease, children with Down syndrome, children with neurological disease or any other severe disease.