FIELD OF THE INVENTION

The invention relates to antibodies specific to human cytomegalovirus (hCMV) interleukin (IL)-10.

BACKGROUND OF THE INVENTION

Human cytomegalovirus (hCMV) is one of the herpesviruses that commonly infects humans. It is estimated that more than 50% to 80% of adult populations are seropositive for hCMV. Most infected individuals are asymptomatic; however, hCMV can cause severe disease in immunocompromised individuals such as transplant patients or patients with AIDS, as well as in congenitally-infected newborns (Crough & Khanna (2009) Clin. Microbiol. Rev. 22(l):76-98; Boeckh & Geballe (2011) J. Clin. Invest. 121(5): 1673-1680). Additionally, many recent studies have implicated hCMV in the development of certain types of cancers such as those of those of the breast, prostate, colon, lung and brain (Michaelis, et al. (2009) Neoplasia 11 (1): 1-9; Lepiller, et al. (2011) The Open Virology Journal 5:60-69; Dziurzynski, et al. (2012) Neuro-Oncology 14(3):246-255; Alibek, et al. (2013) Infectious Agents and Cancer 8(32): 1- 6; Cobbs, et al. (2013) Curr. Opin. Oncol. 25:682-688; Alibek, et al. (2014) Infectious Agents and Cancer 9(3): 1 -8; Chen, et al. (2014) Frontiers in Oncology 4(314): 1 -5; Herbein & Kumar (2014) Frontiers in Oncology 4(230):l-8; Bishop, et al. (2015) Cancer Cell Microenviron. 2(1): 1-12). 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.

Like other herpesviruses, 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. 95:245-262). Among immune evasion mechanisms used by hCMV is the production of a homolog of cellular human IL-10 (hIL-10), which is designated hcmvIL-10 (Kotenko, et al. (2000) PNAS 97(4): 1695-1700; Lockridge, et al. (2000) Virology 268:272-280) and is encoded by the UL111A gene of hCMV. 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. 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. (2014) Frontiers in Microbiology 5(337): 1 -5; Christiaansen, et al. (2015) Current Opinion in Immunology 36:54- 60; Poole and Sinclair (2015) Med. Microbiol. Immunol. 204:421-429; Avdic, et al. (2016) J. Virol. 90(8):3819-3827). 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. 17:4619-4621 ; Dziurzynski, et al. (2011) Clin. Cancer Res. 17(14):4642-9; Valle Oseguera, et al. (2014) PLoS ONE 9(2):e88708; Bishop, et al. (2015) Cancer Cell Microenviron. 2(1):1-12). 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.

The major treatments available for hCMV infection are 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. Currently, there exist neither vaccines nor monoclonal antibodies for use in the prevention and/or treatment of hCMV infections (Plotkin (2015) Med. Microbiol. Immunol. 204:247-254). Targeted 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.

Most vaccine and antibody programs in development target hCMV envelope glycoproteins for prevention of cell infection by the virus. However, 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. Although 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. (2012) PLoS ONE 7(5):e37931 ; Eberhardt, et al. (2013) J. Virol. 87(21):11323-11331 ; WO2012/135177), isolated anti-hcmvIL-10 neutralizing monoclonal antibodies, such as humanized macaque antibodies, have not yet been described. One safety issue in the development of a vaccine or therapeutic antibodies to hcmvIL-10 is the potential for cross-reactivity with endogenous hIL-10, which could generate side effects in treated individuals by inhibiting important physiological functions of hIL-10 (Moore, el al. (2001) Ann. Rev. Immunol. 19:683-765). Furthermore, 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). During lytic infection, 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.

SUMMARY

Described herein are fully-human monoclonal antibodies with specificity to the human interleukin- 10 homolog encoded by the human cytomegalovirus (hCMV) gene UL111A and designated as hcmvIL-10. The antibodies were found to potently and specifically neutralize in vitro biological activities of hcmvIL- 10, including inhibition of TNF-a, IL-I b and IFN-g production by stimulated human peripheral blood mononuclear cells (PBMC).

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.

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. In this capacity, 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. Furthermore, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. The Figures are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

Figure 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 PLATELIA™ 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 9 Cross-reactivity of antibodies VL016-16-4A2, VL016-16-26E8 and VL016-16-31C9 to LAcmvIL-10 is comparable to that of goat anti-hcmvIL-10.

Figure 10 Comparison of hcmvIL-10 and ebvIL-10 inhibitory activity in the PBMC/LPS/TNF-a assay.

Figure 11 Absence of cross-reactivity of anti-hcmvIL-10 antibodies with hIL-10 and ebvIL-10 as measured by ELISA.

Figure 12 Absence of cross-reactivity of anti-hcmvIL-10 antibodies with hIL-10 and ebvIL-10 as measured in the PBMC/LPS/TNF-a assay. Data are expressed as mean +/- SD of triplicates.

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.

Figure 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. 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.

DETAILED DESCRIPTION OF THE INVENTION

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.

The term "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.

As used herein, the term "epitope" 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. 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)).

The term "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. However, the variability is not evenly distributed throughout the variable domains of antibodies ft is concentrated in three segments called complementarity-determining regions (CDRs) or 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. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. For a review of 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.

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. Examples of 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. In an antibody fragment comprising one or more heavy chains, 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). Unless specifically indicated to the contrary, 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. soluble in physiological fluids such as blood, and wherein the heterogeneous molecule is free of any structured aggregate. A conjugate of interest is PEG. In the context of the foregoing definition, the term "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. Accordingly, the term "conjugate" as defined herein 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.

The term "monoclonal antibody" (mAh) 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. Each mAh is directed against a single determinant on the antigen. In addition to their specificity, 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. For example, 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.

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. In some embodiments, 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).

Regarding vaccination with hcmvIL-10, we cannot exclude that 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. Also, 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.

In one aspect, the current disclosure provides an antibody which binds to hcmvIL-10, which does not bind to human cellular IL-10 (hIL-10). In one aspect, the antibody of the current disclosure inhibits or neutralizes one or more biological activities of hcmvIL-10. In one aspect, 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. In one aspect, the specific reduction of the activity of hcmvIL-10 increases overall anti-CMV immune responses. In one aspect, the specific reduction of the immunosuppressive activity of hcmvIL-10, but not hIL-10, results in a safer profde. As used herein,“neutralize” or“inhibit” means to reduce or to abolish, specifically with regard to the biological effects of hcmvIL-10. In one aspect, the antibody of the current disclosure neutralizes or reduces the inhibitory effect of hcmvIL-10 on an immune cell. In one aspect, the anti-hcmvIL-10 antibody of the invention shows various desirable characteristics. In one aspect, 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. In this regard, 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). In a preferred embodiment, 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). In one aspect, 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.

In one aspect, 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). In a preferred aspect, 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).

In one aspect, the anti-hcmvIL-10 antibody of the current disclosure also binds to LAcmvIL-10. In one aspect, the antibody which also binds to LAcmvIL-10 reduces or inhibits one or more biological activity of LAcmvIL-10. In one aspect, the antibody which also binds to LAcmvIL-10 has a broader neutralization activity of hCMV-encoded IL-10 proteins. In one aspect, the antibody which also binds to LAcmvIL-10 is useful for the detection of both hcmvIL-10 and LAcmvIL-10 in a biological sample.

In one aspect, 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. In this regard, the K _D can be measured by any assay well-known in the art. In a preferred embodiment, the K _D of the anti-hcmvIL-10 is measured by surface plasmon resonance (SPR), for example by Biacore. In a preferred aspect, 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. Alternatively, 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.

In a preferred aspect, 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

b. 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

c. 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

d. 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

f. 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

g. 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

h. an antibody that comprises a heavy chain having each of the CDR sequences set forth in SEQ ID NOs: 85 to 87 (Kabat annotation) or SEQ ID NOs: 88 to 90 (IMGT annotation) and a light chain having each of the CDR sequences set forth in SEQ ID NOs: 91 to 93 (Kabat annotation) or SEQ ID NOs: 94 to 96 (IMGT annotation); or

i. 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

j. 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

k. 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

l. 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).

In a preferred aspect, 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

b. a 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

a 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; or

d. a 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

a 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; or

f. a 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

g· a VH having an amino acid sequence set forth in SEQ ID NO: 157 and a VL having an amino acid sequence set forth in SEQ ID NO: 158; or

h. a 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

a 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; or

J· a VH having an amino acid sequence set forth in SEQ ID NO: 163 and a VL having an amino acid sequence set forth in SEQ ID NO: 164; or

k. a 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

1. a 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.

In one aspect, the anti-hcmvIL-10 antibody of the current disclosure is a human antibody. In a preferred aspect, the antibody is a monoclonal antibody. In a preferred aspect, 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. In one aspect, the antibody or a cell producing the antibody is collected from a human blood donor. In one aspect, the human blood donor is an hCMV- positive donor. In some embodiments, the C-terminal lysine residue of the monoclonal antibody is removed to avoid heterogeneity during biopharmaceutical production process(es) or in pharmaceutical preparation(s). In some embodiments, a potential glycosyation site present in the VH/VL region of some antibodies is removed. In one aspect, the anti-hcmvIL-10 antibody of the current disclosure has a human IgGl, IgG2, IgG3, IgG4 or IgA constant region. In a preferred aspect, the variable regions of the antibody are combined with an artificial and/or recombinant Fc region of a particular allotype. In a preferred aspect, deamidation sites are removed within the VH and VL regions to increase stability of the pharmaceutical product. In a preferred aspect, the anti-hcmvIL-10 antibody is produced recombinantly; i.e., as a fully-human recombinant antibody.

In one aspect, 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. In addition to Fabs, smaller antibody 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. For example, 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. Techniques for obtaining a single domain antibody with at least some of the binding specificity of the intact antibody from which they are derived are known in the art. For instance, Ward, et al. in "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted from Escherichia coli", Nature 341 :644-646, disclose a method for screening to obtain an antibody heavy chain variable region (H single domain antibody) with sufficient affinity for its target epitope to bind thereto in isolated form.

In one aspect the anti-hcmvIL-10 antibody of the current disclosure is a bispecific antibody. As used herein,“bispecific” antibodies are defined as antibodies which bind specifically to two distinct targets (epitopes). In a preferred embodiment, the bispecific antibodies bind specifically to both hcmvIL- 10 and another distinct target. In one embodiment, the other distinct target is an immune modulator, such as a cytokine or chemokine. In one embodiment, the other distinct target of the bispecific antibody is another hCMV protein such as an envelope glycoprotein or another immunomodulatory produced by hCMV. In one embodiment, the other distinct target of the bispecific antibody is a cell-surface marker, such as a receptor or tumor-associated antigen. In one embodiment, the cell-surface marker is a receptor expressed or over-expressed by a cancer or immune cell. In a preferred embodiment, the receptor is CD47, CD20, PD-L1 or PD-1, phospatidylserine (PS), EGFR (HER), HER2, MUC1, MUC5AC or SIRP- a.

In one aspect, 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. For recombinant production of the antibody, 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. For prokaryotic host cells that do not recognize and process the native antibody signal sequence, 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. However, 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.

In one aspect, the nucleic acid encoding the anti-hcmvlL-10 antibody of the disclosure is comprised in a host cell. In one aspect 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. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells; Chinese hamster ovary cells/dhFr- (CHO/dhFr-, Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23 :243-251 (1980)); 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. EP 2150275B1); FS4 cells; and a human hepatoma line (Hep G2). 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.

In one aspect, the disclosure provides a pharmaceutical composition comprising an antibody as provided. In one aspect, 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 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 counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURON1CS™ or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

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. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

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. In addition, the anti-hcmvIL-10 antibody or pharmaceutical composition of the disclosure is suitably administered by pulse infusion, particularly with declining doses of the antibody.

For the prevention or treatment of disease, 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.

In another embodiment of the invention, an article of manufacture containing materials useful for the treatment of the disorders described above is provided. The article of manufacture 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.

In one aspect, the antibody as provided or the pharmaceutical composition as provided is used in the treatment or prevention of a disease in a subject. In one aspect, the disease to be treated is hepatitis, esophagitis, colitis, pneumonia, retinitis, myocarditis or ependymitis. In one aspect, the disease to be treated is characterized by hepatosplenomegaly, petechiae, microcephaly, hypotomia, seizures, and/or lethargy. In one aspect, the disease to be treated is CMV infection. In one aspect, the CMV infection affects the lung, liver, spleen, gastrointestinal tract, Central Nervous System (CNS), hematologic system, kidney, adrenal, salivary gland, pancreas, and/or esophagus. In one aspect, the subject has an acute hCMV infection, a latent hCMV infection or an acute CMV reinfection or reactivation. In one aspect, the disease to be treated is a disease associated with CMV infection such as cancer or atherosclerosis. In one aspect, the disease to be treated or prevented is congenital hCMV infection. In one aspect, the disease to be treated arises from hCMV infection of the transplanted organ or tissue. In one aspect, the disease to be treated is another infection or infections co-present with hCMV infection. In one aspect, the other infection(s) compromises the immune function of the subject. In one aspect, the other infection is Human Immunodeficiency Virus (HIV).

In one aspect, the disease to be treated by the antibody or the pharmaceutical composition provided is cancer. In one aspect, the subject to be treated has an hCMV infection. In one aspect, the subject (cancer patient) to be treated suffers from an hCMV-positive cancer. In one aspect, the subject (cancer patient) has a latent hCMV infection. In one aspect, the cancer patient has circulating hcmvIL-10- producing cells, e.g., cancer cells. In one aspect, the subject (cancer patient) has a lytic hCMV infection.

In one aspect, 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. In one aspect, the combination therapy includes other preparations for treatment of hCMV, such as the antiviral drugs which are the standard of care. In a preferred aspect, the combination therapy includes a human hCMV -hyperimmune serum preparation; i.e., Cytotect CP. In one aspect, the antiviral drugs are ganciclovir (cytovene), valganciclovir (valcyte), foscamet or cidofovir. In one aspect, the combination therapy includes drugs targeting another disease or condition which co-exists with hCMV in the subject. In one aspect, the antibodies or pharmaceutical compositions provided are used to treat a human subject. In one aspect, the subject to be treated is an adult or elderly adult. In one aspect, 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 antibody or composition for use of the disclosure, wherein the subject is immunocompromised. In one aspect, the immunocompromised subject has an immature immune system. In one aspect, the subject with an immature immune system is an infant, toddler or small child. In one aspect, the immunocompromised subject is elderly. In one aspect, the immunocompromised subject has poor nutritional status; i.e., malnutrition. In one aspect, the immunocompromised subject has a primary immunodeficiency, e.g., an X-linked or autosomal recessive immunodeficiency disorder. In one aspect, 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. In one aspect, the immunocompromised subject is a transplant patient. In one aspect, the transplant patient is a solid organ transplant patient, e.g., a kidney, lung, liver, pancreas or heart recipient. In one aspect, the transplant patient is a hematopoietic stem cell transplant patient. In one aspect, the transplant patient is a blood transfusion recipient. In one aspect, the transplant patient is on short-term or chronic immunosuppressive therapy. In one aspect, the immunocompromised subject has an infection which reduces or ablates immune function, e.g., Human Immunodeficiency Virus (HIV). In one aspect, the subject is an HIV-positive patient or an AIDS patient. In one aspect, the subject has an autoimmune disorder.

In one aspect, 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.

In one aspect, the disclosure provides a method of treating, preventing or protecting a subject from hCMV infection or complications of an hCMV infection. In one aspect, 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.

In one aspect, a method of treating or preventing cancer in a subject is provided. In one aspect, the method comprises administering to a subject having or at risk of developing cancer an effective amount of the antibody or the pharmaceutical composition provided herein. In one aspect, the subject suffers from a CMV-positive cancer. In one aspect, the subject is infected with hCMV.

In one aspect, the method of treating or preventing cancer in a subject, comprises administering the antibody or the pharmaceutical composition as a monotherapy. In one aspect, the cancer to be treated or prevented is a cancer of the breast, prostate, colon, lung or brain, e.g., glioma.

In one aspect, the method of treating or preventing cancer in a subject, comprises administering the antibody or the pharmaceutical composition as a combination therapy. In one aspect, the cancer to be treated is hCMV positive and/or hcmvIL-10 positive. In one aspect, the combination therapy further comprises an anti-viral or anti-cancer drug. In one aspect, the combination therapy further comprises another therapeutic antibody. In a preferred aspect, the subject to be treated is human. In one aspect, the subject to be treated is immunocompromised as defined above.

In one aspect, a process for producing the antibody of the disclosure is provided, comprising culturing the aforementioned host cell so that the nucleic acid encoding the antibody of the disclosure is expressed. In one aspect, 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. 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 gΐ, 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. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J.T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

Following any preliminary purification step(s), 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). In one aspect, 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. In this regard, 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. In addition, the monoclonal antibodies in these immunoassays can be detectably labeled in various ways. Examples of 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. Examples of well-known 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.

There are many different labels and methods of labeling known to those of ordinary skill in the art, which find use as tracers in therapeutic methods, for use in diagnostic methods, and the like. For diagnostic purposes 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. Furthermore, 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.

In some embodiments 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).

For purposes of the invention, 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.

As a matter of convenience, 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. Where the antibody is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). In addition, 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. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

EXAMPLES

Example 1 : Identification and isolation of human B cells producing antibodies specific for hcmvIL-10

Functional assays for screening and characterization of anti-hcmvIL-10 antibodies Cell-based assays using human peripheral blood mononuclear cells (PBMC) were used to screen and evaluate the ability of the anti-hcmvIL-10 antibodies to inhibit the biological activity of hcmvIL-10. It was expected that 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 ; Nachtwey, et al. (2008) Viral Immunol. 21(4):477-482). Assays measuring the IL-10-mediated inhibition of TNF-a and IL-I b secretion by PBMC activated with the TLR4-ligand LPS, or inhibition of the secretion of IFN-g by PBMC activated with the TLR3 -ligand Poly(I:C) were implemented (Moore, et al. (2001), supra; Perrot, et al. (2010) J. Immunol. 185:2080- 2088). For all cell-based assays, PBMC were purified from healthy donors that were previously screened seronegative for hCMV by using the PLATELIA™ CMV IgG kit (Bio-Rad).

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. 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. The selected neutralizing concentrations of 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.

These results confirmed that hcmvIL-10 displayed strong functional activity similar to the activity of cellular hIL-10 in inhibiting inflammatory cytokine secretion by human PBMC.

Human donor screening

Serum was collected from 519 healthy adult donors and was first tested for the presence of IgG antibodies against hCMV by using the PLATELIA™ 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 (titer comprised between 0.25 and 0.5 AU/mL) was observed in 6 serum samples while the remaining 236 samples were negative (hCMV titer < 0.25 AU/mL). All 277 sera with IgG anti-hCMV positivity, as well as the 6 equivocal sera and a random selection of 86 negative sera, were further assessed for their ability to bind to hcmvIL-10 by ELISA. Briefly, 96-well ELISA plates (half size well plates, Greiner) were coated overnight with 0.25 pg/mL of recombinant hcmvIL-10 (R&D Systems) in 50 pL carbonate buffer. After 3 washings with 200 pL/well of PBS, 0.05% Tween-20, plates were saturated by addition of 200 pL/well of PBS, 1% BSA, 0.05% Tween-20 buffer. After 1 hour incubation at 25°C, plates were washed 3 times with 200 pL/well PBS, 0.05% Tween-20 buffer, and 50 pL/well of 1 :20 to 1 :40 serum dilutions were added and incubated for 1 hour at 25°C. Plates were then washed 4 times with 200 pL/well PBS, 0.05% Tween-20, and 50 pL/well of HRP-conjugated goat anti-human IgG antibody (SIGMA) were added. Plates were incubated for 1 hour at 25°C, then washed with 200 pL/well PBS, 0.05% Tween-20, and the HRP enzymatic activity was revealed by addition of 50 pL/well of TMB substrate (KPL). After 15 minutes incubation with TMB, the reaction was stopped by addition of 50 pL/well orthophosphoric acid and the OD was read at 450 nm on a VersaMax microplate reader (Molecular Devices). To determine the specificity of binding, serum dilutions were tested in parallel by ELISA on plates not coated with hcmvIL-10 (uncoated plates). Moreover, because immobilization of hcmvIL-10 on a plastic surface may affect its conformation and may reveal epitopes not present on the soluble bioactive protein, a competition ELISA was further used to characterize all hcmvIL-10 positive serum samples. In 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.

In total, 17 samples of the 277 hCMV IgG positive and 1 of the 6 hCMV IgG equivocal sera displayed significant binding to coated-hcmvIL-10 that was almost completely inhibited by pre incubation with soluble hcmvIL-10, thus demonstrating the presence of IgG recognizing the soluble bioactive hcmvIL-10 protein in these 18 donors (Table 1). Of note, no specific binding to hcmvIL-10 was observed in the 86 hCMV IgG negative sera tested.

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. Following addition of 5 pg/mL LPS or 20 pg/mL Poly(TC), the cells were incubated for 24 hours at 37°C, 5% CO2 in humidified incubator. Supernatants were then collected and tested for their concentration of TNF-a or IFN-g by using ELISA kits (BD Biosciences). When tested at a 1 :40 final dilution, 12 of the 18 serum samples showing specific recognition of hcmvIL-10 by ELISA also neutralized the inhibitory activity of hcmvIL-10 on TNF-a or IFN-g secretion by human activated PBMC (Table 1).

Table 1. Donor screening for anti-hcmvIL-10 antibodies.

*only the 18 sera positive by hcmvIL-10 competition ELISA were further tested in the hcmvIL-10 inhibition assay; ND: not done.

Comparison of the titer of anti-hCMV IgG measured by the PLATELIA™ CMV IgG kit in the serum of the 18 anti-hcmvIL-10 positive donors with the titer of anti-hCMV IgG measured in all the 283 hCMV seropositive donors, indicated that there was no correlation between the presence of anti-hcmvIL-10 IgG and the titer of anti-hCMV IgG. In particular, donor sera that displayed measurable anti-hcmvIL-10 neutralizing activity did not show the highest titer of anti-hCMV IgG (Figure 2). PBMC were isolated from 5 donors displaying hcmvIL-10 neutralizing activity and further used for the antibody screening campaigns.

VIVA\Screen antibody screening campaigns

Human anti-hcmvIL-10 monoclonal antibodies were obtained by implementing the VIVA| Screen ^® method described in WO2013/000982. Briefly, B lymphocytes from donor PBMC previously depleted of IgM ⁺ and IgD ⁺ B cells were activated and expanded in vitro for 14 days in 96-well culture plates at a cell density of 100 to 300 IgG ⁺ B cells/well by using the combination of EBV and CD40Ligand-expressing feeder cells. Supernatants from activated B cell pools were then screened for IgG anti-hcmvIL-10 in a primary assay by using the previously described ELISA against hcmvIL-10, and positive supernatants were further confirmed in a secondary ELISA against hcmvIL-10, hIL-10 and uncoated plates. A total of 185 supernatants (hits) containing IgG binding specifically to hcmvIL-10 were identified starting from a total of about 10 x 10 ⁶ IgG ⁺ B cells isolated from 5 donors (Table 2). None of the 185 hits were positive on hIL-10 coated wells. 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. Following addition of 25 pL of 4 pg/mL LPS or 20 pg/mL Poly(I:C), the cells were incubated for 24 hours at 37°C, 5% CO2 in a humidified incubator. Supernatants were then collected and tested for their concentration of TNF-a or IFN-g by using ELISA kits (BD-Biosciences). Among the 185 hits tested, 88 were found to neutralize the biological activity of hcmvIL-10 as measured by the hcmvIL-10-mediated inhibition of IFN-g secretion by Poly(I:C)-activated PBMC or of TNF-a secretion by LPS-activated PBMC (Table 2).

Table 2. Summary of VIVAScreen campaigns for human IgG anti-hcmvIL-10.

* Neutralization of hcmvIL-10-induced inhibition of cytokine secretion by human activated PBMC.

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. 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. 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.

From this procedure, a total of 32 antibodies were confirmed by ELISA to bind to hcmvlL-10. Further re-cloning and sequence analysis confirmed 31 antibodies with original VH and VL sequences among those 32 antibodies. Out of these, twenty-three were produced at larger scale by transient transfection of CHO cells and purified on a protein A column for further characterization.

The names and clone sequence families of the 12 antibody candidates which were selected according to their functional activity and binding domain recognition on hcmvlL-10 as described herein below are shown in Table 3.

Table 3: Names and clone sequence families of the twelve selected candidates for which the sequences are disclosed.

Example 2: Characterization of selected antibody candidates

Equilibrium binding constant by ELISA Serial dilutions of the purified antibodies were tested for their capacity to bind to purified hcmvIL-10 immobilized on ELISA plates (data not shown) and the equilibrium binding constant was calculated for each antibody by using the one-site binding model with GraphPad Prism software. As shown in Table 4, the equilibrium binding constant of all 12 selected anti-hcmvIL-10 antibodies was estimated in the range of about 8 to about 82 pM.

Table 4: Equilibrium binding constants of selected anti-hcmvIL-10 antibodies as determined by ELISA.

Binding affinity of anti-hcmvIL-10 antibodies by surface plasmon resonance (SPR)

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. 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.

Table 5. Association, dissociation and affinity constants of anti-hcmvIL-10 antibodies as measured by SPR (mean of duplicate determinations by Biacore).

Neutralization of hcmvIL- 10 -induced inhibition of cytokine secretion by LPS-activated PBMC

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. Cell-free supernatants were then collected from each well and the concentration of TNF-a or IL-I b was determined by using an ELISA kit (BD-Biosciences). The percentage of hcmvIL- 10 neutralization was calculated by applying the following equation: % 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 tested. The percent inhibition was plotted against antibody concentration (see Fig. 3) and the IC50 and IC90 values were calculated by using a nonlinear regression analysis model of GraphPad Prism software (see Table 6).

Eleven of the 12 anti-hcmvIL-10 antibodies were capable of neutralizing the activity of hcvmlL- 10 on TNF-a secretion with an IC50 ranging from 70 pM to 1.2 nM (Figure 3 and Table 6). The antibodies 4-20A9, 6-11 Cl 1, 6-28A2, 16-4A2 and 16-31C9 inhibited hcmvIL-10 activity on TNF-a secretion with an IC50 lower than 150 pM and with an IC90 lower than 2.5 nM. The antibody 8-9F5 did not display significant neutralizing activity in this assay (IC50 > 70 nM).

Table 6: IC50 and IC90 values of anti-hcmvIL-10 antibodies in the PBMC/LPS/TNF-a assay.

Furthermore, with exception of antibody 8-9F5, all antibodies tested at 3 pg/mL (~20 nM) were also capable of restoring secretion of IL-Ib by LPS-stimulated PBMC in the presence of 1.5 ng/mL of hcmvIL-10 (Figure 3).

Neutralization of hcmvIL- 10 -induced inhibition of IFN-g secretion by Poly(I:C)-activated PBMC

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. 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). The percentage of hcmvIL- 10 neutralization was calculated by applying the following equation: % 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 nonlinear regression analysis model of GraphPad Prism.

Eleven of the 12 anti-hcmvIL-10 antibodies tested neutralized the activity of hcvmIL-10 on IFN-g secretion, with an IC50 ranging from 50 pM to 1.7 nM (Figure 4 and Table 7). The antibodies 4-6D2, 4- 20A9, 6-11 Cl 1, 6-28A2, 9-6E5, 9-7F10, 16-4A2 and 16-31C9 inhibited hcmvIL-10 activity on IFN-g secretion by Poly(I:C)-stimulated PBMC with an IC50 lower than 600 pM and an IC90 lower than 5 nM. The antibody 8-9F5 did not show strong neutralization of hcmvIL-10 on IFN-g secretion (IC50 > 70 nM).

Table 7: IC50 and IC90 values of anti-hcmvIL-10 mAbs in the PBMC/ Poly-IC/IFN-g assay.

Example 3: Characterizing the recognition of LAcmvIL-10 by candidate antibodies

Anti-hcmvIL-10 antibodies do not recognize LAcmvIL-10

A second isoform of hcmvIL-10 has been described which is produced during latent infection with hCMV and has been designated as latency associated cmvIL-10 (LAcmvIL-10; SEQ ID NO: 170) (Jenkins, et al. (2004), supra). 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. To determine if the anti-hcvmIL-10 antibodies also recognized LAcmvIL-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. As controls, 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. In order to confirm the presence of LAcmvIL-10 in CHO cell supernatants, 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. As expected, the anti-hcmvIL-10 polyclonal antibody strongly recognized the supernatants of CHO-cells transfected with hcmvIL-10 vector, but not the supernatant of CHO-cells transfected with the empty vector (Figure 6). A positive but much weaker signal was observed with the wells coated with the supernatant of CHO-cells transfected with LAcmvIL-10 vector, indicating that the anti-hcmvIL-10 polyclonal antibody cross-reacted with LAcmvIL-10 (Figure 6). These results thus indicated the presence of LAcmvIL-10 in the supernatants of CHO-cells transfected with the LAcmvIL- 10 expression vector.

To measure the biological activity of LAcmvIL-10, serial dilutions of the CHO cell supernatants were tested for their capacity to block the secretion of TNF-a by LPS-stimulated human PBMC. As shown in Figure 7, the supernatant of CHO cells transfected with the LAcmvIL-10 expression vector did not inhibit the production of TNF-a, nor did the supernatant of CHO cells transfected with the empty vector. In contrast, the supernatant of CHO cells transfected with the hcmvIL-10 vector strongly inhibited TNF-a secretion for the same tested dilutions. These results confirmed that 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. To this end, 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. After washings in PBS, 0.05% Tween-20 and 1 hour saturation (with PBS, 1% BSA, 0.05% Tween-20), antibodies (1 pg/mL) were incubated for 1 hour at 25 °C. After washings, the binding of antibodies was detected by addition of an HRP -conjugated secondary antibody. The HRP enzymatic activity was then revealed by addition of 50 pL/wcll of TMB substrate (KPL). After 15 minutes incubation with TMB, the reaction was stopped by addition of 50 pL/well orthophosphoric acid and the OD was read at 450 nm on a VersaMax microplate reader (Molecular Devices).

As shown in Figure 8, a specific signal was observed on supernatant from LAcmvIL-10- transfected cells compared to supernatant of not-transfected cells with three antibodies: 16-4A2, 16-26E8 and 16-31C9. The nine other antibodies did not show binding to LAcmvIL-10 by ELISA, while they strongly bound to hcmvIL-10 contained in the supernatant of transfected CHO cells. As a positive control, the goat anti-hcmvIL-10 polyclonal antibody recognized both the hcmvIL-10 and LAcmvIL-10 contained in the supernatants of CHO cells transfected with the corresponding vectors.

In order to confirm the observed binding of 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. As shown in Figure 9, 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. In contrast, the nine other anti-hcmvIL-10 antibodies did not show specific binding to LAcmvIL-10-containing supernatant at any of the tested dilutions (VL016-4-6D2 and VL016-8-9F5 shown in Fig. 9; all others, data not shown). These results indicate that VL016-16-4A2, VL016-16-26E8 and 16-31C9 all belong to the same clone sequence family (see Table 3) which cross-react with LAcmvIL-10.

Example 4: Assessing the cross reactivity of candidate antibodies

Specific recognition ofhcmvIL-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). By contrast, the 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). Comparison of ebvIL-10 and hcmvIL-10 biological activity also showed that 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). To address whether the anti-hcmvIL-10 antibodies of the current disclosure can also recognize hIL-10 and/or ebvIL-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). None of the anti-hcmvlL-10 mAbs tested at 3 pg/mL were found to bind to hlL-10 or to ebvlL-10 by ELISA, while they all recognized hcmvlL-10 (Figure 11). Similar binding specificity was observed for a goat anti- hcmvlL-10 antibody (R&D Systems) used as a positive control. In contrast, and as expected from the strong sequence homology between hlL-10 and ebvlL-10, goat and mouse antibodies against hlL-10 or ebvlL-10 (both from R&D Systems) recognized both hlL-10 and ebvlL-10, but not hcmvlL-10. A non- related human monoclonal IgGl antibody, produced and purified as for the anti-hcmvlL-10 antibodies, was used as a negative control (control hlgGl) and did not show specific binding to hcmvlL-10, hlL-10 and ebvlL-10. 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.

Moreover, none of the anti-hcmvlL-10 mAbs tested at 3 pg/mL (—20 nM) neutralized the biological activity of hlL-10 (3 ng/mL, NT90) or ebvlL-10 (90 ng/mL, NT90), while l lof them inhibited hcmvlL-10, as measured by inhibition of TNF-a production by LPS-activated PBMC (Figure 12). Similar hcmvlL-10 specific inhibition was observed for a goat anti-hcmvlL-10 antibody (R&D Systems) used as a positive control. In contrast, and as expected from the strong sequence homology between hlL- 10 and ebvlL-10, mouse antibodies against hlL-10 or ebvlL-10 (both from R&D Systems) inhibited both hlL-10 and ebvlL-10, but not hcmvlL-10. A non -related human monoclonal IgGl antibody, produced and purified as for the anti-hcmvlL-10 antibodies, was used as negative control (control hlgGl) and did not show any specific inhibition of hcmvlL-10, hlL-10 or ebvlL-10 activity ft is notable that, in spite of the low level of binding shown in Figure 11 above, no inhibitory activity on hcmvlL-10, hlL-10 or ebvlL-10 by the pharmaceutical preparation of human IgG purified from hCMV hyper-immune donors (Cytotect CP, Biotest, 100 pg/mL) was observed, suggesting low levels of anti-hcmvlL-10 antibodies present in the preparation and also that these antibodies are probably not neutralizing. This observation may indicate that the therapeutic action of Cytotect CP is effected by a different mechanism other than hcmvlL-10 binding and/or neutralization.

Altogether, these results show that the anti-hcmvlL-10 monoclonal antibodies provided in the disclosure specifically recognize and inhibit hcmvlL-10, but not hlL-10 and ebvlL-10.

Anti-hcmvIL-10 antibodies do not recognize RhcmvIL-10

Like hCMV, the Rhesus macaque CMV genome also encodes an lL-10 homolog designated as RhcmvlL- 10 (Lockridge, et al. (2000), supra). The RhcmvlL-10 sequence shares only 30% amino-acid identity with hcmvIL-10. To determine if the anti-hcvmIL-10 antibodies also recognized the RhcmvIL-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. As controls, 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. In contrast, the supernatant of CHO cells transfected with an empty vector did not inhibit the production of TNF-a. This indicated the presence of an IL- 10-like activity in the supernatant of CHO cells transfected with the RhcmvIL-10 expressing vector and confirmed the strong activity of RhcmvIL-10 on human PBMC already reported (Spencer, et al. (2002), supra).

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. As shown in Figure 14, none of the anti-hcmvIL-10 antibodies specifically recognized the RhcmvIL-10 present in the corresponding transfected CHO cell supernatant; however, as expected, they bound strongly to the hcmvIL-10 present in the corresponding CHO cell supernatant. The binding observed with antibody 16- 31C9 on RhcmvIL-10 was likely to be non-specific because a similar binding was observed in wells coated with the supernatant of cells transfected with an empty vector. As a control, a rabbit anti-hcmvIL- 10 antibody was also used and revealed by use of an HRP-conjugated anti -rabbit antibody. Similar to the human anti-hcmvIL-10 monoclonal antibodies, the rabbit anti-hcmvIL-10 antibody displayed specific binding to hcmvIL-10 but not to RhcmvIL-10.

Moreover, none of the anti-hcmvIL-10 mAbs tested at 3 pg/mL inhibited the IL- 10-like biological activity contained in the 1 :80,000 dilution of supernatant of CHO cells transfected with RhcmvIL-10 vector, while 11 of them inhibited the activity contained in the 1 :80,000 dilution of supernatant of CHO cells transfected with the hcmvIL-10 vector, as determined by neutralization of inhibition of TNF-a secretion by LPS-activated human PBMC (Figure 15). Altogether, these results indicate that the anti-hcmvIL-10 antibodies specifically recognized the IL-10 protein homolog encoded by hCMV, but not the IL-10 homolog encoded by RhCMV.

Example 5: Determining binding specificity of candidate antibodies

Epitope / domain recognition using a competition ELISA

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. Plates were then washed 3 times with PBS, 0.05% Tween-20 and 50 pL/wcll of 2 pg/mL HRP-conjugated anti-hcmvlL-10 antibodies were added. Conjugation of the antibodies to HRP was carried out by using the EZ-Link Plus Activated Peroxidase kit (Thermo Scientific). After 1 hour incubation at RT and 3 washings, the HRP activity was revealed by adding 50 pL/well of TMB substrate, blocking with orthophosphoric acid and measured by OD reading at 450 nm. Six families of antibodies were identified according to their capacity to compete to each other for binding to hcmvlL-10. The results of the competition ELISA are summarized in Table 8 below. Briefly:

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.

Finally, 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

Epitope mapping using linear peptides

An ELISA with linear peptides was used to identify the amino-acid sequence recognized by the 12 anti- hcmvIL-10 candidates. Briefly, 22 amino-acid long peptides covering the entire sequence of hcmvIL-10 (SEQ ID NO: 169) and overlapping each other by 5 amino-acids, were generated (Eurogentec) and coated on ELISA plates. After washings, the anti-hcmvIL-10 antibodies were tested for their capacity to recognized one or several coated peptides by ELISA. The results revealed that none of the tested anti- hcmvIL-10 antibodies recognized a specific linear peptide from hcmvIL-10 (data not shown). As a control, a goat polyclonal anti-hcmvIL-10 antibody (R&D systems) recognized 8 of the 9 linear peptides tested. This finding suggests that, most likely, all anti-hcmvIL-10 candidates recognize a conformational epitope on hcmvIL-10 and not linear epitopes.

Example 6: Recognition of native hcmvIL-10 by antibodies of the invention

The anti-hcmvIL-10 antibodies were tested for their capacity to recognize hcmvIL-10 naturally produced by cells infected with hCMV. To this end, 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. The presence of hCMV -infected cells was analyzed by staining of CMV immediate early non-structural antigens (IE antigens) using an Alexa-Fluor 488-coupled mouse anti-CMV IE antigen antibody (MAB810X, Millipore). As expected, 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). A strong red PE-fluorescence was observed in the cytoplasm of MRC-5 and ARPE-19 cells stained with antibody 6-28A2 and infected with hCMV (indicated by cells tagged with a white star in Figure 16), while only background fluorescence was observed in the cytoplasm of non-infected cells stained with antibody 6-28A2. This demonstrated that the anti-hcmvIL-10 antibodies are capable of recognizing hcmvIL-10 protein produced by cells infected by hCMV.

SEQUENCES

SEQ ID NO: 1

VLO 16-4-6D2-P3G2; heavy chain; CDRl-Kabat

SYAMH

SEQ ID NO: 2

VLO 16-4-6D2-P3G2; heavy chain; CDR2-Kabat

FLSHDGNIKNYADSVKG

SEQ ID NO: 3

VLO 16-4-6D2-P3G2; heavy chain; CDR3-Kabat

RDPDGFDV

SEQ ID NO: 4

VLO 16-4-6D2-P3G2; heavy chain; CDR1-IMGT

GFTFSSYA

SEQ ID NO: 5

VLO 16-4-6D2-P3G2; heavy chain; CDR2-IMGT

LSHDGNIK

SEQ ID NO: 6

VLO 16-4-6D2-P3G2; heavy chain; CDR3-IMGT

AVRDPDGFDV

SEQ ID NO: 7

VLO 16-4-6D2-P2L2; light chain; CDRl-Kabat

AGTSSDVGAYNYVS

SEQ ID NO: 8

VLO 16-4-6D2-P2L2; light chain; CDR2-Kabat

DVSNRPS

SEQ ID NO: 9

VLO 16-4-6D2-P2L2; light chain; CDR3-Kabat

SSYTNRRTWV

SEQ ID NO: 10

VLO 16-4-6D2-P2L2; light chain; CDR1-IMGT

SSDVGAYNY

SEQ ID NO: 11

VLO 16-4-6D2-P2L2; light chain; CDR2-IMGT

DVS

SEQ ID NO: 12

VLO 16-4-6D2-P2L2; light chain; CDR3-IMGT

SSYTNRRTWV

SEQ ID NO: 13

VL016-4-20A9-1G1 ; heavy chain; CDRl -Kabat

SHAMS

SEQ ID NO: 14 VL016-4-20A9-1G1 ; heavy chain; CDR2-Kabat SISGGGVSTYYADSVKG

SEQ ID NO: 15

VL016-4-20A9-1G1 ; heavy chain; CDR3-Kabat GIGLNWNYVGS

SEQ ID NO: 16

VL016-4-20A9-1G1 ; heavy chain; CDR1 -IMGT GFMFDSHA

SEQ ID NO: 17

VL016-4-20A9-1G1 ; heavy chain; CDR2-IMGT ISGGGVST

SEQ ID NO: 18

VL016-4-20A9-1G1 ; heavy chain; CDR3-IMGT SKGIGLNWNYVGS

SEQ ID NO: 19

VLO 16-4-20A9- 1 K3 ; light chain; CDRl-Kabat QASQDINKFVN

SEQ ID NO: 20

VLO 16-4-20A9- 1 K3 ; light chain; CDR2-Kabat DATNVET

SEQ ID NO: 21

VLO 16-4-20A9- 1 K3 ; light chain; CDR3-Kabat QQFDDLPVT

SEQ ID NO: 22

VLO 16-4-20A9- 1 K3 ; light chain; CDR1-IMGT QDINKF

SEQ ID NO: 23

VLO 16-4-20A9- 1 K3 ; light chain; CDR2-IMGT DAT

SEQ ID NO: 24

VLO 16-4-20A9- 1 K3 ; light chain; CDR3-IMGT QQFDDLPVT

SEQ ID NO: 25

VLO 16-9-7F 10- 1 G 1 ; heavy chain; CDRl-Kabat SHAMS

SEQ ID NO: 26

VLO 16-9-7F 10- 1 G 1 ; heavy chain; CDR2-Kabat SISGGGVSTYYADSVKG

SEQ ID NO: 27

VLO 16-9-7F 10- 1 G 1 ; heavy chain; CDR3-Kabat GIGLNWNYVGS

SEQ ID NO: 28 VLO 16-9-7F10-1G1 ; heavy chain; CDR1-IMGT GFMFDSHA

SEQ ID NO: 29

VLO 16-9-7F10-1G1 ; heavy chain; CDR2-IMGT ISGGGVST

SEQ ID NO: 30

VLO 16-9-7F10-1G1 ; heavy chain; CDR3-IMGT SRGIGLNWNYVGS

SEQ ID NO: 31

VLO 16-9-7F 10- 1 R1 ; light chain; CDRl -Rabat QASQDISKFLN

SEQ ID NO: 32

VLO 16-9-7F 10- 1 R1 ; light chain; CDR2 -Rabat DASNVET

SEQ ID NO: 33

VLO 16-9-7F 10- 1 K1 ; light chain; CDR3 -Rabat QQFDVLPVT

SEQ ID NO: 34

VLO 16-9-7F 10- 1 R1 ; light chain; CDR1 -IMGT QDISRF

SEQ ID NO: 35

VLO 16-9-7F 10- 1 R1 ; light chain; CDR2-IMGT DAS

SEQ ID NO: 36

VLO 16-9-7F 10- 1 R1 ; light chain; CDR3-IMGT QQFDVLPVT

SEQ ID NO: 37

VL016-6-11C1 1-30G2; heavy chain; CDRl-Rabat RYVIS

SEQ ID NO: 38

VL016-6-11C1 1-30G2; heavy chain; CDR2-Rabat RILPRHDIRNYAQRF QG

SEQ ID NO: 39

VL016-6-11C1 1-30G2; heavy chain; CDR3-Rabat HYDS SGYNMEGGRYWYFDL

SEQ ID NO: 40

VL016-6-11C11 -30G2; heavy chain; CDR1-IMGT GDTFNRYV

SEQ ID NO: 41

VL016-6-11C11 -30G2; heavy chain; CDR2-IMGT ILPRHDIR

SEQ ID NO: 42 VL016-6-11C11 -30G2; heavy chain; CDR3-IMGT ARH YD S S GYNMEGGRY W YFDL

SEQ ID NO: 43

VL016-6-11C1 1-30L5; light chain; CDRl-Kabat SGGDSNIGSNYIS

SEQ ID NO: 44

VL016-6-11C1 1-30L5; light chain; CDR2-Kabat ENDDRPS

SEQ ID NO: 45

VL016-6-11C1 1-30L5; light chain; CDR3-Kabat GAWDTTLTAMV

SEQ ID NO: 46

VL016-6-11C1 1-30L5; light chain; CDR1-IMGT DSNIGSNY

SEQ ID NO: 47

VL016-6-11C1 1-30L5; light chain; CDR2-IMGT END

SEQ ID NO: 48

VL016-6-11C1 1-30L5; light chain; CDR3-IMGT GAWDTTLTAMV

SEQ ID NO: 49

VL016-4-11D11-Gl ; heavy chain; CDRl-Kabat TYVIS

SEQ ID NO: 50

VL016-4-11D11-Gl ; heavy chain; CDR2-Kabat RIIPRLGITNYAQKF QG

SEQ ID NO: 51

VL016-4-11D11-Gl ; heavy chain; CDR3-Kabat HYDS SGYNMEGGRYWYFDL

SEQ ID NO: 52

VL016-4-11D1 1-Gl ; heavy chain; CDR1 -IMGT GDTFRTYV

SEQ ID NO: 53

VL016-4-11D1 1-Gl ; heavy chain; CDR2-IMGT IIPRLGIT

SEQ ID NO: 54

VLO 16-4-11D1 1-Gl ; heavy chain; CDR3-IMGT ARH YD S S GYNMEGGRY W YFDL

SEQ ID NO: 55

VL016-6-4- 11D1 1-L16; light chain; CDRl-Kabat SGSDSNIGNNYIS

SEQ ID NO: 56 VL016-6-4- 11D1 1-L16; light chain; CDR2-Kabat ENNDRPS

SEQ ID NO: 57

VL016-6-4- 11D1 1-L16; light chain; CDR3-Kabat GTWDSTLTAVL

SEQ ID NO: 58

VL016-6-4- 11D1 1-L16; light chain; CDR1-IMGT DSNIGNNY

SEQ ID NO: 59

VL016-6-4- 11D1 1-L16; light chain; CDR2-IMGT ENN

SEQ ID NO: 60

VL016-6-4- 11D1 1-L16; light chain; CDR3-IMGT GTWDSTLTAVL

SEQ ID NO: 61

VLO 16-9-6E5-P4G4; heavy chain; CDRl -Kabat TYVIS

SEQ ID NO: 62

VLO 16-9-6E5-P4G4; heavy chain; CDR2 -Rabat RIIPRHEITNNAQKF QG

SEQ ID NO: 63

VLO 16-9-6E5-P4G4; heavy chain; CDR3-Kabat HHDS SGY QMEGGRYWYFDL

SEQ ID NO: 64

VLO 16-9-6E5-P4G4; heavy chain; CDR1 -IMGT GDIFSTYV

SEQ ID NO: 65

VLO 16-9-6E5-P4G4; heavy chain; CDR2-IMGT IIPRHEIT

SEQ ID NO: 66

VLO 16-9-6E5-P4G4; heavy chain; CDR3-IMGT AS HHDS SGY QMEGGRYWYFDL

SEQ ID NO: 67

VL016-9-6E5-L1 ; light chain; CDRl-Kabat SGSDSNIGNSHVS

SEQ ID NO: 68

VL016-9-6E5-L1 ; light chain; CDR2-Kabat ANDKRPS

SEQ ID NO: 69

VL016-9-6E5-L1 ; light chain; CDR3-Kabat GT WDHTP SAW

SEQ ID NO: 70 VL016-9-6E5-L1 ; light chain; CDR1-IMGT DSNIGNSH

SEQ ID NO: 71

VL016-9-6E5-L1 ; light chain; CDR2-IMGT AND

SEQ ID NO: 72

VL016-9-6E5-L1 ; light chain; CDR3-IMGT GT WDHTP SAW

SEQ ID NO: 73

VL016-6-28A2-3G15; heavy chain; CDRl-Kabat SGAYYWA

SEQ ID NO: 74

VL016-6-28A2-3G15; heavy chain; CDR2-Kabat YIYNTGRPYYNPSLKS

SEQ ID NO: 75

VL016-6-28A2-3G15; heavy chain; CDR3-Kabat DGGLYGLDV

SEQ ID NO: 76

VL016-6-28A2-3G15; heavy chain; CDR1-IMGT GDSVSSGAYY

SEQ ID NO: 77

VL016-6-28A2-3G15; heavy chain; CDR2-IMGT IYNTGRP

SEQ ID NO: 78

VL016-6-28A2-3G15; heavy chain; CDR3-IMGT ARDGGLY GLDV

SEQ ID NO: 79

VL016-6-28A2-3K4; light chain; CDRl-Kabat RASQSVSTSLA

SEQ ID NO: 80

VL016-6-28A2-3K4; light chain; CDR2-Kabat DASNRAT

SEQ ID NO: 81

VL016-6-28A2-3K4; light chain; CDR3-Kabat QQRSNWPPT

SEQ ID NO: 82

VL016-6-28A2-3K4; light chain; CDR1-IMGT QSVSTS

SEQ ID NO: 83

VL016-6-28A2-3K4; light chain; CDR2-IMGT DAS

SEQ ID NO: 84 VL016-6-28A2-3K4; light chain; CDR3-IMGT QQRSNWPPT

SEQ ID NO: 85

VLO 16-9-32E3-G3 ; heavy chain; CDRl-Kabat SGPYYWS

SEQ ID NO: 86

VLO 16-9-32E3-G3 ; heavy chain; CDR2-Kabat YIYYTGRPYYNPSLKS

SEQ ID NO: 87

VLO 16-9-32E3-G3 ; heavy chain; CDR3-Kabat DGGLYGLDV

SEQ ID NO: 88

VLO 16-9-32E3-G3 ; heavy chain; CDR1-IMGT GDSVTSGPYY

SEQ ID NO: 89

VLO 16-9-32E3-G3 ; heavy chain; CDR2-IMGT IYYTGRP

SEQ ID NO: 90

VLO 16-9-32E3-G3 ; heavy chain; CDR3-IMGT ARDGGLY GLDV

SEQ ID NO: 91

VL016-6-9-32E3-K1 ; light chain; CDRl-Kabat RASQSVSTSLA

SEQ ID NO: 92

VL016-6-9-32E3-K1 ; light chain; CDR2-Kabat DASKRAT

SEQ ID NO: 93

VL016-6-9-32E3-K1 ; light chain; CDR3-Kabat QQRSNWPPT

SEQ ID NO: 94

VL016-6-9-32E3-K1 ; light chain; CDR1-IMGT QSVSTS

SEQ ID NO: 95

VL016-6-9-32E3-K1 ; light chain; CDR2-IMGT DAS

SEQ ID NO: 96

VL016-6-9-32E3-K1 ; light chain; CDR3-IMGT QQRSNWPPT

SEQ ID NO: 97

VLO 16-16-4A2 -P2G5 ; heavy chain; CDRl-Kabat GDSYYWG

SEQ ID NO: 98 VLO 16-16-4A2 -P2G5 ; heavy chain; CDR2-Kabat YIYHSGNINYNPSLKS

SEQ ID NO: 99

VLO 16-16-4A2 -P2G5 ; heavy chain; CDR3-Kabat IFGGSVFDY

SEQ ID NO: 100

VLO 16-16-4A2 -P2G5 ; heavy chain; CDR1-IMGT GGSVSGDSYY

SEQ ID NO: 101

VLO 16-16-4A2 -P2G5 ; heavy chain; CDR2-IMGT IYHSGNI

SEQ ID NO: 102

VLO 16-16-4A2 -P2G5 ; heavy chain; CDR3-IMGT ARIF GGS VFDY

SEQ ID NO: 103

VLO 16-16-4A2 -P2K3 ; light chain; CDRl-Kabat RASQSIDIWLA

SEQ ID NO: 104

VLO 16-16-4A2 -P2K3 ; light chain; CDR2-Kabat KASTLE

SEQ ID NO: 105

VLO 16-16-4A2 -P2K3 ; light chain; CDR3-Kabat QQSDTFPWT

SEQ ID NO: 106

VLO 16-16-4A2 -P2K3 ; light chain; CDR1-IMGT QSIDIW

SEQ ID NO: 107

VLO 16-16-4A2 -P2K3 ; light chain; CDR2-IMGT KAS

SEQ ID NO: 108

VLO 16-16-4A2 -P2K3 ; light chain; CDR3-IMGT QQSDTFPWT

SEQ ID NO: 109

VLO 16-16-26E8 -G3 ; heavy chain; CDRl-Kabat SPHYHWS

SEQ ID NO: 110

VLO 16-16-26E8 -G3 ; heavy chain; CDR2-Kabat YIQYSRTTKQNPSFRS

SEQ ID NO: 11 1

VLO 16-16-26E8 -G3 ; heavy chain; CDR3-Kabat VFGDSVFDS

SEQ ID NO: 112 VLO 16-16-26E8 -G3 ; heavy chain; CDR1-IMGT GGSVSSPHYH

SEQ ID NO: 113

VLO 16-16-26E8 -G3 ; heavy chain; CDR2-IMGT IQYSRTT

SEQ ID NO: 114

VLO 16-16-26E8 -G3 ; heavy chain; CDR3-IMGT ARVFGDSVFDS

SEQ ID NO: 115

VL016-16-26E8-K1 ; light chain; CDRl-Kabat RASQSIDIWVA

SEQ ID NO: 116

VL016-16-26E8-K1 ; light chain; CDR2-Kabat KASSLES

SEQ ID NO: 117

VL016-16-26E8-K1 ; light chain; CDR3-Kabat QQYNTFPWT

SEQ ID NO: 118

VL016-16-26E8-K1 ; light chain; CDR1-IMGT QSIDIW

SEQ ID NO: 119

VL016-16-26E8-K1 ; light chain; CDR2-IMGT KAS

SEQ ID NO: 120

VL016-16-26E8-K1 ; light chain; CDR3-IMGT QQYNTFPWT

SEQ ID NO: 121

VLO 16-16-31 C9-P 1 G2 ; heavy chain; CDRl -Kabat SPHYHWS

SEQ ID NO: 122

VLO 16-16-31 C9-P 1 G2 ; heavy chain; CDR2-Kabat YIQYRRTTKQNPSFGS

SEQ ID NO: 123

VLO 16-16-31 C9-P 1 G2 ; heavy chain; CDR3 -Rabat VFGDSVFDS

SEQ ID NO: 124

VLO 16-16-31 C9-P 1 G2 ; heavy chain; CDR1 -IMGT GGSVSSPHYH

SEQ ID NO: 125

VLO 16-16-31 C9-P 1 G2 ; heavy chain; CDR2-IMGT IQYRRTT

SEQ ID NO: 126 VLO 16-16-31 C9-P 1 G2 ; heavy chain; CDR3-IMGT ARVFGDSVFDS

SEQ ID NO: 127

VL016-16-31C9-P1K2; light chain; CDRl-Kabat RASQNIDIWVA

SEQ ID NO: 128

VL016-16-31C9-P1K2; light chain; CDR2-Kabat KASSLES

SEQ ID NO: 129

VL016-16-31C9-P1K2; light chain; CDR3-Kabat QQYNSFPWT

SEQ ID NO: 130

VL016-16-31C9-P1K2; light chain; CDR1-IMGT QNIDIW

SEQ ID NO: 131

VL016-16-31C9-P1K2; light chain; CDR2-IMGT KAS

SEQ ID NO: 132

VL016-16-31C9-P1K2; light chain; CDR3-IMGT QQYNSFPWT

SEQ ID NO: 133

VL016-8-9F5-G1 ; heavy chain; CDRl-Kabat DYSMS

SEQ ID NO: 134

VL016-8-9F5-G1 ; heavy chain; CDR2-Kabat FIRNKV Y GGTTE Y AAS VKG

SEQ ID NO: 135

VL016-8-9F5-G1 ; heavy chain; CDR3-Kabat HIGYCSSTSCYNPNYFDN

SEQ ID NO: 136

VL016-8-9F5-G1 ; heavy chain; CDR1-IMGT GFTLGDYS

SEQ ID NO: 137

VL016-8-9F5-G1 ; heavy chain; CDR2-IMGT IRNKVYGGTT

SEQ ID NO: 138

VL016-8-9F5-G1 ; heavy chain; CDR3-IMGT TRHIGY CSSTSCYNPNYFDN

SEQ ID NO: 139

VL016-68-9F5-K4; light chain; CDRl-Kabat QASQVISNYLN

SEQ ID NO: 140 VL016-68-9F5-K4; light chain; CDR2-Kabat

GASNLET

SEQ ID NO: 141

VL016-68-9F5-K4; light chain; CDR3-Kabat

QQYDTLPPT

SEQ ID NO: 142

VL016-68-9F5-K4; light chain; CDR1-IMGT

QVISNY

SEQ ID NO: 143

VL016-68-9F5-K4; light chain; CDR2-IMGT

GAS

SEQ ID NO: 144

VL016-68-9F5-K4; light chain;CDR3-IMGT

QQYDTLPPT

SEQ ID NO: 145

VL016-4-6D2-P3G2; variable region heavy chain

QVQLVESGGGWQPGRSLRISCAASGFTFSSYAMHWVRQAPGRGLEWVTFLSHDGNIKNYA DSVKGRFTI

SRDNSENTLYLQMNSLRPDDTALYYCAVRDPDGFDVWGQGTMVTVSS

SEQ ID NO: 146

VL016-4-6D2-P2L2; variable region light chain

QSALTQPASVSGSPGQSITISCAGTSSDVGAYNYVSWYQQHPGNAPKLIIYDVSNRPSGF SNRFSGSKSGNA

ASLTISGLQTEDEADYYCSSYTNRRTWVFGGGTKVTVL

SEQ ID NO: 147

VL016-4-20A9-1G1 ; variable region heavy chain

EVQLLESGGVLIQPGRSLRLSCAASGFMFDSHAMSWVRQAPGMGLEWVSSISGGGVSTYY ADSVKGRFTIS

RDNSKNALYLDMSSLTAEDTAVYFCSKGIGLNWNYVGSWGQGTLVTVSS

SEQ ID NO: 148

VL016-4-20A9-1K3; variable region light chain

DIQMTQSPSSLSASVGDRVTITCQASQDINKFVNWHQQKPGKAPQLLIYDATNVETGVPS RFSGSGSGTHFT

LTISSLQPEDVATYYCQQFDDLPVTFGGGTKVEIK

SEQ ID NO: 149

VL016-9-7F10-1G1 ; variable region heavy chain

EVQLLESGGVLIQPGRSLRLSCAASGFMFDSHAMSWVRQAPGMGLEWVSSISGGGVSTYY ADSVKGRFTIS RDN S KNAL YLDMS S LTAEDT AVYFCS KGIGLN WN YV GS W GQGTL VT V S S

SEQ ID NO: 150

VL016-9-7F10-1K1 ; variable region light chain

DIQMTQSPSSLSASVGDRVSFTCQASQDISKFLNWHQQKPGKAPQLLIYDASNVETGVSS RFIGSGSGTHFT

LTINNLQPEDSATYYCQQFDVLPVTFGGGTKVEIK

SEQ ID NO: 151

VL016-6-11C1 1-30G2; variable region heavy chain

QVQLVQSGAEVKKPGSSVKVSCKASGDTFNKYVISWVRQAPGQGLEWMGRILPRHDIRNY AQRFQGRVTI

NADKSTGTAYMELSSLRFDDTAVYYCARHYDSSGYNMEGGRYWYFDLWGRGTLVTVS S

SEQ ID NO: 152

VLO 16-6- 11 C 1 1 -30L5 QSVLTQPPSVSAAPGQKVTISCSGGDSNIGSNYISWYQQIPGTAPRLIIYENDDRPSGIP DRFSGSKSGTSATL GIT GLQT GDE AD YY CG A WDTTLT AM VF GGGTKLT VL

SEQ ID NO: 153

VLO 16-4-11D1 1-Gl ; variable region heavy chain

QVQLVQSGAEVKKPGSSVKVSCTASGDTFRTYVISWVRQAPGQGLEWMGRIIPRLGITNY AQKFQGRVTL

TADKSTSTGYMELSSLRFEDSAVYYCARHYDSSGYNMEGGRYWYFDLWGRGTLVTVS S

SEQ ID NO: 154

VL016-6-4- 11D1 1-L16; variable region light chain

QSVLTQPPSVSAAPGQKVTISCSGSDSNIGNNYISWYQHFPGTAPKLLIYENNDRPSGIP DRFSGSKSGTSATL AIT GLQT GDE AT YY CGT WDS TLTA VLF GGGTKLT VL

SEQ ID NO: 155

VL016-9-6E5-P4G4; variable region heavy chain

QVQLMQSGPEVKKPGSSVKISCEASGDIFSTYVISWVRQSPGQGLEWMGRIIPRHEITNN AQKFQGRVTIIAD

KSTSTVYMELSSLRFEDTAVYYCASHHDSSGYQMEGGRYWYFDLWGRGTLVTVSS

SEQ ID NO: 156

VL016-9-6E5-L1 ; variable region light chain

QSVLTQPPSVSAAPGQKVTISCSGSDSNIGNSHVSWYQQFPGTAPKLLIYANDKRPSGVS GRFSGSKSGTSA SLGIT GLQTGDEAD YF CGT WDHTP S A WF GGGTKLT VL

SEQ ID NO: 157

VL016-6-28A2-3G15; variable region heavy chain

QVQLQGSGPGLVKPSQTLSLTCTVSGDSVSSGAYYWAWIRQHSGKGLEWIGYIYNTGRPY YNPSLKSGVSI

SIDTSQNHFSLNLSSVTAADTAVYYCARDGGLYGLDVWGRGTPVTVSS

SEQ ID NO: 158

VL016-6-28A2-3K4; variable region light chain

EIVLTQSPATLSLSPGERATLSCRASQSVSTSLAWYLQKPGQAPRLLIYDASNRATGIPT RFSGSGSGTDFTLT IS SLEAEDF WYYCQQRSNWPPTF GQGTKVEIK

SEQ ID NO: 159

VL016-9-32E3-G3; variable region heavy chain

QVQLQDSGPGLVKPSQTLSLTCTVSGDSVTSGPYYWSWIRQHPGKGLEWIGYIYYTGRPY YNPSLKSRVSIS VDTSQNQFSLNLSSVTAADTAVYY CARDGGLY GLDVWGRGTPVTVSS

SEQ ID NO: 160

VL016-6-9-32E3-K1 ; variable region light chain

ENVLTQSPVTLSLSPGERATLSCRASQSVSTSLAWYQQKPGQAPRLLIYDASKRATGIPA RFSASGSGTDFTL TIS SLEPEDF AVYY CQQRSNWPPTF GRGTKVEIK

SEQ ID NO: 161

VL016-16-4A2-P2G5; variable region heavy chain

QVQLQESGPGLVKPSETLSLTCSVSGGSVSGDSYYWGWIRQTPGKGLECLGYIYHSGNIN YNPSLKSRVTM

SKDTSKNQFSLKLTSVTAADTALYYCARIFGGSVFDYWGPGTWSVSS

SEQ ID NO: 162

VL016-16-4A2-P2K3; variable region light chain

DIQMTQSPSTLSASVGDRVTITCRASQSIDIWLAWYQQIPGKAPKFLIYKASTLESGVPS RFSGSGSGTDFTLT

ISSLQPDDFATYYCQQSDTFPWTFGQGTKVEIK

SEQ ID NO: 163

VL016-16-26E8-G3; variable region heavy chain QVQLQESGPGLVKPSETLSLICTVSGGSVSSPHYHWSWIRQSPGKGLEWIGYIQYSRTTK QNPSFRSRVTISV

DTSKNQFSLELKSVTAADTAIYYCARVFGDSVFDSWGQGTLVSVSS

SEQ ID NO: 164

VL016-16-26E8-K1 ; variable region light chain

DIQMTQSPSTESASVGDRVTITCRASQSIDIWVAWYQQKAGKAPKFEIYKASSEESGVPS RFRGSGSGTEFTE

TISGLQLDDFATYYCQQYNTFPWTFGQGTKVEIN

SEQ ID NO: 165

VLO 16-16-31 C9-P 1 G2 ; variable region heavy chain

QVQLQESGPGLVKPSETLSLICTVSGGSVSSPHYHWSWIRQSPGKGLEWIAYIQYRRTTK QNPSFGSRVTISA

DTSKNQFSLELKSVTAADTAIYYCARVFGDSVFDSWGQGTLVSVSS

SEQ ID NO: 166

VLO 16-16-31 C9-P 1 K2 ; variable region light chain

DIQMTQSPSTLSASVGDRVTITCRASQNIDIWVAWYQQKAGKAPKFLIYKASSLESGVPS RFRGSGSGTEFT

LTISSLQLDDFATYYCQQYNSFPWTFGQGTKVEIN

SEQ ID NO: 167

VL016-8-9F5-G1 ; variable region heavy chain

EVQLVESGGGLVQPGRSLRLSCTASGFTLGDYSMSWIRQAPGKGLEWIGFIRNKVYGGTT EYAASVKGRFS

ISRDDTKSIAYLQMNSLQTEDTAVYYCTRHIGYCSSTSCYNPNYFDNWGQGTLVIVS S

SEQ ID NO: 168

VL016-68-9F5-K4; variable region light chain

DIQMTQSPSSLSASVGDRVTITCQASQVISNYLNWYQQKPGRAPQLLIYGASNLETGVPS RFSGSGSGTDFT

FTISSLQPEDIATYYCQQYDTLPPTFGPGTKVDIK

SEQ ID NO: 169

hcmvIL-10: Genbank Accession No. AAF63437

MLS VMVSS SLVLIVFFLGASEEAKP ATTTTIKNTKPQCRPEDY ATRLQDLRVTFHRVKPTLQREDDY S V WL DGTWKGCWGCSVMDWLLRRYLEIVFPAGDHVYPGLKTELHSMRSTLESIYKDMRQCPLLG CGDKSVISR LS QE AERKS DNGTRKGLS ELDTLF S RLEE YLHS RK

SEQ ID NO: 170

Latency-associated cmvIL-10 (LAcmvIL-10); Genbank Accession No. ACR49217

MLSVMVSSSLVLIVFFLGASEEAKPATTTIKNTKPQCRPEDYATRLQDLRVTFHRVKPTL QREDDYSVWLD

GTWKGCWGCSVMDWLLRRYLEIVFPAGDHVYPGLKTELHSMRSTLESIYKDMRQCVS VSVAALSAQR

SEQ ID NO: 171

Macacine cmvIL-10 (RhcmvIL-10); Genbank Accession No. AAF59907

MRRRRRSFGIIVAGAIGTLLMMAVWLSAHDHEHKEVPPACDPVHGNLAGIFKELRATYAS IREGLQKKDT

VYYTSLFNDRVLHEMLSPMGCRVTNELMEHYLDGVLPRASHLDYDNSTLNGLHVFAS SMQALYQHMLK

CPALACTGKTPAWMYFLEVEHKLNPWRGTAKAAAEADLLLNYLETFLLQF

SEQ ID NO: 172

Interleukin 10, Homo sapiens (human cellular IL-10; hIL-10); Genbank Accession No. AAI04253

MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQM KDQLDNLLLKESL LEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFL PCENKSKAVEQ VKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

SEQ ID NO: 173

Heavy chain constant hu-IgGl

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVP

SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCV

WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDG

SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 174

Light chain constant hu-kappa

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSST

LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 175

Light chain constant hu-lambda

SQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSK QSNNKYAASSY

LSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

SEQ ID NO: 176

Light chain constant hu-lambda used only for VL016-4-6D2-P2L2

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSK QSNNKYAASSY

LSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS