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WO2020108735A1 - Anticorps dirigés contre l'interleukine-10 du cytomégalovirus - Google Patents

Anticorps dirigés contre l'interleukine-10 du cytomégalovirus Download PDF

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WO2020108735A1
WO2020108735A1 PCT/EP2018/082603 EP2018082603W WO2020108735A1 WO 2020108735 A1 WO2020108735 A1 WO 2020108735A1 EP 2018082603 W EP2018082603 W EP 2018082603W WO 2020108735 A1 WO2020108735 A1 WO 2020108735A1
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antibody
set forth
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annotation
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Nicola Beltraminelli
Pierre Garrone
Adrianne MARTIN
Virginie MANDRON
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Blink Biomedical Sas
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244Interleukins [IL]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/70Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/90Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to antibodies specific to human cytomegalovirus (hCMV) interleukin (IL)-10.
  • hCMV human cytomegalovirus
  • IL interleukin
  • hCMV Human cytomegalovirus
  • hCMV infections can be acute (lytic) or latent (inactive).
  • the lytic phase can occur upon first infection or can result from the reactivation of an existing infection.
  • the hCMV genome contains a variety of genes which provide different mechanisms for hCMV to evade host immune responses, which can lead to the establishment of viral latency in healthy individuals and/or to chronic infections and diseases in immunodeficient patients (Alcami, et al. (2003) Nature Rev. Immunol. 3:36-50; McSharry, et al. (2012) Viruses 4:2448-2470; Spencer, et al. (2012) Herpesviridae-A Look Into This Unique Family of Viruses, doi: 10.5772/27095; Goodrum, et al. (2012) Cell. Microbiol. 14(5):644-655; Ouyang, et al. (2014) J. Gen. Virol.
  • hCMV human IL-10
  • hcmvIL-10 a homolog of cellular human IL-10
  • the hcmvIL- 10 protein binds to the same cell surface receptors as hIL-10 and possesses most of the biological activities of hIL-10, including its immunosuppressive activities on anti-viral immune responses.
  • hcmvIL- 10 The production of hcmvIL- 10 during hCMV infection is thought to contribute to the establishment of latency and/or of chronic infections and diseases through modulation of host immune responses (Jones, et al. (2002) PNAS 99(14):9404-9409; Slobedman, et al. (2009) J. Virol. 83(19):9618-9629; Chang, et al. (2009) Virology 390:330-337; Chang & Barry (2010) PNAS 107(52):22647-22652; Avdic, et al. (2013) J. Virol. 87(18): 10273-10282; Sinclair & Reeves (2013) Viruses 5:2803-2824; Avdic, et al.
  • hcmvIL-10 The production of hcmvIL-10 by tumor cells or by hCMV-infected normal cells in the microenvironment of a tumor may inhibit the establishment of efficient anti-tumoral immune responses, thus favoring the development of the tumor. Also hcmvIL-10 may act directly on tumor cells to increase cancer cell proliferation, migration and invasiveness (Sampson & Mitchell (2011) Clin. Cancer Res.
  • hcmvIL-10 The production of hcmvIL-10 during gestation may also contribute to hCMV-mediated impairment of placental development, which results in intrauterine growth restriction (Yamamoto-Tabata, et al. (2004) J. Virol. 78(6):2831-2840; Pereira, et al. (2014) J. Infect. Dis. 209:1573-84).
  • Targeting hcmvIL-10 with antagonists such as anti-hcmvIL-10 neutralizing antibodies may represent a way to limit the modulation of immune responses during hCMV infections and/or reactivation, and thus reduce disease severity in at- risk patients.
  • antiviral drugs which inhibit viral replication, but use of these drugs can lead to toxicity and to the development of resistance. Moreover, due to their toxicity, antivirals cannot be prescribed in certain indications; for example, for administration to pregnant women to prevent congenital infections in the fetus.
  • vaccines nor monoclonal antibodies for use in the prevention and/or treatment of hCMV infections (Plotkin (2015) Med. Microbiol. Immunol. 204:247-254).
  • hCMV treatments are available in the form of polyclonal antibodies from hCMV hyperimmune donors, e.g., Cytotect CP and IVIG from healthy donors, but these are approved for only some indications and are available only in limited countries.
  • hCMV Most vaccine and antibody programs in development target hCMV envelope glycoproteins for prevention of cell infection by the virus.
  • hCMV uses multiple glycoprotein complexes to infect a large variety of target cells and has developed strategies to escape anti-envelope immune responses.
  • Targeting one of the viral mechanisms of immunomodulation, such as hcmvIL-10, represents a new and promising approach for preventing, treating or reducing the severity of hCMV diseases.
  • a vaccine to induce antibodies against cmvIL-10 is currently under development in the Rhesus macaque model (Logsdon, et al. (2011) PLoS ONE 6(l l):e28127; Eberhardt, et al.
  • hCMV produces at least two splice variants of hcmvIL-10, resulting in production of the full -length hcmvIL-10 as well as a truncated isoform designated latency associated hcmvIL-10 (LAcmvIL-10; Jenkins, 2004 J. Virol. 78(3): 1440-1447; Lin, et al. (2008) Virus Res. 131(2): 213-223).
  • LAcmvIL-10 a truncated isoform designated latency associated hcmvIL-10
  • hcmvIL-10 and the truncated LAcmvIL-10 are the main viral IL-10 analogs produced; whereas, during the latent phase of infection, LAcmvIL-10 is the predominant form.
  • the truncated LAcmvIL-10 retains only a limited range of the hcmvIL-10 functional activities and it is unclear whether this protein can interact with IL-10 receptors (Christiaansen, 2015, supra). Further study is required to understand the function of this protein in hCMV disease states.
  • the present invention discloses fully-human antibodies with high affinity and strong binding specificity to hcmvIL-10. Most of the provided antibodies also potently neutralize in vitro biological effects of hcmvIL-10, but not hIL-10, thus suggesting a safe profde in this regard. Further, a subset of the antibodies provided also bind to the latency associated form of the hcmvIL-10 (LAcmvIL-10), which may be advantageous in certain therapeutic and diagnostic settings.
  • hCMV human cytomegalovirus
  • the disclosed antibodies specifically bind to hcmvIL-10, but not to cellular human IL-10 (hIL-10), Epstein-Barr Virus (EBV)-encoded IL-10 (ebvIL-10) or macacine (rhesus macaque) CMV-encoded IL-10 (RhcmvIL-10). All but one of the antibodies (VL016-8-9F5) has the capacity to neutralize biological activities of hcmvIL-10 in vitro. In addition, one out of the six binding variant families of antibodies identified also bind to the truncated latency-associated form of hcvmIL-10 (LAcmvIL-10), as assessed by ELISA.
  • LAcmvIL-10 truncated latency-associated form of hcvmIL-10
  • the anti-hcmvIL-10 neutralizing antibodies disclosed herein possess an IC50 in the range of 50- 200 pM, as measured by their capacity to neutralize the hcmvIL-10-mediated inhibition of TNF-a, IL-I b or IFN-g production by human PBMC activated with LPS or Poly(LC).
  • the equilibrium binding constant of the antibodies for hcmvIL-10 is in the 10 to 100 pM range as determined by ELISA.
  • the affinity constant (K D ) of the antibodies for hcmvIL-10 is in the range of 35 to 270 pM as determined by surface plasmon resonance (SPR) on Biacore.
  • Antibodies neutralizing hcmvIL-10 may be of potential value to specifically inhibit the immunomodulatory effects of hcmvIL-10 produced by hCMV-infected cells.
  • the antibodies may be of use for preventing, treating and/or decreasing the severity of hCMV-mediated diseases in at-risk individuals such as immunodeficient patients, transplant patients and pregnant women and/or infants at risk of hCMV primary infection.
  • Neutralizing anti-hcmvIL-10 antibodies may also be of great value for treating cancer patients with hCMV infection and/or with hCMV-positive tumors.
  • the antibodies may be used for the detection of hcmvIL-10 in patients or patient samples and may serve as diagnostic reagents or as biomarkers for hCMV infections and/or be used to track progression of diseases marked by hCMV infection.
  • the antibodies of the current disclosure have distinct safety advantages due to at least: 1) the folly-human nature of the antibodies, 2) less side effects due to non-cross-reactivity with other forms of IL-10, and 3) a reduced risk of IL-10 cross-reactivity which may be triggered by active immunization against an hcmvIL-10 antigen.
  • FIG. 1 Comparison of hcmvIL-10 and hIL-10 biological activity on cytokine secretion by activated human PBMC.
  • A-C PBMC from Donor#l were stimulated with LPS (A, B) or Poly(I:C) (C) in the presence of increasing concentrations of hcmvIL-10 or hIL-10.
  • D PBMC from Donor#2 were stimulated with Poly(I:C) in the presence of increasing concentrations of hcmvIL-10 or hIL-10. After 24 hours of treatment, supernatants were collected and concentrations of TNF-a (A), IL-I b (B) or IFN-g (C, D) were determined.
  • Figure 2 Titers of anti-hCMV IgG in the sera of hCMV-seropositive donors and of anti-hcmvIL- 10-positive donors, as determined by the PLATELIATM CMV IgG kit. Also shown is the geometric mean with a 95% confidence interval“eq.” in the figure is for equivocal sera;“+” is for positive sera.
  • Figure 3 Inhibition of hcmvIL-10 activity by anti-hcmvIL-10 antibodies of the disclosure, as measured by the PBMC/LPS/TNF-a assay.
  • Human PBMC were cultured with 1.5 ng/mL hcmvIL-10 in the presence of serial dilutions of anti-hcmvIL-10 antibodies and were stimulated with LPS. After 24 hours treatment, supernatants were collected and concentrations of TNF-a were determined. The results are expressed as percent inhibition of hcmvIL-10 activity. Data shown are representative of 3 individual experiments.
  • Figure 4 Inhibition of hcmvIL-10 activity by anti-hcmvIL-10 antibodies of the disclosure, as measured by the PBMC/Poly(I:C)/IFN-y assay.
  • Human PBMC were cultured with 1.5 ng/mL hcmvIL-10 in the presence of serial dilutions of anti-hcmvIL-10 antibodies and were stimulated with Poly(I:C). After 24 hours treatment, supernatants were collected and concentrations of IFN-g were determined. The results are expressed as percent inhibition of hcmvIL-10 activity. Data shown are representative of 3 individual experiments.
  • Figure 5 Inhibition of hcmvIL-10 activity by anti-hcmvIL-10 antibodies at 3 pg/mL (20nM) as measured by the PBMC/LPS/IL-Ib assay.
  • the dashed line in the figure represents the concentration of IL-Ib produced by LPS-activated PBMC in the absence of hcmvIL-10.
  • Figure 6 Detection of hcmvIL-10 and LAcmvIL-10 by ELISA with an anti-hcmvIL-10 polyclonal antibody. Serial dilutions of supernatants of CHO cells transfected or not with expression plasmids encoding LAcmvIL-10 or hcmvIL-10 were coated onto ELISA plates. The presence of LAcmvIL-10 was detected by a rabbit anti-hcvmIL-10 antibody.
  • Figure 7 Inhibition of TNF-a secretion by LPS-stimulated human PBMC by supernatants of CHO cells transfected with a control vector or expression plasmids encoding LAcmvIL-10 or hcmvIL-10.
  • Figure 8 Cross-reactivity of anti-hcmvIL-10 antibodies with LAcmvIL-10 as measured by ELISA.
  • Supernatants of CHO cells transfected or not with expression plasmids encoding LAcmvIL-10 or hcmvIL-10 were coated at 1/20 dilution onto ELISA plates.
  • Anti-hcmvIL-10 antibodies or control antibody were tested at 1 pg/mL. Data are expressed as mean +/- SD of triplicate determinations.
  • Figure 13 Inhibition of TNF-a secretion by LPS-activated human PBMC in the presence of serial dilutions of supernatants of CHO cells transfected with a control vector or expression plasmids encoding RhcmvIL-10 and hcmvIL-10.
  • Figure 14 Specific recognition of hcmvIL-10 but not of RhcmvIL-10 by anti-hcmvIL-10 antibodies as determined by ELISA.
  • Figure 15 Specific inhibition of hcmvIL-10 but not of RhcmvIL-10 by anti-hcmvIL-10 antibodies as determined by the PBMC/LPS/TNF-a assay.
  • FIG. 16 Detection of hcmvIL-10 produced by hCMV-infected cells with the anti-hcmvIL-10 antibody 6-28A2.
  • Fibroblastic MRC-5 and epithelial ARPE-19 cells infected or not with the hCMV strain AD 169 or VR1814 were stained for the presence of CMV immediate early non-structural antigens (IE antigens) and hcmvIL-10.
  • IE antigens CMV immediate early non-structural antigens
  • Cells showing staining of both nuclear IE antigens and cytoplasmic hcmvIL-10 are tagged with a white star.
  • Cells showing only nuclear IE antigen staining are tagged with a black star.
  • the present invention relates to fully -human monoclonal antibodies which are specific for hcmvIL-10. Also disclosed are amino acid sequences of such antibodies.
  • the antibodies find use in therapeutic and diagnostic methods associated with hCMV infection and associated diseases.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. "Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multi-specific antibodies (e.g., bi- specific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
  • Antibodies (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • epitopic determinants means any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • “Native antibodies and immunoglobulins” are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
  • VH variable domain
  • Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains (Clothia et al., J. Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985)).
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • CDRs complementarity-determining regions
  • hypervariable regions both in the light-chain and the heavy-chain variable domains.
  • the more highly conserved portions of variable domains are called the framework (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a b-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the b-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (for RABAT annotation see Rabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, MD (1991) or for 1MGT annotation, see http://www.imgt.org).
  • the constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity (ADCC).
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen recognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species (scFv), one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that of a two-chain Fv species ft is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody.
  • variable domain or half of an Fv comprising only three CDRs specific for an antigen
  • scFv see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Spring er-Verlag, New York, pp. 269-315 (1994).
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGl, lgG2, lgG3, lgG4, IgAl, lgA2.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • Antibody fragment and all grammatical variants thereof as used herein are defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody.
  • constant heavy chain domains i.e. CH2, CH3, and CH4, depending on antibody isotype
  • antibody fragments include Fab, Fab', Fab'- SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment” or “single chain polypeptide”), including without limitation (1) single-chain Fv (scFv) molecules; (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety; (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and (4) multispecific or multivalent structures formed from antibody fragments.
  • single-chain antibody fragment single chain polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues
  • the heavy chain(s) can contain any constant domain sequence (e.g. CHI in the IgG isotype) found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s).
  • the term "conjugate” as described and claimed herein is defined as a heterogeneous molecule formed by the covalent attachment of one or more antibody fragment(s) to one or more polymer molecule(s), wherein the heterogeneous molecule is water soluble, i.e.
  • structured aggregate refers to (1) any aggregate of molecules in aqueous solution having a spheroid or spheroid shell structure, such that the heterogeneous molecule is not in a micelle or other emulsion structure, and is not anchored to a lipid bilayer, vesicle or liposome; and (2) any aggregate of molecules in solid or insolubilized form, such as a chromatography bead matrix, that does not release the heterogeneous molecule into solution upon contact with an aqueous phase.
  • conjugate encompasses the aforementioned heterogeneous molecule in a precipitate, sediment, bioerodible matrix or other solid capable of releasing the heterogeneous molecule into aqueous solution upon hydration of the solid.
  • mAh monoclonal antibody
  • monoclonal antibody 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. Each mAh is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture or mammalian cell lines, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made in an immortalized B cell or hybridoma thereof, or may be made by recombinant DNA methods.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) 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.
  • chimeric antibodies immunoglobulins 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.
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 75% by weight of antibody as determined by the Lowry method, and most preferably more than 80%, 90% or 99% by weight, or (2) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • the antibodies or pharmaceutical compositions of the present invention may be used in combination with the anti-hCMV standard of care (SOC) and/or with the hCMV-targeted therapies currently in development. Also, the antibodies of the present invention may be used in combination with other therapeutics aimed at stimulating immune responses such as immune checkpoint inhibitors, as suggested for the use of anti-IL-10 antagonists for the prevention/treatment of chronic viral infections (Martinic, et al. (2008) Trends Immunol. 29(3): 116-24; Filippi, et al. (2008) J. Pathol. 214:224-230).
  • the antibodies are specific to the viral hcmvIL-10 and do not recognize the endogenous cellular human IL-10. Therefore, the treatment with anti-hcmvIL-10 antibodies will not interfere with the physiological and pleiotropic activities mediated by human cellular IL-10 (Moore, et al. (2001), supra).
  • a vaccine with a modified hcmvIL-10 may induce antibodies cross-reacting with human IL-10, even if there is only 27% amino acid identity between the two proteins, and may thus induce persistent side effects in the vaccinated individuals due to interference with human cellular IL-10 activities.
  • an hcmvIL-10 vaccine may induce non-neutralizing antibodies that may interfere with the binding of neutralizing antibodies to hcmvIL-10 and may thus decrease their efficacy.
  • the current disclosure provides an antibody which binds to hcmvIL-10, which does not bind to human cellular IL-10 (hIL-10).
  • the antibody of the current disclosure inhibits or neutralizes one or more biological activities of hcmvIL-10.
  • the inhibition or neutralization of hcmvIL-10 by the antibody of the disclosure results in reduction of the activity of hcmvIL-10, with concomitantly little or no reduction of the activity of hIL-10.
  • the specific reduction of the activity of hcmvIL-10 increases overall anti-CMV immune responses.
  • the specific reduction of the immunosuppressive activity of hcmvIL-10, but not hIL-10 results in a safer profde.
  • “neutralize” or“inhibit” means to reduce or to abolish, specifically with regard to the biological effects of hcmvIL-10.
  • the antibody of the current disclosure neutralizes or reduces the inhibitory effect of hcmvIL-10 on an immune cell.
  • the anti-hcmvIL-10 antibody of the invention shows various desirable characteristics.
  • the anti-hcmvIL-10 antibody of the invention reduces or neutralizes at least one or preferably several of the effects of hcmvIL-10 on immunoregulation and/or inflammation.
  • the biological activities of hcmvIL- 10 include, but are not limited to, inhibition of TNF-a, IFN-g and/or IL-Ib release from LPS- or poly (I:C)-stimulated cells such as peripheral blood mononuclear cells (PBMC).
  • the inhibitory capacity of the anti-hcmvIL-10 antibody is assessed by neutralization of hcmvIL-10- mediated inhibition of TNF-a, IL-Ib or IFN-g release by human PBMC stimulated with LPS or Poly(LC).
  • the inhibitory capacity (IC) is reported as the“IC50” (or alternatively, the “IC90”) value, which is reported as the concentration of antibody at which 50% (or 90%) of the biological activity being measured is inhibited.
  • the anti-hcmvIL-10 antibody of the current disclosure is highly specific to hcmvIL- 10 and, as such, does not bind or significantly neutralize the biological activity of the splice variant of hcmvIL-10 referred to as latency associated hcmvIL-10 (LAcmvIL-10).
  • the antibody of the current invention does not bind to or interfere with the biological activity of other forms of IL-10 not produced by hCMV, such as human cellular IL-10 (hIL-10) or the IL-10 homologue produced by Epstein-Barr Virus (ebvIL-10).
  • the anti-hcmvIL-10 antibody of the current disclosure also binds to LAcmvIL-10.
  • the antibody which also binds to LAcmvIL-10 reduces or inhibits one or more biological activity of LAcmvIL-10.
  • the antibody which also binds to LAcmvIL-10 has a broader neutralization activity of hCMV-encoded IL-10 proteins.
  • the antibody which also binds to LAcmvIL-10 is useful for the detection of both hcmvIL-10 and LAcmvIL-10 in a biological sample.
  • the anti-hcmvIL-10 antibody of the current disclosure has a high affinity for hcmvIL-10; that is, the anti-hcmvIL-10 antibody preferably has a K D value of less than 1 mM, preferably less than 500 nM, preferably less than 100 nM, more preferably less than about 10 nM, more preferably less than 2 nM, especially less than 1 nM, more preferably less than 600 pM, 500 pM, 400 pM, 300 pM, 200 pM or 100 pM. In a preferred embodiment, the anti-hcmvIL-10 antibody has a K D of about 300 pM or less.
  • the K D can be measured by any assay well-known in the art.
  • the K D of the anti-hcmvIL-10 is measured by surface plasmon resonance (SPR), for example by Biacore.
  • the antibody of the disclosure has a k off value of about 1 x 10 4 s 1 (1/s) or less as measured by surface plasmon resonance (SPR).
  • Preferred anti-hcmvIL-10 antibody candidates are antibodies 1-12, as set forth in Tables A1-A3.
  • the variable regions of the heavy and light chains (VH and VL, respectively) as well as the CDR sequences according to KABAT and IMGT schemes of exemplary anti-hcmvIL-10 heavy and light chain combinations are set forth in the sequence listing, including SEQ ID NOs: 1-168; also see Tables A-l, A- 2 and A-3.
  • Antibodies of interest include these provided combinations, as well as fusions of the variable regions to appropriate constant regions or fragments of constant regions, e.g. to generate F(ab)' antibodies.
  • Variable regions of interest include at least one CDR sequence of the provided anti-hcmvIL- 10 antibody, where a CDR may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids.
  • antibodies of interest include a variable region as set forth in the provided antibodies, or pairs of variable regions sequences as set forth herein.
  • Table A-l Selected candidate antibodies with SEQ ID NOs of the variable regions of the heavy chains (VH) and light chains (VL).
  • Table A-2 Selected candidate antibodies with SEQ ID NOs of the CDRs of heavy (VH) and light (VL) chains of the selected candidates according to KABAT annotation.
  • Table A-3 Selected candidate antibodies with SEQ ID NOs of the CDRs of heavy (VH) and light (VL) chains of the selected candidates according to IMGT annotation.
  • the anti-hcmvIL-10 antibody of the current disclosure is selected from the group of antibodies consisting of: a. an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 1 to 3 (Rabat annotation) or SEQ ID NOs: 4 to 6 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 7 to 9 (Rabat annotation) or SEQ ID NOs: 10 to 12 (IMGT annotation); or
  • an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 13 to 15 (Rabat annotation) or SEQ ID NOs: 16 to 18 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 19 to 21 (Rabat annotation) or SEQ ID NOs: 22 to 24 (IMGT annotation); or
  • an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 25 to 27 (Rabat annotation) or SEQ ID NOs: 28 to 30 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 31 to 33 (Rabat annotation) or SEQ ID NOs: 34 to 36 (IMGT annotation); or
  • an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 37 to 39 (Rabat annotation) or SEQ ID NOs: 40 to 42 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 43 to 45 (Rabat annotation) or SEQ ID NOs: 46 to 48 (IMGT annotation); or e.
  • an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 49 to 51 (Kabat annotation) or SEQ ID NOs: 52 to 54 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 55 to 57 (Kabat annotation) or SEQ ID NOs: 58 to 60 (IMGT annotation); or
  • an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 61 to 63 (Kabat annotation) or SEQ ID NOs: 64 to 66 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 67 to 69 (Kabat annotation) or SEQ ID NOs: 70 to 72 (IMGT annotation); or
  • an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 73 to 75 (Kabat annotation) or SEQ ID NOs: 76 to 78 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 79 to 81 (Kabat annotation) or SEQ ID NOs: 82 to 84 (IMGT annotation); or
  • an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 97 to 99 (Kabat annotation) or SEQ ID NOs: 100 to 102 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 103 to 105 (Kabat annotation) or SEQ ID NOs: 106 to 108 (IMGT annotation); or
  • an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 109 to 111 (Kabat annotation) or SEQ ID NOs: 112 to 114 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 115 to 117 (Kabat annotation) or SEQ ID NOs: 118 to 120 (IMGT annotation); or
  • an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 121 to 123 (Kabat annotation) or SEQ ID NOs: 124 to 126 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 127 to 129 (Kabat annotation) or SEQ ID NOs: 130 to 132 (IMGT annotation); or
  • an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 133 to 135 (Kabat annotation) or SEQ ID NOs: 136 to 138 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 139 to 141 (Kabat annotation) or SEQ ID NOs: 142 to 144 (IMGT annotation).
  • the anti-hcmvIL-10 antibody of the current disclosure comprises: a. a VH having an amino acid sequence set forth in SEQ ID NO: 145 and a VL having an amino acid sequence set forth in SEQ ID NO: 146; or
  • VH having an amino acid sequence set forth in SEQ ID NO: 147 and a VL having an amino acid sequence set forth in SEQ ID NO: 148; or
  • VH having an amino acid sequence set forth in SEQ ID NO: 149 and a VL having an amino acid sequence set forth in SEQ ID NO: 150;
  • VH having an amino acid sequence set forth in SEQ ID NO: 151 and a VL having an amino acid sequence set forth in SEQ ID NO: 152; or
  • VH having an amino acid sequence set forth in SEQ ID NO: 153 and a VL having an amino acid sequence set forth in SEQ ID NO: 154;
  • VH having an amino acid sequence set forth in SEQ ID NO: 155 and a VL having an amino acid sequence set forth in SEQ ID NO: 156; or
  • VH having an amino acid sequence set forth in SEQ ID NO: 159 and a VL having an amino acid sequence set forth in SEQ ID NO: 160; or
  • VH having an amino acid sequence set forth in SEQ ID NO: 161 and a VL having an amino acid sequence set forth in SEQ ID NO: 162;
  • VH having an amino acid sequence set forth in SEQ ID NO: 165 and a VL having an amino acid sequence set forth in SEQ ID NO: 166; or
  • VH having an amino acid sequence set forth in SEQ ID NO: 167 and a VL having an amino acid sequence set forth in SEQ ID NO: 168.
  • the anti-hcmvIL-10 antibody of the current disclosure is a human antibody.
  • the antibody is a monoclonal antibody.
  • the anti-hcmvIL-10 antibody of the current disclosure is a folly-human antibody, i.e., VH and VL amino acid sequences are folly human.
  • Advantages to folly-human antibodies are well-known in the art and include weak immunogenicity of said antibodies when administered to a human subject. Additionally, folly-human antibodies have undergone affinity maturation by the antigen in the human subject (donor); therefore, selected antibodies can have very high antigen specificity.
  • the antibody or a cell producing the antibody is collected from a human blood donor.
  • the human blood donor is an hCMV- positive donor.
  • the C-terminal lysine residue of the monoclonal antibody is removed to avoid heterogeneity during biopharmaceutical production process(es) or in pharmaceutical preparation(s).
  • a potential glycosyation site present in the VH/VL region of some antibodies is removed.
  • the anti-hcmvIL-10 antibody of the current disclosure has a human IgGl, IgG2, IgG3, IgG4 or IgA constant region.
  • the variable regions of the antibody are combined with an artificial and/or recombinant Fc region of a particular allotype.
  • the anti-hcmvIL-10 antibody is produced recombinantly; i.e., as a fully-human recombinant antibody.
  • the anti-hcmvIL-10 antibody of the current disclosure is an antibody fragment, such as, for example, an F(ab’) 2 or an Fab fragment.
  • an antibody fragment such as, for example, an F(ab’) 2 or an Fab fragment.
  • Fabs fragments and epitope-binding peptides having binding specificity for at least one epitope of hcmvIL-10 are also contemplated by the present invention and can also be used in the methods of the invention.
  • single chain antibodies can be constructed according to the method of U.S. Pat. No. 4,946,778, which is incorporated herein by reference in its entirety. Single chain antibodies comprise the variable regions of the light and heavy chains joined by a flexible linker moiety. Yet smaller is the antibody fragment known as the single domain antibody, which comprises an isolated VH single domain.
  • the anti-hcmvIL-10 antibody of the current disclosure is a bispecific antibody.
  • “bispecific” antibodies are defined as antibodies which bind specifically to two distinct targets (epitopes).
  • the bispecific antibodies bind specifically to both hcmvIL- 10 and another distinct target.
  • the other distinct target is an immune modulator, such as a cytokine or chemokine.
  • the other distinct target of the bispecific antibody is another hCMV protein such as an envelope glycoprotein or another immunomodulatory produced by hCMV.
  • the other distinct target of the bispecific antibody is a cell-surface marker, such as a receptor or tumor-associated antigen.
  • the cell-surface marker is a receptor expressed or over-expressed by a cancer or immune cell.
  • the receptor is CD47, CD20, PD-L1 or PD-1, phospatidylserine (PS), EGFR (HER), HER2, MUC1, MUC5AC or SIRP- a.
  • the anti-hcmvIL-10 antibody of the disclosure is provided as a nucleic acid encoding an anti-hcmvIL-10 antibody of any of the aspects of the disclosure.
  • a nucleic acid encoding it is inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • the anti-hcmvlL-10 antibody of this invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous or homologous polypeptide, which includes a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide, an immunoglobulin constant region sequence, and the like.
  • a heterologous signal sequence selected preferably may be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected.
  • an "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid.
  • An isolated nucleic acid molecule is different than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • the nucleic acid encoding the anti-hcmvlL-10 antibody of the disclosure is comprised in a host cell.
  • the host cell produces the anti-hcmvlL-10 antibody of the disclosure.
  • Suitable host cells for cloning or expressing the DNA are the prokaryote, yeast, or higher eukaryote cells. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cells (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J. Gen Virol.
  • monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TR1 cells (Mather et al, Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; EB66 cells (see e.g.
  • Host cells are transformed with the above-described expression or cloning vectors for anti-hcmvIL-10 antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the anti-hcmvlL-10 antibodies may also be produced from B cells or immortalized B cells, such as a hybridoma cell.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody as provided.
  • the pharmaceutical composition optionally further comprises a pharmaceutically acceptable excipient.
  • Acceptable carriers, excipients, or stabilizers are non-toxic 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 octadecyidimethylbenzyl 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 immunoglobulin
  • Therapeutic formulations comprising one or more antibodies of the invention are prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • the antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the "therapeutically effective amount" of the antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent the CMV-associated disease.
  • the therapeutic dose may be at least about 0.01 mg per kg body weight, at least about 0.05 mg per kg body weight; at least about 0.1 mg per kg body weight, at least about 0.5 mg per kg body weight, at least about 1 mg per kg body weight, at least about 2.5 mg per kg body weight, at least about 5 mg per kg body weight, at least about 10 mg per kg body weight, and not more than about 100 mg per kg body weight with a preference of 0.1 to 10 mg per kg body weight. It will be understood by one of skill in the art that such guidelines will be adjusted for the molecular weight of the active agent, e.g. in the use of antibody fragments, or in the use of antibody conjugates.
  • the dosage may also be varied for localized administration, e.g. intranasal, inhalation, etc., or for systemic administration, e.g., i.m., i.p., i.v., and the like.
  • the antibody need not be, but is optionally formulated with one or more agents that potentiate activity, or that otherwise increase the therapeutic effect. These are generally used in the same dosages and with administration routes as used hereinbefore or about from 1 to 99% of the heretofore employed dosages.
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the anti-hcmvIL-10 antibody or pharmaceutical composition of the disclosure is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the anti-hcmvIL-10 antibody or pharmaceutical composition of the disclosure is suitably administered by pulse infusion, particularly with declining doses of the antibody.
  • the appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody or pharmaceutical composition is administered for preventive purposes, previous therapy, clinical history of the patient and response to the antibody or pharmaceutical composition of the disclosure, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • an article of manufacture containing materials useful for the treatment of the disorders described above comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the active agent in the composition is the anti-hcmvIL-10 antibody.
  • the label on, or associated with, the container indicates that the composition is used for treating the condition of choice.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate -buffered saline, Ringer's solution and/or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a pharmaceutically- acceptable buffer such as phosphate -buffered saline, Ringer's solution and/or dextrose solution.
  • It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the antibody as provided or the pharmaceutical composition as provided is used in the treatment or prevention of a disease in a subject.
  • the disease to be treated is hepatitis, esophagitis, colitis, pneumonia, retinitis, myocarditis or ependymitis.
  • the disease to be treated is characterized by hepatosplenomegaly, petechiae, microcephaly, hypotomia, seizures, and/or lethargy.
  • the disease to be treated is CMV infection.
  • the CMV infection affects the lung, liver, spleen, gastrointestinal tract, Central Nervous System (CNS), hematologic system, kidney, adrenal, salivary gland, pancreas, and/or esophagus.
  • the subject has an acute hCMV infection, a latent hCMV infection or an acute CMV reinfection or reactivation.
  • the disease to be treated is a disease associated with CMV infection such as cancer or atherosclerosis.
  • the disease to be treated or prevented is congenital hCMV infection.
  • the disease to be treated arises from hCMV infection of the transplanted organ or tissue.
  • the disease to be treated is another infection or infections co-present with hCMV infection.
  • the other infection(s) compromises the immune function of the subject.
  • the other infection is Human Immunodeficiency Virus (HIV).
  • the disease to be treated by the antibody or the pharmaceutical composition provided is cancer.
  • the subject to be treated has an hCMV infection.
  • the subject (cancer patient) to be treated suffers from an hCMV-positive cancer.
  • the subject (cancer patient) has a latent hCMV infection.
  • the cancer patient has circulating hcmvIL-10- producing cells, e.g., cancer cells.
  • the subject (cancer patient) has a lytic hCMV infection.
  • the antibody or the pharmaceutical composition provided is used as a monotherapy. In one aspect, the antibody or the pharmaceutical composition provided is used in combination therapy. In a preferred embodiment, the antibodies of the invention can be used against hematological and solid tumors, as a monotherapy, or in combinations with other anti-cancer agent(s). Preferred combinations are combinations of a hcmvIL-10 antibody of the invention and i) an immune check-point inhibitor or ii) an antibody against a tumor associated antigen. In one aspect, the co-administered antibody is Herceptin®, Erbitux® anti-CTLA-4, anti-PD-1 and/or anti-PD-Ll. In one aspect, the co-administered antibody is specific to another tumor target.
  • the combination therapy includes other preparations for treatment of hCMV, such as the antiviral drugs which are the standard of care.
  • the combination therapy includes a human hCMV -hyperimmune serum preparation; i.e., Cytotect CP.
  • the antiviral drugs are ganciclovir (cytovene), valganciclovir (valcyte), foscamet or cidofovir.
  • the combination therapy includes drugs targeting another disease or condition which co-exists with hCMV in the subject.
  • the antibodies or pharmaceutical compositions provided are used to treat a human subject.
  • the subject to be treated is an adult or elderly adult.
  • the subject to be treated is a child or youth. In one aspect, the subject to be treated is a transplant patient. In one aspect, the subject to be treated is a pregnant woman. In one aspect, the subject to be treated is a fetus or newborn.
  • the immunocompromised subject has an immature immune system.
  • the subject with an immature immune system is an infant, toddler or small child.
  • the immunocompromised subject is elderly.
  • the immunocompromised subject has poor nutritional status; i.e., malnutrition.
  • the immunocompromised subject has a primary immunodeficiency, e.g., an X-linked or autosomal recessive immunodeficiency disorder.
  • the immunocompromised patient has a secondary (acquired) immunodeficiency; e.g., from use of chemotherapy, immunosuppressive drugs, e.g., glucocorticoids or from diseases such as cancer or chronic infections.
  • the immunocompromised subject is a transplant patient.
  • the transplant patient is a solid organ transplant patient, e.g., a kidney, lung, liver, pancreas or heart recipient.
  • the transplant patient is a hematopoietic stem cell transplant patient.
  • the transplant patient is a blood transfusion recipient.
  • the transplant patient is on short-term or chronic immunosuppressive therapy.
  • the immunocompromised subject has an infection which reduces or ablates immune function, e.g., Human Immunodeficiency Virus (HIV).
  • HIV Human Immunodeficiency Virus
  • the subject is an HIV-positive patient or an AIDS patient.
  • the subject has an autoimmune disorder.
  • the disclosure provides a method of detecting the presence of hcmvIL-10 in a biological sample or tissue, the method comprising (i) contacting said sample or tissue with the hcmvIL- 10 antibody of the disclosure, and (ii) determining the presence of antibody bound to said tissue or sample.
  • the disclosure provides a method of treating, preventing or protecting a subject from hCMV infection or complications of an hCMV infection.
  • the method provided comprises administering to said subject having or at risk of contracting an hCMV infection an effective amount of the antibody or the pharmaceutical composition provided herein.
  • a method of treating or preventing cancer in a subject comprises administering to a subject having or at risk of developing cancer an effective amount of the antibody or the pharmaceutical composition provided herein.
  • the subject suffers from a CMV-positive cancer.
  • the subject is infected with hCMV.
  • the method of treating or preventing cancer in a subject comprises administering the antibody or the pharmaceutical composition as a monotherapy.
  • the cancer to be treated or prevented is a cancer of the breast, prostate, colon, lung or brain, e.g., glioma.
  • the method of treating or preventing cancer in a subject comprises administering the antibody or the pharmaceutical composition as a combination therapy.
  • the cancer to be treated is hCMV positive and/or hcmvIL-10 positive.
  • the combination therapy further comprises an anti-viral or anti-cancer drug.
  • the combination therapy further comprises another therapeutic antibody.
  • the subject to be treated is human.
  • the subject to be treated is immunocompromised as defined above.
  • a process for producing the antibody of the disclosure comprising culturing the aforementioned host cell so that the nucleic acid encoding the antibody of the disclosure is expressed.
  • the process optionally includes the recovery of the antibody from the cell culture.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique.
  • affinity chromatography being the preferred purification technique.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • Protein A can be used to purify antibodies that are based on human g ⁇ , g2, or g4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for human g3 (Guss el al., EMBO J. 5:1567-1575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the antibody comprises a CH3 domain
  • the Bakerbond ABXTM resin J.T.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0- 0.25M salt).
  • the current disclosure provides a method of monitoring progression of disease, wherein progression is assessed by detecting levels of hcmvIL-10 or, alternatively, hcmvIL-10 and LAcmvIL-10, in a patient biological sample or tissue by use of the antibody according to previous aspects of the current disclosure.
  • the monoclonal antibodies of the invention may be used in vitro in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • the monoclonal antibodies in these immunoassays can be detectably labeled in various ways.
  • types of immunoassays which can utilize monoclonal antibodies of the invention are flow cytometry, e.g. FACS, MACS, immunohistochemistry, competitive and non-competitive immunoassays in either direct or indirect formats; and the like.
  • Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples.
  • Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.
  • the monoclonal antibodies of the invention can be bound to many different carriers and used to detect the presence of hcmvIL- 10-expressing cells.
  • carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation.
  • a label may be covalently or non-covalently attached to an antibody of the invention or a fragment thereof, including fragments consisting or comprising of CDR sequences.
  • Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bio-luminescent compounds.
  • Those of ordinary skill in the art will know of other suitable labels for binding to the monoclonal antibodies of the invention, or will be able to ascertain such, using routine experimentation.
  • the binding of these labels to the monoclonal antibodies of the invention can be done using standard techniques common to those of ordinary skill in the art.
  • the antibody or a fragment thereof is attached to a nanoparticle, e.g. for use in imaging.
  • Useful nanoparticles are those known in the art, for example including without limitation, Raman-silica-gold-nanoparticles (R-Si-Au-NP).
  • the R- Si-Au-NPs consist of a Raman organic molecule, with a narrow-band spectral signature, adsorbed onto a gold core. Because the Raman organic molecule can be changed, each nanoparticle can carry its own signature, thereby allowing multiple nanoparticles to be independently detected simultaneously by multiplexing.
  • the entire nanoparticle is encapsulated in a silica shell to hold the Raman organic molecule on the gold nanocore.
  • Optional polyethylene glycol (PEG)-ylation of R-Si-Au-NPs increases their bioavailability and provides functional "handles" for attaching targeting moieties (see Thakor et al. (2011) Sci. Transl. Med. 3(79):79ra33; Jokerst et al. (2011) Small. 7(5):625-33; Gao et al. (2011) Biomaterials 32(8):2141-8; each herein specifically incorporated by reference).
  • hcmvIL-10 may be detected by the monoclonal antibodies of the invention when present in biological fluids and on tissues, in vivo or in vitro.
  • Any sample containing a detectable amount of hcmvIL-10 can be used.
  • a sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, semen, amniotic fluid, serum and the like, or a solid or semi-solid such as tissues, feces, and the like, or, alternatively, a solid tissue such as those commonly used in histological diagnosis.
  • Another labeling technique which may result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies.
  • the antibody of the present invention can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay.
  • the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore).
  • substrates and cofactors required by the enzyme e.g., a substrate precursor which provides the detectable chromophore or fluorophore
  • other additives may be included such as stabilizers, buffers (e.g., a blocking buffer or lysis buffer) and the like.
  • the relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay.
  • the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.
  • Example 1 Identification and isolation of human B cells producing antibodies specific for hcmvIL-10
  • hcmvIL-10 displayed biological activities similar than human cellular IL-10 (hIL-10), one of the IL-10-mediated activities being the inhibition of inflammatory cytokine production by activated peripheral blood mononuclear cells (Spencer, et al. (2002) J. Virol. 76(3): 1285-1292; Chang, et al. (2004) J. Virol. 78(16):8720-8731 ; demowey, et al.
  • hcmvIL-10 In order to standardize the functional assays, the biological activity of purified hcmvIL-10 (R&D Systems) was compared to that of purified hIL-10 (R&D Systems).
  • Various dilutions of recombinant hlL- 10 or hcmvIL-10 (in 10% FBS supplemented RPMI-1640 culture medium) were prepared in 96-well microtiter plates (50 pL/well), and 50 pL of LPS (from E. coli 0111 :B4; Invivogen) at 20 pg/mL or 50 pL of Poly(I:C) (Invivogen) at 80 pg/mL was added to each well except in non-stimulated control wells.
  • PBMC Purified human PBMC were then added to each well at 2 x 10 5 cells/well in 100 pL culture medium and the 96-well plates were incubated at 37°C, 5% CO2 overnight. Cell-free supernatants were then collected from each well and concentrations of TNF-a, IL-I b or IFN-g were determined by using an ELISA kit (BD-Biosciences). Concentrations of hcmvIL-10 and hIL-10 inhibiting more than 90% (NT90) of TNF-a and IL-Ib secretion by LPS-activated PBMC and of IFN-g secretion by Poly(I:C)-activated PBMC were determined and selected for further experiments.
  • hcmvIL-10 and hIL-10 were 1.5 ng/mL and 3 ng/mL, respectively.
  • Examples of hcmvIL-10 and hIL-10 titration are shown in Figure 1 for PBMCs isolated from 2 different donors.
  • hcmvIL-10 displayed strong functional activity similar to the activity of cellular hIL-10 in inhibiting inflammatory cytokine secretion by human PBMC.
  • Serum was collected from 519 healthy adult donors and was first tested for the presence of IgG antibodies against hCMV by using the PLATELIATM CMV IgG kit (Bio-Rad).
  • hCMV IgG positivity was observed in 277 donors (53.4 % prevalence) with titer ranging from to 0.5 to 65.8 AU/mL (Table 1).
  • Equivocal positivity was observed in 6 serum samples while the remaining 236 samples were negative (hCMV titer ⁇ 0.25 AU/mL).
  • a competition ELISA was further used to characterize all hcmvIL-10 positive serum samples.
  • this competition ELISA the 1 :20 and 1 :40 serum dilutions of sera from donors showing specific binding to hcmvIL-10-coated plates but not to uncoated plates, were first pre-incubated with or without 1 pg/mL soluble (uncoated) hcmvIL-10 before incubation on hcmvIL-10-coated plates, as previously described.
  • the 18 sera positive in the hcmvIL-10 competition ELISA were further tested for their capacity to inhibit the biological activity of hcmvIL-10, as measured by the hcmvIL-10-induced inhibition of cytokine production by LPS- or Poly(I:C)-activated human PBMC. Briefly, 25 pL of 1 :5 to 1 :10 dilution (1 :20 to 1 :40 final dilution) of heat-inactivated serum samples were pre-incubated for 30 minutes with 25 pL of 6 ng/mL hcvmIL-10, and the mixture was then added to PBMC (2 x 10 5 cells/well) in a 25 pi volume.
  • Human anti-hcmvIL-10 monoclonal antibodies were obtained by implementing the VIVA
  • ELISA -positive supernatants were then tested in a functional assay in order to select hits capable of neutralizing the inhibitory activity of hcmvIL-10 on cytokine secretion by activated human PBMC. Briefly, 30 pL of activated B cell pool supernatants were incubated with 30 pL of 6 ng/mL hcmvIL-10 for 30 minutes at 37°C, and 50 pL of the mixture was then added to PBMC (2 x 10 5 cells/well) previously distributed in a 25 pi volume in 96-half size well plates.
  • Table 2 Summary of VIVAScreen campaigns for human IgG anti-hcmvIL-10.
  • Activated B cell pools corresponding to the neutralizing hits were collected from 96-well culture plates, deposited on microarray chips previously coated with 10 pg/mL of hcmvIL-10, and single B cells secreting IgG against hcmvIL-10 were identified and isolated by the ImmunoSpot Array Assay on a Chip (ISAAC) method essentially as described in Jin et al. (Jin, et al. (2009) Nature Medicine 15(9): 1088- 1092) and in WO2013/000982. Activated B cell pools from a selection of non-neutralizing hits were also processed in parallel by ISAAC.
  • ISAAC ImmunoSpot Array Assay on a Chip
  • the sequences of the VH and VL regions of the selected monoclonal antibodies were amplified by single cell reverse transcriptase polymerase chain reaction (RT-PCR) starting from the isolated single B cells retrieved from ISAAC microarrays.
  • RT-PCR single cell reverse transcriptase polymerase chain reaction
  • the resulting cDNAs were subsequently cloned into expression vectors containing the constant domains of the heavy and light chains of a human IgGl (human heavy chain: SEQ ID NO: 173; human kappa: SEQ ID NO: 174 or human lambda: SEQ ID NOs: 175 or 176).
  • the vectors thus obtained for heavy and light chains were co-transfected into CHO cells, and the CHO cell supernatants were tested for the presence of human IgG binding to hcmvlL-10 by ELISA.
  • Table 3 Names and clone sequence families of the twelve selected candidates for which the sequences are disclosed.
  • Table 4 Equilibrium binding constants of selected anti-hcmvIL-10 antibodies as determined by ELISA.
  • the on rate (Kon) and off rate (Koff) kinetic constants were measured for the 12 selected antibodies by SPR on a Biacore T200 (GE Healthcare), and the equilibrium dissociation constant (K D ) was calculated. Briefly, the binding affinity of the antibodies to hcmvIL-10 was measured using the single-cycle kinetics protocol of the Biacore T200 instrument at +20°C. An anti-human IgG (Fc) was first immobilized on the surface of Serie S Sensor Chips CM5 using the human antibody capture and amine coupling kits, following the manufacturer’s instructions (GE Healthcare). This resulted in approximately 10,000 response units (RU) immobilized on the surface.
  • RU response units
  • the antibodies to be tested were then captured onto anti human IgG surface in all flow cells except flow cell 1 (which was used as control), at a concentration and contact time optimized so that—400 RU were captured on the surface.
  • Binding kinetics were studied by passing increasing concentrations of hcmvIL-10 in a series of 5 minute injections through all flow cells at a rate of 30 pL/min. Three-fold dilutions were used up to a maximum concentration of 270 nM. Following the final injection, buffer was passed across each flow cell for 30 minutes to monitor the dissociation of bound antigen.
  • Regeneration of the binding surface was carried out at the end of the cycle by flowing 3 M MgCE for 60 s at 20 pL/min followed by 10 mM glycine-HCl, pH 1.7 for 180 s at 10 pl/min.
  • a second cycle with the same antibodies but no antigen was run as a control.
  • Kinetic binding constants were estimated by non-linear fitting of the sensogram data to the 1 : 1 binding model provided by the Biacore T200 evaluation software.
  • the thus determined Kon, Koff and K D values of the 12 selected anti-hcmvIL-10 antibodies are provided in Table 5.
  • the K D of the 12 selected anti-hcmvIL-10 antibodies was found in the range of 35 to 270 pM, indicating that the 12 antibodies were of high affinity for hcmvIL- 10.
  • Serial dilutions of the anti-hcmvIL-10 antibodies were prepared in 10% FBS supplemented RPMI-1640 culture medium (PBMC medium) and deposited in microtiter culture plates (50 pL/well). Then 50pL/well of hcmvIL- 10 at 6 ng/mL (4x NT90) in PBMC medium were added and the plates were incubated for 15 minutes at 37°C. Purified PBMC (2 x 10 5 cells/well, 50 pL/well) were then added to the wells, followed by addition of 50 pL/well of LPS at 20 pg/mL. Microtiter plates were incubated overnight at 37°C, 5% CO2.
  • % inhibition (1- (([TNFLPS] - [TNFLPs+iL-10+Ab]) / ([TNFLPS] - ([TNFLPS+IL-IO]))) X 100, where [TNFLPS] is the concentration of TNF-a measured in the supernatant of PBMC activated with LPS only, [TNFLPS+IL-IO] is the concentration of TNF-a measured in the supernatant of PBMC activated with LPS in the presence of hcmvIL- 10, and [TNF L ps+iL-io+Ab] is the concentration of TNF-a measured in the supernatant of PBMC activated with LPS in the presence of hcmvIL- 10 and the antibody to be
  • Table 6 IC50 and IC90 values of anti-hcmvIL-10 antibodies in the PBMC/LPS/TNF-a assay.
  • the 12 anti-hcmvIL-10 antibodies were further tested for their capacity to neutralize the hcmvIL- 10 inhibitory activity on IFN-g secretion by Poly(I:C)-activated PBMC (PBMC/Poly(I:C)/IFN-y assay).
  • Serial dilutions of the anti-hcmvIL-10 antibodies were prepared in 10% FBS supplemented RPMI-1640 culture medium (PBMC medium) and deposited in culture microtiter plates (50 pL/wcll). Then 50 pL/wcll of hcmvIL- 10 at 6 ng/mL (4x NT90) in PBMC medium were added and the plates were incubated for 15 minutes at 37°C.
  • PBMC Purified PBMC (2 x 10 5 cells/well, 50 pL/wcll) were then added to the wells, followed by addition of 50 pL/wcll of Poly(LC) at 80 pg/mL. Microtiter plates were incubated overnight at 37°C, 5% CO2. Cell-free supernatants were then collected from each well and the concentration of IFN-g was determined by using an ELISA kit (BD-Biosciences).
  • % inhibition (1- (([IFNpic] - [IFNpic+i L -io+ Ab ]) / ([IFNpic] - ([IFN P I C +IL-IO]))) X 100, where [IFNpic] is the concentration of IFN-g measured in the supernatant of PBMC activated with Poly(I:C) only, [IFNHC+IL IO] is the concentration of IFN-g measured in the supernatant of PBMC activated with Poly(I:C) in the presence of hcmvIL-10, and [IFNpic +iL -io +Ab ] is the concentration of IFN-g measured in the supernatant of PBMC activated with Poly(I:C) in the presence of hcmvIL-10 and the antibody to be tested. The percent inhibition was plotted against antibody concentration and the IC50 and IC90 values were calculated by using a nonline
  • Table 7 IC50 and IC90 values of anti-hcmvIL-10 mAbs in the PBMC/ Poly-IC/IFN-g assay.
  • LAcmvIL-10 is a truncated isoform of hcmvIL-10 which, like full-length hcmvIL-10, is encoded by the UL111A gene, but retains only the first two introns as a result of alternative splicing.
  • LAcmvIL-10 shares the first 127 amino acids with hcmvIL-10, but diverges for its final 12 amino acids at the C-terminus, thus resulting in a truncated protein 139 amino acids in length instead of 175 amino acids for hcmvIL-10.
  • mammalian CHO cells were transiently transfected with an expression plasmid containing the sequence of LAcmvIL-10 (GenBank ® Database Accession No. ACR49217; SEQ ID NO: 170) and their supernatants were collected after 7 days of culture.
  • CHO cells were also transfected with a plasmid vector encoding hcmvIL-10 (GenBank ® Database Accession No. AAF63437; SEQ ID NO: 169) or with an empty vector, and their culture supernatants were collected as for LAcmvIL-10-transfected cells.
  • serial dilutions of the CHO-cell supernatants were first coated on ELISA plates and the reactivity of a goat polyclonal antibody against hcmvIL-10 (R&D Systems) was analyzed.
  • LAcmvIL-10 is less potent than hcmvIL-10 for inhibiting the secretion of inflammatory cytokines by human mononuclear cells and that LAcmvIL-10 possesses only a small subset of the biological activities of hcmvIL-10 and hIL-10 (Spencer, et al. (2008) Virology 374:164-169; Jenkins, et al. (2008) J. Virol. 82(7):3736-3750; Slobedman, et al. (2009), supra, Christiaansen, et al. 2015, supra).
  • Anti-hcvmIL-10 monoclonal antibodies were additionally tested for their capacity to recognize LAcmvIL-10 by ELISA.
  • a supernatant of LAcmvIL-10-transfected CHO cells was coated onto wells of ELISA plates (1 :20 dilution in carbonate buffer).
  • Supernatants of CHO cells transfected with an hcmvIL-10 expression vector or with an empty vector were coated at same dilutions on same ELISA plates and were used as positive control and for background measurement, respectively.
  • VL016-16-4A2 VL016-16-26E8 and VL016-16- 31C9 to LAcmvIL-10 in Fig. 8
  • the antibodies were tested at 2 pg/mL in an ELISA in which serial dilutions (1/10 to 1/lxlO 8 ) of CHO cell supernatant containing LAcmvIL-10, hcmvIL-10 or from not- transfected CHO cells were used.
  • antibodies VL016-16-4A2 and VL016-16-26E8 showed weak but specific binding to LAcmvIL-10-containing supernatants from 1/10 to 1/1,000 dilution, similar to what was observed with the goat anti-hcmvIL-10 antibody.
  • the antibody VL016-16-31C9 bound to LAcmvIL-10 also at the 1/10 to 1/1,000 supernatant dilutions, although non-specific binding to the supernatant of non-transfected cells was also observed at high supernatant concentrations.
  • Example 4 Assessing the cross reactivity of candidate antibodies
  • hcmvIL-10 specific recognition of hcmvIL-10, but not ofhIL-lO and ebvIL-10, by anti-hcmvIL-10 antibodies hcmvIL-10 shares only 27% amino-acid sequence identity with human cellular IL-10 (hIL-10), but binds with even higher affinity to human IL-10 receptors than hIL-10 and possesses almost identical biological activities as hIL-10 (Kotenko, et al. (2000); Lockridge, et al. (2000); Ouyang, et al. (2014), supra).
  • EBV-encoded IL-10 homolog (ebvIL-10) produced by EBV-infected human cells, shares 83% amino-acid sequence identity with hIL-10, but binds with a lower affinity to the hIL-10 receptors as hIL-10 and possesses only part of the hIL-10 biological activities (Yoon, et al. (2012) J. Biol. Chem. 287(32):26586-26595).
  • hcmvIL-10 was much more potent than ebvIL-10 (purified protein, R&D systems) for inhibiting cytokine secretion by human PBMC, as measured in the PBMC/LPS/TNF-a assay ( Figure 10).
  • the purified mAbs were tested for their capacity to bind to and neutralize the biological activity of purified recombinant hlL-10 or ebvlL-10 (both from R&D Systems).
  • ft is notable that Cytotect CP (Biotest), a pharmaceutical preparation of human IgG purified from pools of plasma from hCMV hyper-immune donors, displayed weak (but specific) binding to hcmvlL-10 when tested at 100 pg/mL.
  • mouse antibodies against hlL-10 or ebvlL-10 inhibited both hlL-10 and ebvlL-10, but not hcmvlL-10.
  • RhcmvlL- 10 the Rhesus macaque CMV genome also encodes an lL-10 homolog designated as RhcmvlL- 10 (Lockridge, et al. (2000), supra).
  • RhcmvlL-10 sequence shares only 30% amino-acid identity with hcmvIL-10.
  • CHO cells were transiently transfected with an expression plasmid expressing the sequence of RhcmvIL-10 (GenBank ® Database Accession No. AAF59907; SEQ ID NO: 171) and their supernatants were collected after 7 days of culture.
  • CHO cells were also transfected with a plasmid vector encoding hcmvIL-10 (GenBank ® Database Accession No. AAF63437; SEQ ID NO: 169) or with an empty vector, and their culture supernatants were collected as for RhcmvIL-10-transfected cells. The supernatants were further tested for their capacity to inhibit the production of cytokines by human activated PBMC. As shown in Figure 13, serial dilutions of the supernatant of CHO cells transfected with RhcmvIL-10 strongly inhibited the production of TNF-a by LPS-activated human PBMC as did the supernatant of CHO cells transfected with hcmvIL-10.
  • the anti-hcmvIL-10 antibodies were tested for their capacity to recognize RhcmvIL-10 by ELISA. Plates were coated with 1 : 100 dilution of the supernatant of CHO cells transfected with RhcmvIL-10, hcmvIL-10 or empty vector (Control Medium), and incubated with the anti-hcmvIL-10 antibodies or a negative control hlgGl antibody at 3 pg/mL. After washings, the binding of the tested antibodies was revealed by the use of an HRP-conjugated anti-human IgG antibody, followed by the addition of TMB, blocking with orthophosphoric acid and OD reading at 450 nm.
  • a competitive sandwich ELISA was further used to address whether the anti-hcmvlL-10 antibodies recognized the same or close epitopes / domains on hcmvlL-10.
  • Purified anti-hcmvlL-10 antibodies were coated at 1 pg/mL on 96 half-well ELISA plates overnight at +4°C. After 3 washings with PBS, 0.05% Tween-20 and saturation with 150 pL/wcll PBS, 0.05% Tween-20, 1% BSA, 50 pL/wcll of hcmvlL-10 at 3 ng/mL was added and incubated for 1 hour at room temperature.
  • Family- 1 contains 3 antibodies (6- 11 C 11 , 4- 11 D 11 , and 9-6E5) that strongly competed with mAb 6-28A2 of family-2 and mAb 8-9F5 of family-5, but not with mAb 16-4A2 of family -3, mAb 9-7F10 of family-4, and with mAb 4-6D2 of family-6.
  • Family-2 contains 2 antibodies (6-28A2 and 9-32E3) that strongly competed with mAb 6-11 Cl 1 of family-1, mAb 16-4A2 of family-3 and mAb 8-9F5 of family-5, but not with mAb 9-7F10 of family-4, and with mAb 4-6D2 of family-6.
  • Family-3 contains 3 antibodies (16-4A2, 16-26E8 and 16-31C9) that strongly competed with mAb 6-28A2 from family-2, mAb 9-7F10 from family-4 and mAb 8-9F5 of family-5, but not with mAb 11C11 of family-1, and with mAb 4-6D2 of family-6.
  • Family-4 contains 2 mAbs (4-20A9 and 9-7F10) that strongly competed with mAb 16-4A2 of family-3 and mAb 8-9F5 of family-5, but not with mAb 11C11 of family-1, mAb 6-28A2 of family-2 and with mAb 4-6D2 of family-6.
  • Family-5 contains only 1 antibody (8-9F5) that competed with mAbs from families- 1, -2, -3 and - 4, but not with mAb 4-6D2 of family-6.
  • family-6 contains 1 antibody (4-6D2) that did not compete with any mAb of families- 1, - 2, -3, -4 or -5.
  • Table 8 Antibody binding competition to hcmvIL-10 as determined by ELISA. Crossed cases denote
  • the anti-hcmvIL-10 antibodies were tested for their capacity to recognize hcmvIL-10 naturally produced by cells infected with hCMV.
  • ARPE-19 epithelial cells (ATCC, CRL-2302) and MRC-5 fibroblastic cells (ATCC, CCL-171) were infected in 96-well culture plates with the hCMV strains AD 169 or VR1814 that display fibroblast-restricted or broad tropism, respectively. After 2 days of incubation at +37°C, 5% CO2, virus supernatants were removed, and the cells were washed with cold PBS and fixed/permeabilized with 100% cold ethanol.
  • IE antigens CMV immediate early non-structural antigens
  • MAB810X Alexa-Fluor 488-coupled mouse anti-CMV IE antigen antibody
  • IE antigens were detected in the nuclei of MRC-5 cells infected with both AD 169 and VR1814 strains, whereas IE antigens were detected only in VR1814-infected ARPE-19 cells (staining indicated by black and white stars in Figure 16).
  • the cells were stained in parallel with human anti-hcmvIL-10 antibodies that were further detected using a goat anti-human IgG antibody coupled to phycoerythrin (PE).
  • PE phycoerythrin
  • VL016-4-20A9-1G1 heavy chain; CDRl -Kabat
  • VL016-4-20A9-1G1 heavy chain; CDR3-Kabat GIGLNWNYVGS
  • VL016-4-20A9-1G1 heavy chain; CDR1 -IMGT GFMFDSHA
  • VL016-4-20A9-1G1 heavy chain; CDR2-IMGT ISGGGVST
  • VL016-4-20A9-1G1 heavy chain; CDR3-IMGT SKGIGLNWNYVGS
  • VLO 16-4-20A9- 1 K3 light chain; CDRl-Kabat QASQDINKFVN
  • VLO 16-4-20A9- 1 K3 light chain; CDR2-Kabat DATNVET
  • VLO 16-4-20A9- 1 K3 light chain; CDR1-IMGT QDINKF
  • VLO 16-4-20A9- 1 K3 light chain; CDR2-IMGT DAT
  • VLO 16-4-20A9- 1 K3 light chain; CDR3-IMGT QQFDDLPVT
  • VLO 16-9-7F 10- 1 G 1 ; heavy chain; CDR2-Kabat SISGGGVSTYYADSVKG
  • VLO 16-9-7F 10- 1 G 1 ; heavy chain; CDR3-Kabat GIGLNWNYVGS
  • VLO 16-9-7F10-1G1 heavy chain; CDR2-IMGT ISGGGVST
  • VLO 16-9-7F10-1G1 heavy chain; CDR3-IMGT SRGIGLNWNYVGS
  • VLO 16-9-7F 10- 1 R1 ; light chain; CDRl -Rabat QASQDISKFLN
  • VLO 16-9-7F 10- 1 R1 ; light chain; CDR2 -Rabat DASNVET
  • VLO 16-9-7F 10- 1 R1 ; light chain; CDR1 -IMGT QDISRF
  • VLO 16-9-7F 10- 1 R1 ; light chain; CDR2-IMGT DAS
  • VL016-4-11D11-Gl heavy chain; CDR2-Kabat RIIPRLGITNYAQKF QG
  • VL016-4-11D11-Gl heavy chain; CDR3-Kabat HYDS SGYNMEGGRYWYFDL
  • VL016-9-6E5-L1 light chain; CDRl-Kabat SGSDSNIGNSHVS
  • VL016-9-6E5-L1 light chain
  • VL016-9-6E5-L1 light chain; CDR3-Kabat GT WDHTP SAW
  • VL016-9-6E5-L1 light chain; CDR3-IMGT GT WDHTP SAW
  • VL016-6-28A2-3G15 heavy chain
  • VL016-6-28A2-3G15 heavy chain
  • VL016-6-28A2-3G15 heavy chain
  • VL016-6-28A2-3G15 heavy chain
  • VL016-6-28A2-3K4 light chain
  • VL016-6-28A2-3K4 light chain; CDR3-Kabat QQRSNWPPT
  • VL016-6-28A2-3K4 light chain
  • CDR1-IMGT QSVSTS CDR1-IMGT QSVSTS
  • VLO 16-9-32E3-G3 heavy chain; CDRl-Kabat SGPYYWS
  • VLO 16-9-32E3-G3 heavy chain; CDR2-Kabat YIYYTGRPYYNPSLKS
  • VLO 16-9-32E3-G3 heavy chain; CDR3-Kabat DGGLYGLDV
  • VLO 16-9-32E3-G3 heavy chain; CDR1-IMGT GDSVTSGPYY
  • VLO 16-9-32E3-G3 heavy chain; CDR2-IMGT IYYTGRP
  • VL016-6-9-32E3-K1 light chain; CDRl-Kabat RASQSVSTSLA
  • VL016-6-9-32E3-K1 light chain
  • VL016-6-9-32E3-K1 light chain; CDR3-Kabat QQRSNWPPT
  • VL016-6-9-32E3-K1 light chain;
  • VL016-6-9-32E3-K1 light chain; CDR3-IMGT QQRSNWPPT
  • VLO 16-16-4A2 -P2G5 heavy chain; CDRl-Kabat GDSYYWG
  • VLO 16-16-4A2 -P2G5 heavy chain; CDR3-Kabat IFGGSVFDY
  • VLO 16-16-4A2 -P2G5 heavy chain; CDR1-IMGT GGSVSGDSYY;
  • VLO 16-16-4A2 -P2G5 heavy chain; CDR2-IMGT IYHSGNI
  • VLO 16-16-4A2 -P2G5 heavy chain; CDR3-IMGT ARIF GGS VFDY
  • VLO 16-16-4A2 -P2K3 light chain; CDRl-Kabat RASQSIDIWLA
  • VLO 16-16-4A2 -P2K3 light chain; CDR2-Kabat KASTLE
  • VLO 16-16-4A2 -P2K3 light chain; CDR3-Kabat QQSDTFPWT
  • VLO 16-16-4A2 -P2K3 light chain; CDR1-IMGT QSIDIW
  • VLO 16-16-4A2 -P2K3 light chain; CDR2-IMGT KAS
  • VLO 16-16-4A2 -P2K3 light chain; CDR3-IMGT QQSDTFPWT
  • VLO 16-16-26E8 -G3 heavy chain; CDRl-Kabat SPHYHWS
  • VLO 16-16-26E8 -G3 heavy chain; CDR2-Kabat YIQYSRTTKQNPSFRS
  • VLO 16-16-26E8 -G3 heavy chain; CDR3-Kabat VFGDSVFDS
  • VLO 16-16-26E8 -G3 heavy chain; CDR2-IMGT IQYSRTT
  • VLO 16-16-26E8 -G3 heavy chain; CDR3-IMGT ARVFGDSVFDS
  • VL016-16-26E8-K1 light chain; CDRl-Kabat RASQSIDIWVA
  • VL016-16-26E8-K1 light chain
  • VL016-16-26E8-K1 light chain; CDR3-Kabat QQYNTFPWT
  • VL016-16-26E8-K1 light chain
  • VL016-16-26E8-K1 light chain
  • CDR2-IMGT KAS CDR2-IMGT KAS
  • VL016-16-26E8-K1 light chain
  • VLO 16-16-31 C9-P 1 G2 ; heavy chain; CDR2-Kabat YIQYRRTTKQNPSFGS
  • VL016-16-31C9-P1K2 light chain; CDR3-Kabat QQYNSFPWT
  • VL016-16-31C9-P1K2 light chain; CDR1-IMGT QNIDIW
  • VL016-16-31C9-P1K2 light chain
  • CDR3-IMGT QQYNSFPWT QQYNSFPWT
  • VL016-8-9F5-G1 heavy chain
  • VL016-8-9F5-G1 heavy chain; CDR2-Kabat FIRNKV Y GGTTE Y AAS VKG
  • VL016-8-9F5-G1 heavy chain; CDR3-Kabat HIGYCSSTSCYNPNYFDN
  • VL016-8-9F5-G1 heavy chain; CDR1-IMGT GFTLGDYS
  • VL016-8-9F5-G1 heavy chain; CDR2-IMGT IRNKVYGGTT
  • VL016-8-9F5-G1 heavy chain; CDR3-IMGT TRHIGY CSSTSCYNPNYFDN
  • VL016-68-9F5-K4 light chain
  • CDR3-Kabat CDR3-Kabat
  • VL016-68-9F5-K4 light chain
  • CDR1-IMGT CDR1-IMGT
  • variable region light chain VL016-4-6D2-P2L2; variable region light chain
  • variable region heavy chain VL016-9-7F10-1G1 ; variable region heavy chain
  • variable region light chain VL016-9-7F10-1K1 ; variable region light chain
  • VLO 16-6- 11 C 1 1 -30L5 QSVLTQPPSVSAAPGQKVTISCSGGDSNIGSNYISWYQQIPGTAPRLIIYENDDRPSGIPDRFSGSKSGTSATL GIT GLQT GDE AD YY CG A WDTTLT AM VF GGGTKLT VL
  • VLO 16-4-11D1 1-Gl variable region heavy chain
  • variable region light chain VL016-6-4- 11D1 1-L16; variable region light chain
  • variable region light chain VL016-9-6E5-L1 ; variable region light chain
  • variable region light chain VL016-6-28A2-3K4; variable region light chain
  • variable region light chain VL016-6-9-32E3-K1 ; variable region light chain
  • variable region light chain VL016-16-4A2-P2K3; variable region light chain
  • variable region light chain VL016-16-26E8-K1 ; variable region light chain
  • variable region light chain VLO 16-16-31 C9-P 1 K2 ; variable region light chain
  • hcmvIL-10 Genbank Accession No. AAF63437
  • Latency-associated cmvIL-10 (LAcmvIL-10); Genbank Accession No. ACR49217
  • Interleukin 10 Homo sapiens (human cellular IL-10; hIL-10); Genbank Accession No. AAI04253

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  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne des anticorps spécifiques de l'interleukine (IL)-10 du cytomégalovirus (hCMV) humain (hcmvIL-10) qui n'ont pas de réactions croisées avec l'IL-10 cellulaire humaine (hIL-10), le virus d'Epstein-Barr IL-10 (ebvIL-10) ou le macaque rhésus CMV IL-10 (RhcmvIL-10). Un sous-ensemble des anticorps selon l'invention a une réaction croisée avec hcmvIL-10 associé à la latence (LAcmvIL-10). Les anticorps selon l'invention sont capables d'inhiber des activités biologiques de hcmvIL-10 et peuvent être utilisés à des fins diagnostiques ou thérapeutiques.
PCT/EP2018/082603 2018-11-26 2018-11-26 Anticorps dirigés contre l'interleukine-10 du cytomégalovirus WO2020108735A1 (fr)

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