CD163 binding proteins

Abstract

The present disclosure provides an antibody that binds to porcine CD163 wherein the antibody binds to porcine CD163 in a cell membrane binding form and does not significantly bind to porcine CD163 in a soluble form. The disclosure also provides combinations of the antibodies with one or more other anti-porcine CD163 antibodies or binding proteins. A preferred combination is a combination in which each antibody or binding protein binds to a different epitope in porcine CD163, and wherein the combination of anti-porcine CD163 antibodies or binding proteins is co-present in a single construct. Nucleic acid molecules, expression vectors and compositions are also provided.

Description

[0001] The present invention generally relates to the field of binding proteins that bind to CD163 (cluster of differentiation 163), particularly antibodies, and more particularly to binding proteins and antibodies that bind to membrane-bound porcine CD163 and do not significantly bind to the soluble form of porcine CD163. Such anti-CD163 binding proteins and antibodies have therapeutic and prophylactic uses, particularly when used in combination with other anti-CD163 binding proteins, for example in the treatment or prevention of infections such as porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) infection, for example to reduce its incidence and severity. Binding proteins and antibody-based compositions, methods, and kits are also provided.

[0002] Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating swine viral diseases worldwide, causing huge economic losses to the swine industry. The pathogen is PRRSV, an enveloped RNA virus belonging to the family Arteriviridae, order Nidovirales. PRRSV has a restricted host and cell tropism, with porcine alveolar macrophages (PAM) being important target cells. Clinical symptoms are diverse but include respiratory distress and respiratory diseases in piglets and young pigs, late-term abortions and stillbirths in gilts and sows, fetal resorption in early pregnancy, reduced viability of surviving piglets, and growth retardation in finishing pigs. Due to reduced pregnancy or abortions, piglet deaths, and decreased growth rates in all pigs infected with PRRSV, it is estimated that pork producers in the United States alone lose more than $650 million annually (Holtkamp et al., 2013, Journal of Swine Health and Production, 21(2)72 - 84). A 2021 study of PRRSV-endemic farms in Germany (Renken et al., 2021, Porcine Health Management, Jan 4; 7(1):3) calculated an average loss of €74,181 per farm (€255 per sow).

[0003] All currently known PRRSV isolates belong to one of two species: PRRSV-1 or PRRSV-2. Although both cause persistent infections and produce similar clinical symptoms, their nucleotide sequence homology is only about 60%. PRRSV-1 was initially discovered in Europe and is common in European PRRSV isolates or strains; while PRRSV-2 was first discovered in North America and is more common in Asian or American isolates or strains (see the review by Stoian and Rowland in Vet. Sci., 6, 9 in 2019).

[0004] In each species, there is significant diversity, and a large number of strains / subtypes have been identified, including new highly pathogenic strains that have emerged since 2006, especially in China and Vietnam. Similar highly pathogenic strains have also emerged in other regions, with a distribution range extending from the Malay Peninsula to southern Russia, posing an increasing threat to pig populations (An et al., 2011, Emerging Infect Dis 17(9):1782). In China alone, more than 20 million pigs were culled annually due to PRRSV infection between 2006 and 2007 (An et al., 2010, Emerging Infect Dis 16(2):365). Recently, reports of cases of highly virulent strains in Europe have indicated an increasing threat from PRRSV (Sinn et al., 2016, Porcine Health Management(2):28).

[0005] The scavenger receptor CD163 is a key entry mediator for PRRSV infection and thus plays a crucial role in PRRSV infection. CD163 is a 130 kDa type I transmembrane protein that has a signal peptide followed by 9 scavenger receptor cysteine-rich (SRCR) domains, each approximately 100 amino acids long, with a proline-serine-threonine (PST)-rich region (PST-1) of 35 amino acids that separates SRCR domain 6 (SRCR6) and SRCR7. A second PST-rich region (PST-2) links SRCR9 to the transmembrane domain and a short cytoplasmic tail that contains a functional internalization motif. Surface expression of CD163 is restricted to cells of the monocyte-macrophage lineage. The SRCR5 domain of CD163 has been shown to play an important role in PRRSV infection of porcine alveolar macrophages (Gorp et al., 2010, J. of Virology, March, 3101-3105).

[0006] The mechanism of entry of PRRSV is not yet clear. However, as part of this mechanism, PRRSV is thought to enter the endosomal compartment of the cell. Here, CD163 interacts with the GP2-GP3-GP4 heterotrimer of PRRSV, mediating viral uncoating and release of the viral genome into the cytoplasm.

[0007] A proposed treatment for PRRSV involves some form of gene knockout or gene editing of CD163 to render pigs resistant to PRRSV infection, followed by breeding these pigs to spread the genetic modification (Burkard et al., 2017, PLOS Pathogens 13(2):e1006206). Although this approach has proven to be very effective, implementing this therapy across a large portion of the swine population would be extremely complex and time-consuming. Additionally, and importantly, there is strong resistance in many markets to technologies involving animal genetic modification, such as the acceptability of animal products derived from such animals being highly controversial.

[0008] The most common medical intervention used to limit the economic impact of PRRS is vaccination. Vaccines are used in all regions where the disease is endemic. However, for safety reasons, they are only used in specific scenarios. The vaccines used are of two types, inactivated vaccines or (in most cases) live attenuated vaccines (MLV). However, currently the vaccines are only partially effective and their greatest value is seen when deployed in an integrated disease management approach that is tightly coupled with biosecurity and husbandry management decisions. The reasons for vaccine ineffectiveness are complex, but due to the high genetic diversity of the PRRSV population, combined with the biological properties of the virus (alveolar macrophage tropism and high variability), the best results are seen when the vaccine strain and the circulating strain are highly matched in terms of immunogenicity (review by Nan et al. in Front. Immunol. 8:1635 in 2017). Additionally, live vaccine strains can recombine with wild strains, generating new wild strains that may be pathogenic. Therefore, MLV can only be used in specific situations, which further limits its use.

[0009] There is currently no effective antiviral treatment for PRRSV infection.

[0010] Recently, alternative treatment or prevention options in the form of binding proteins and antibodies against porcine CD163 have been developed that can reduce or prevent PRRSV infection.

[0011] However, there is still a need to develop alternative and superior treatment and prevention options for PRRSV infection (or other CD163-mediated infections) so that they can be easily used to treat or prevent infections in large numbers of animals.

[0012] The present invention provides an alternative treatment or prophylaxis in the form of an antibody capable of binding to porcine CD163 in its cell membrane-bound form, which antibody has the advantage of not significantly binding to the soluble form of porcine CD163. Such antibodies (or binding proteins comprising such antibodies) are sometimes referred to herein as membrane-specific CD163 antibodies (or binding proteins). Such antibodies (or binding proteins) are capable of targeting only the membrane-bound form of CD163. In other words, they do not target or do not significantly target the soluble form of CD163. This advantage is relevant because the soluble form of CD163 is cleaved from the cell surface and high concentrations of the soluble form of CD163 can be found in serum during some infections in pigs. Thus, the soluble form of CD163 has the potential to act as a reservoir for therapeutic anti-CD163 antibodies, thereby interfering with their efficacy. However, the antibodies of the present invention, being able to distinguish or discriminate between the membrane-bound (cell surface) form and the soluble form of CD163, should not be interfered with by such soluble (shed) antigen, but rather target cells expressing CD163 - the same cells that PRRSV targets during infection. Indeed, the antibodies of the present invention have been shown to be resistant to such soluble forms of CD163, for example, compositions have been shown to be highly effective at inhibiting PRRSV infection in the presence of soluble CD163.

[0013] In particular, the antibodies of the present invention have been shown to have excellent activity in inhibiting PRRSV infection when combined with other CD163 antibodies. Without being bound by theory, it is believed that the antibodies of the present invention can assist other CD163 antibodies in targeting cells expressing CD163, thereby enabling a reduction or blockade of PRRSV infection in an efficient manner.

[0014] When two anti-CD163 antibodies are paired together (e.g., one antibody of the present invention paired with a second, different anti-CD163 antibody), excellent effects can be observed. Such antibodies are sometimes referred to herein as bi-paratopic anti-CD163 antibodies. These bi-paratopic constructs exhibit good inhibitory effects against PRRSV-1 infection, although the effect is reduced in the presence of high concentrations of soluble CD163, and also have some inhibitory effect on PRRSV-2 infection. When three anti-CD163 antibodies are paired together (i.e., one antibody of the present invention paired with a second and a third, different anti-CD163 antibody), even better effects can be observed. Such antibodies are sometimes referred to herein as tri-paratopic anti-CD163 antibodies. Advantageously, the tri-paratopic constructs of the present invention are capable of extremely effectively inhibiting PRRSV-1 and PRRSV-2 infections, even in the presence of high concentrations of soluble CD163, such as may be found in infected pigs described elsewhere herein, e.g., pigs suffering from viral or bacterial infections, e.g., non-PRRSV infections, e.g., Lawsonia intracellularis infection, or pigs suffering from complex (multi-pathogen) diseases, or pigs suffering from multiple (or mixed) infections, or pigs suffering from severe infections. Such pigs are frequently encountered in the field.

[0015] Accordingly, the antibodies and constructs of the present invention (e.g., other binding proteins comprising the CD163 antigen-binding domains described herein) are considered to provide a novel therapeutic molecule that can preferentially target the cell membrane-bound form of CD163, thereby providing a highly effective treatment option for treating PRRSV-1 and PRRSV-2 infections. Since such antibodies do not significantly bind to the soluble form of CD163, they also have the potential to function at lower doses, which is a further advantage both for the animals receiving treatment and from a cost perspective.

[0016] In one embodiment, the present invention provides a binding protein, such as an antibody, that binds to CD163 (e.g., porcine CD163), wherein the binding protein or antibody:

[0017] (i) binds to the cell membrane-bound form of CD163, such as porcine CD163; and

[0018] (ii) does not significantly bind to the soluble form of CD163, such as porcine CD163.

[0019] In one embodiment, the present invention provides a binding protein, such as an antibody, that binds to porcine CD163, wherein the binding protein or antibody:

[0020] (i) binds to porcine CD163 in a membrane-bound form; and

[0021] (ii) does not significantly bind to soluble porcine CD163.

[0022] As described in other parts of this document, the antibodies (or binding proteins) of the present invention that are preferred and suitable for the treatment methods described herein are capable of binding to the SRCR5 domain of CD163 (such as porcine CD163), for example, having an epitope (or a part of an epitope) in the SRCR5 domain of CD163. In addition, the preferred antibodies (or binding proteins) are capable of inhibiting PRRSV-2 infection.

[0023] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising at least one antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises a heavy-chain variable region, and the heavy-chain variable region comprises three complementarity-determining regions (CDRs), wherein the heavy-chain variable region comprises:

[0024] (i) a variable heavy-chain (VH) CDR1 comprising the amino acid sequence GRTFSSYA (SEQ ID NO: 2) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, or 3 amino acid substitutions compared to the given CDR sequence,

[0025] (ii) a variable heavy-chain (VH) CDR2 comprising the amino acid sequence IGWTGGTT (SEQ ID NO: 3) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, or 3 amino acid substitutions compared to the given CDR sequence; and

[0026] (iii) a variable heavy-chain (VH) CDR3 comprising the amino acid sequence AADQAGWRTAGVRNTYEYDY (SEQ ID NO: 4) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence.

[0027] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising at least one antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises a heavy-chain variable region, and the heavy-chain variable region comprises three complementarity-determining regions (CDRs), wherein the heavy-chain variable region comprises:

[0028] (i) a variable heavy-chain (VH) CDR1 comprising the amino acid sequence GRTFSSYA (SEQ ID NO: 2),

[0029] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence IGWTGGTT (SEQ ID NO:3), and

[0030] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence AADQAGWRTAGVRNTYEYDY (SEQ ID NO:4).

[0031] As described elsewhere herein, the antibody (or binding protein) preferably is capable of binding to the membrane-bound form of CD163, such as porcine CD163, on cells, but does not significantly bind to the soluble form of CD163, such as porcine CD163.

[0032] Certain preferred embodiments of the invention provide an antibody (or binding protein) that binds to CD163, such as porcine CD163, comprising a VH domain having or comprising the amino acid sequence of SEQ ID NO:1 or a sequence substantially homologous thereto. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises up to three light chain CDRs, preferably three light chain CDRs.

[0033] In a preferred embodiment, the invention provides an antibody (or binding protein) that binds to CD163, such as porcine CD163, comprising a VH domain having or comprising the amino acid sequence of SEQ ID NO:1 or a sequence having at least 70%, 75% or 80% sequence identity (such as at least 85%, 90%, 95% or 98% identity) thereto. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises up to three light chain CDRs, preferably three light chain CDRs.

[0034] In a preferred embodiment, the invention provides an antibody (or binding protein) that binds to CD163, such as porcine CD163, comprising a VH domain having or comprising the amino acid sequence of SEQ ID NO:1. In some embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises up to three light chain CDRs, preferably three light chain CDRs.

[0035] As described elsewhere herein, the antibody (or binding protein) preferably is capable of binding to the membrane-bound form of CD163, such as porcine CD163, on cells, but does not significantly bind to the soluble form of CD163, such as porcine CD163.

[0036] As described above, the present invention provides binding proteins, such as antibodies or binding proteins comprising an antigen-binding domain, which bind (or specifically recognize or specifically bind) to CD163 (preferably porcine CD163). CD163 is also known as M130, MM130, SCAR1, macrophage-associated antigen, hemoglobin scavenger receptor, or cysteine-rich scavenger receptor type 1 protein M130.

[0037] CD163 is a 130 kDa type I transmembrane protein that has a signal peptide followed by nine scavenger receptor cysteine-rich (SRCR) domains, each approximately 100 amino acids in length, with a proline-serine-threonine (PST)-rich region (PST-1) of 35 amino acids that separates SRCR domain 6 (SRCR6) and SRCR7. A second PST-rich region (PST-2) links SRCR9 to the transmembrane domain and a short cytoplasmic tail that contains a functional internalization motif. Surface expression of CD163 is restricted to cells of the monocyte-macrophage lineage.

[0038] Thus, the binding protein or antibody of the present invention binds or is capable of binding to CD163. Thus, the binding protein or antibody of the present invention is sometimes also referred to as an anti-CD163 binding protein or antibody. According to the present invention, CD163 can be from any species, such as any mammalian species, such as porcine, human, bovine, canine, feline, ovine, equine, murine, and simian. In a preferred embodiment, CD163 is porcine CD163, and the antibody (or binding protein) binds or is capable of binding to porcine CD163 (or specifically recognizes or specifically binds), and can be referred to, for example, as an anti-porcine CD163 antibody.

[0039] Particularly relevant to the present invention is that CD163 is expressed on the surface of porcine alveolar macrophages (PAM) and is thought to play a crucial role in the ability of various pathogens, including viral pathogens, especially PRRSV, to cause disease in pigs.

[0040] As described above, the present invention also provides an antibody or binding protein that binds to CD163 (preferably porcine CD163), preferably an isolated antibody or binding protein, wherein the antibody (or binding protein) binds to the cell membrane-bound form of CD163 and does not significantly bind to the soluble form of CD163. Such an antibody (or binding protein) of the present invention is sometimes also referred to as a membrane-specific antibody (or binding protein).

[0041] The term "membrane-bound form of CD163" or "cell membrane-bound form of CD163" or other equivalent terms refers to CD163 that is attached to, associated with, embedded in, or otherwise bound to the cell membrane, or is a component of the cell membrane. Thus, the membrane-bound form of CD163 can be referred to as, for example, the cell surface form of CD163 or the cell surface CD163 molecule or the cell surface-associated CD163 molecule, or the CD163 molecule expressed by the cell, or the full-length CD163 (e.g., including the transmembrane domain and optional cytoplasmic tail region). Thus, this membrane-bound form represents (or corresponds to) in many cases the native form of CD163, such as the form found on cells that naturally express or overexpress CD163.

[0042] Unless otherwise specified, in the context of the present invention, the term "cell" is used to refer to CD163-positive (expressing CD163) cells. In the context of the present invention, the term "cell" is used to refer to nucleated cells.

[0043] Suitable cell types that naturally express CD163 are well known to those skilled in the art and include monocytes and macrophages. A preferred cell type is porcine alveolar macrophages (PAM). Alternatively, CD163 can be expressed or overexpressed in a membrane-bound form, for example, by recombinant means (or other engineering means) in cell types that do not normally express CD163. In other words, cells expressing a recombinant membrane-bound form of CD163, such as full-length CD163, can be used.

[0044] Thus, in certain embodiments, the membrane-bound form of CD163 refers to the membrane-bound form of CD163 on PAM, and the antibodies (or binding proteins) of the present invention have the ability to bind to PAM, which is sometimes also referred to herein as porcine PAM (or pPAM). In other words, in certain embodiments, the membrane-bound form of CD163 is CD163 associated with PAM. In certain embodiments, the membrane-bound form of CD163 refers to the membrane-bound form of CD163 that has been transfected (and thus expresses or overexpresses) CD163, such as a recombinant form of CD163, preferably porcine CD163. Thus, antibodies (or binding proteins) that bind to recombinant cells expressing CD163 are also included. Suitable cells for transfection are well known to those skilled in the art and are described elsewhere herein, such as HEK293 and CHO cells. CD163-negative cells, i.e., cells that do not express CD163 prior to transfection, are typically used.

[0045] The preferred cell membrane-bound form of CD163 is the native membrane-bound form of 130 kDa or the corresponding recombinant form. Such membrane-bound forms of CD163 contain all CD163 regions except for the signal sequence (which is removed during intracellular processing and subsequent cell surface expression), for example, containing all nine SRCR domains as well as two PST-rich regions, a transmembrane region, and a cytoplasmic tail region.

[0046] The preferred membrane-bound form of CD163 on cells comprises (or consists of) amino acid residues 47 to 1044 of SEQ ID NO:42 (porcine CD163). Accordingly, the antibodies of the present invention preferably bind to this membrane-bound form of CD163 on cells. The antibodies of the present invention can bind to a membrane-bound form of CD163 corresponding to this membrane-bound form of CD163 on cells (e.g., different CD163 subtypes or CD163 of different species).

[0047] The antibodies of the present invention preferably bind to the SRCR5 domain of CD163, such as porcine CD163 (or bind to an epitope containing one or more residues in the SRCR5 domain). Accordingly, the preferred membrane-specific anti-CD163 binding protein or antibody of the present invention has the ability to bind to an epitope (or a part of an epitope) in the SRCR5 domain of CD163 or the SRCR5 domain (preferably the porcine SRCR5 domain).

[0048] Accordingly, in certain embodiments, the membrane-specific anti-CD163 antibodies of the present invention do not bind (or do not significantly bind) to CD163 molecules containing a deletion or mutation in the SRCR5 domain. Accordingly, in certain embodiments, the membrane-specific antibodies of the present invention do not bind (or do not significantly bind) to the PST-2 domain of CD163 (e.g., porcine CD163) or an epitope (or a part of an epitope) in the PST-2 domain.

[0049] The porcine CD163 form is preferably used to evaluate the binding ability of the antibodies of the present invention, although equivalent forms from other species (e.g., other mammalian species) can also be used, for example, for evaluating cross-reactivity.

[0050] CD163 sequences of various species are well-known and described in the art and can be obtained from, for example, various sequence databases (e.g., Uniprot). For convenience of reference, the Uniprot number of porcine CD163 is Q2VL90, which is reproduced below for reference.

[0051] The sequence of the porcine SRCR5 domain is shown below, corresponding to residues 477 - 577 of Uniprot Q2VL90: PRLVGGDIPCSGRVEVQHGDTWGTVCDSDFSLEAASVLCRELQCGTVVSLLGGAHFG EGSGQIWAEEFQCEGHESHLSLCPVAPRPDGTCSHSRDVGVVCS (SEQ ID NO:41).

[0052]

[0053] The ability of an antibody (or binding protein) to bind to CD163 in the form bound to the cell membrane (or CD163 expressed on the cell surface) can be easily detected by conventional methods well known in the art, and any suitable method can be used. For example, flow cytometry (such as FACS) can be used. In an exemplary flow cytometry method, cells expressing CD163 (such as PAM cells, such as pPAM cells, or cells expressing a recombinant form of CD163, such as cells transfected with CD163, such as full-length CD163, such as cells transfected with a construct containing all regions of CD163, such as the signal sequence, all 9 SRCR domains plus two PST-rich regions, the transmembrane region, and the cytoplasmic tail region) are incubated with the antibody (or binding protein) under study, and the antibody (or binding protein) bound to CD163 on the cells is detected by fluorescence, such as by fluorescently labeling the antibody. For example, such labeling can be carried out by incubating the cell-antibody mixture with a secondary antibody that recognizes the antibody under study (such as an anti-myc antibody if the antibody under study is myc-tagged) and another fluorescently labeled antibody (a third antibody) that recognizes the second antibody. Alternatively, the second antibody can also carry a fluorescent label. Thus, if the antibody (or binding protein) under study binds to CD163 in the form bound to the cell membrane, the cells are fluorescently labeled, and such cells, and thus the antibody (or binding protein) capable of binding to CD163 in the form bound to the cell membrane, can be easily identified using a flow cytometer. A particularly preferred flow cytometry assay for detecting the ability of an antibody (or binding protein) to bind to CD163 in the form bound to the cell membrane is described in the Examples.

[0054] In certain embodiments, PAM cells lacking the SRCR5 domain can be used for these binding assays (e.g., as described in Burkard et al., 2017, PLoS Pathogens, 13(2):e1006206) to evaluate whether the antibody (or binding protein) has the ability to bind to the SRCR5 domain.

[0055] Another method for testing the ability of an antibody (or binding protein) to bind to membrane-bound CD163 is immunohistochemistry. Another method for detecting the ability of an antibody (or binding protein) to bind to membrane-bound CD163 is microscopy (such as confocal microscopy), i.e., microscopy of cells fluorescently labeled due to the binding of the antibody (or binding protein) to membrane-bound CD163.

[0056] As described in other parts of this document, the antibodies (or binding proteins) of the present invention do not significantly bind (or do not bind) to the soluble form of CD163, such as porcine CD163.

[0057] The soluble form of CD163 refers to the form of CD163 that exists in solution or the soluble phase. Thus, this form of CD163 is not membrane-bound, does not exist in particulate form, nor in insoluble aggregates or precipitates. A preferred soluble form of CD163 is (or corresponds to) CD163 that is bound to the surface of a cell (such as a PAM or other macrophage or monocyte), and then converted to the soluble form by shedding or loss from the cell membrane, for example, by cleavage means such as proteolytic cleavage (such a form of CD163 can also be referred to as the "shed form" or the "shed form" of CD163). Thus, the soluble form of CD163 can be a form derived from the membrane-bound form by cleavage, for example, by natural cleavage occurring within the PST2 domain (possibly the cleavage site between residues HATG (residue 1041) and RSS).

[0058] A preferred soluble form of CD163 to which the antibodies of the present invention do not significantly bind is the soluble form of membrane-bound CD163 that has been shed (cleaved from the cell). Thus, the soluble form of CD163 can comprise (or consist of) the same primary amino acid sequence as (or contained within) the membrane-bound form of CD163, or can comprise (or consist of) a substantial portion (fragment) of the primary amino acid sequence of the membrane-bound form of CD163. For example, the soluble form of CD163 can comprise (or consist of) a sequence having at least 100, at least 200, at least 300, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 950 amino acids, which amino acid sequences correspond to the amino acid sequence of the membrane-bound form of CD163. Such forms typically comprise or consist of a fragment of the extracellular domain of CD163, for example, comprising the amino acid sequence from SRCR1-6, PST-1, SRCR7-9, and PST-2 in sequential order from the N-terminus to the C-terminus of the CD163 molecule, or a fragment consisting of or comprising amino acid residues 51 to 1044 or 51 to 1041 taken from SEQ ID NO:42. In some embodiments, the soluble form of CD163 has a corresponding sequence (such as a different CD163 subtype or CD163 from a different species).

[0059] In a preferred embodiment of the present invention, the soluble form of CD163 maintains the tertiary structure that naturally forms under physiological conditions, such as the physiological conditions present in a mammal (e.g., a pig). Thus, the soluble form of CD163 can be a form in which the native tertiary structure is present, i.e., for example, the soluble form of CD163 is not denatured. Thus, the soluble form of CD163 can be a non-denatured protein that retains its two-dimensional and / or three-dimensional structure. The two-dimensional and / or three-dimensional structure of the soluble form of CD163 can be a folded structure. Suitable soluble forms can also be prepared recombinantly, for example, they can be recombinant molecules or synthetic molecules.

[0060] A preferred soluble form of CD163 (e.g., a recombinant soluble form) comprises (or consists of) CD163-SRCR5-6, which corresponds to amino acid residues 477-682 of SEQ ID NO:42. In some embodiments, the soluble form of CD163 has a corresponding sequence (e.g., a different CD163 subtype or CD163 from a different species).

[0061] Another preferred soluble form of CD163 (e.g., a recombinant soluble form) comprises (or consists of) CD163-SRCR4-7, which corresponds to amino acid residues 372-818 of SEQ ID NO:42 and contains the PST-1 domain. In some embodiments, the soluble form of CD163 has a corresponding sequence (e.g., a different CD163 subtype or CD163 from a different species).

[0062] Another preferred soluble form of CD163 (e.g., a recombinant soluble form) comprises (or consists of) CD163-SRCR1-9, which corresponds to amino acid residues 51-1028 of SEQ ID NO:42. In some embodiments, the soluble form of CD163 has a corresponding sequence (e.g., a different CD163 subtype or CD163 from a different species).

[0063] Another preferred soluble form of CD163 (e.g., a recombinant soluble form) comprises (or consists of) CD163-SRCR1-PST2, which corresponds to amino acid residues 51-1044 or 51-1041 of SEQ ID NO:42. In some embodiments, the soluble form of CD163 has a corresponding sequence (e.g., a different CD163 subtype or CD163 from a different species).

[0064] The soluble (or shed) form of CD163 may be present in the blood of a subject (e.g., a pig) (serum CD163), but may also be present in the interstitial space of tissues. Thus, the soluble form of CD163 can occur naturally, or correspond to the naturally occurring form or native form of soluble CD163.

[0065] The soluble form of CD163 can be derived from any suitable source, i.e., any sample or source in which the soluble form of CD163 is present.

[0066] A suitable and preferred source is recombinant CD163 (e.g., recombinant porcine CD163). The relevant recombinant soluble form of CD163 (e.g., porcine CD163) can be readily prepared or synthesized using standard techniques, such as by using the sequence information provided herein and in the prior art. For example, a recombinant CD163 construct encoding the desired soluble form of CD163 (e.g., as described elsewhere herein) can be easily constructed and expressed in a suitable cell line, such that the soluble form of the CD163 protein can be isolated / purified.

[0067] If CD163 is shed from cells into the circulation, a suitable source may be the blood or serum of the relevant subject (e.g., a pig or porcine subject). For example, relatively low levels of the soluble form of CD163 (e.g., about or up to 0.5 mg / ml) can be found in the serum of healthy pigs. In addition, higher levels of the soluble form of CD163 (e.g., about or up to 4.5 mg / ml) can be detected in the serum of infected pigs (pigs suffering from an infection) or pigs showing an inflammatory response. Such high levels of the soluble form of CD163 can be detected in pigs suffering from any infection (e.g., any bacterial or viral infection, such as a non-PRRSV infection), e.g., which induces an inflammatory response or inflammation in the animal. However, typical and exemplary infections that commonly occur in the art are Lawsonia intracellularis infection, as well as Mycoplasma hyopneumoniae (M hyo), Pasteurella multocida (P. multocida), and Streptococcus suis (S. Suis) infections. It is also common for multiple infections (complex / pathogen diseases) to occur simultaneously in the art, and the level of the soluble form of CD163 in such subjects (e.g., pigs or porcine subjects) or pigs with more severe infections may also be higher than that in healthy subjects (e.g., pigs or porcine subjects). Thus, whole blood or serum from such healthy or infected pigs (e.g., the serum of pigs infected with Lawsonia intracellularis or suffering from other infections) can serve as a source of the soluble form of CD163.

[0068] Thus, the antibody (or binding protein) of the present invention that preferably does not significantly bind to the soluble form of CD163 (or a soluble form of CD163-related peptide) can be CD163 in a physiological fluid (e.g., serum), such as a physiological fluid (e.g., serum) from a healthy pig or an infected pig.

[0069] Exemplary soluble forms of CD163 (such as recombinant CD163) as described above and elsewhere in this text can be used to evaluate the ability of binding proteins and antibodies to bind to soluble forms of CD163. Exemplary constructs contain appropriate extracellular portions of CD163, such as subsets of different CD163 SRCR domains, such as CD163-SRCR1-PST2, CD163-SRCR1-9, CD163-SRCR4-7, or CD163-SRCR5-6. Similarly, other combinations of CD163 SRCR domains with fragments of the PST-1 and PST-2 domains containing subsets of different CD163 SRCR domains can also be used as long as only extracellular portions are present. The porcine form is preferably used to evaluate the antibodies of the present invention, although equivalent forms from other species (such as other mammalian species) can also be used, for example, for evaluating cross-reactivity.

[0070] Methods for evaluating binding (or binding ability) to an appropriate soluble form of CD163 are well known to those skilled in the art and any appropriate method can be used.

[0071] Convenient and appropriate methods for evaluating binding include in vitro binding assays, such as ELISA assays, to evaluate the binding of an antibody or binding protein to a fixed antigen (such as a fixed form of soluble CD163 as described herein). ELISA assays are familiar to those skilled in the art and they are able to easily establish suitable conditions to evaluate the ability of a binding protein or antibody to bind to CD163 in such assays. A particularly preferred ELISA assay method is described in the Examples section.

[0072] As evaluated by ELISA, the preferred antibodies (or binding proteins) of the present invention do not bind, do not significantly bind, or bind immeasurably to soluble forms of CD163 (preferably porcine CD163). Exemplary soluble forms are as described elsewhere in this text. Preferred soluble forms are pCD163 SRCR1-PST2 and pCD163-SRCR1-9, for example, as described in the Examples section.

[0073] A preferred method for assessing binding (or binding ability) to a suitable soluble form of CD163, such as porcine CD163, is surface plasmon resonance (SPR) assay (such as BIACore assay). Suitable SPR assays are known in the art and are preferred because they can more easily and consistently quantify binding. In some preferred SPR assays, a suitable form of soluble CD163 is captured (or immobilized) on a solid support (such as a sensor chip), for example by amine coupling (such as immobilizing 2000 to 2500 or 2500 to 3500 response units (RU) of CD163), and then different concentrations (such as serial dilutions, such as two-fold or three-fold dilutions) of the binding protein or antibody to be tested are injected. The preferred injection concentrations and flow rates are described in the Examples section. The preferred pH value for assessment is pH 7.4. Exemplary soluble forms of CD163 for such SPR assays are as described elsewhere herein. Preferred soluble forms are CD163-SRCR1-PST2, CD163-SRCR1-9 or CD163-SRCR4-7.

[0074] Such SPR assay methods can also be conveniently used to measure the binding kinetics of antibody-antigen interactions, such as determining kinetic parameters such as the association rate (ka), dissociation rate (kd) and affinity (KD). In some embodiments, the measurement can be carried out at 25 °C in a suitable buffer, such as standard HEPES-EDTA buffer, such as HBS-EP (sold by GE Healthcare Life Sciences, 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.0005% surfactant P20), with a pH of 7.4. The kinetic parameters can be determined or calculated by any suitable model or software, such as by fitting the experimental data of the sensorgram, assuming a 1:1 interaction, such as using BIAevaluation software. A particularly preferred SPR assay is described in the Examples section herein.

[0075] Thus, in a particularly preferred embodiment, the binding protein or antibody of the present invention is considered not to bind, or not to significantly bind (as determined at the time of assessment), to the soluble form of CD163 (such as porcine CD163) in a surface plasmon resonance (SPR) assay (such as BIACore assay).

[0076] In certain preferred embodiments, the antibodies of the invention that do not bind or do not significantly bind to the soluble form of CD163 (e.g., when in the VHH or VH form) have undetectable or substantially undetectable binding affinity for the soluble form of CD163 (e.g., porcine CD163), e.g., having a K D (equilibrium dissociation constant affinity) (poor / weak binding).

[0077] Thus, preferably, the antibodies of the invention (e.g., when in the VHH or VH form) have a binding affinity for the soluble form of CD163 (e.g., soluble porcine CD163) that corresponds to a K D greater than 20 μM, 50 μM, 100 μM, 150 μM, 200 μM or 250 μM (measured at the time of evaluation), or is undetectable by SPR, or is substantially too low to be measured or measured reliably, e.g., unable to be fitted or correctly fitted to a hypothetical 1:1 interaction. Specific exemplary binding affinities are disclosed in the Examples. Thus, for example, the H17B11 VHH antibody of the invention (e.g., as shown in Table A) has a binding affinity of at least 100 μM, e.g., at least 200 or 250 μM, as evaluated in a surface plasmon resonance (SPR) assay / BIACore assay.

[0078] Exemplary forms of the soluble form of CD163 that can be used to evaluate such binding affinities are recombinant soluble forms of CD163, such as porcine CD163 as described herein, e.g., forms containing SRCR4-7 or SRCR1-9, e.g., recombinant porcine CD163 containing SRCR4-7 or SRCR1-9. The Examples section describes suitable exemplary forms, such as construct pCD163-SRCR4-7 (optionally with human (hu) Fc) or pCD163-SRCR1-9 (optionally with huFc) or pCD163-SRCR1-PST2 (optionally with His tag), preferably pCD163-SRCR1-PST2 or pCD163-SRCR1-9 or pCD163-SRCR4-7 (optionally with huFc). pCD163-SRCR-FL-PST2, pCD163-SRCR-1-PST2 and pCD163-1-PST2 can be used interchangeably to refer to a porcine CD163 construct containing the full-length CD163 sequence from SRCR1 to PST2. Thus, when detecting the antibodies of the invention (e.g., in an SPR assay) using these constructs (e.g., when in the VHH or VH form), the above-described binding affinities can be observed.

[0079] Importantly, the antibodies (or binding proteins) of the present invention bind to the membrane-bound form of CD163 on cells, and do not significantly bind to the soluble form of CD163. From the perspective of antibody-based therapies (such as treating or preventing PRRSV infection), this combination of properties may be particularly important. As described above, without wishing to be bound by theory, antibodies that bind to the membrane-bound form of CD163 on cells (such as PAM, porcine PAM) but not to the soluble form will not be interfered with by the soluble form (shed) of CD163 in the circulation or interstitial space of the subject being treated, but will directly target the cell membrane of the appropriate disease-related cells (such as PAM expressing CD163 targeted by PRRSV).

[0080] The preferred antibodies of the present invention retain the ability to bind to the membrane-bound form of CD163 and do not significantly bind to the soluble form of CD163 in the presence of any physiological concentration (such as any concentration observed in the human or animal body) of the soluble form of CD163. For example, even in the presence of a high concentration of the soluble form of CD163, such as a concentration reaching or at least reaching 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 mg / l, this binding ability is still retained.

[0081] Therefore, the preferred antibodies of the present invention are able to distinguish the membrane-bound form of CD163 on cells from the soluble form of CD163. Therefore, the antibodies of the present invention can accurately distinguish the membrane-bound form of CD163. Therefore, the antibodies of the present invention can be considered specific for the membrane-bound form of CD163.

[0082] Therefore, the antibodies of the present invention can bind to conformational epitopes on CD163. Such conformational epitopes of CD163 are present on the membrane-bound form of CD163 but not on (or are significantly reduced or altered in) the soluble form of CD163. Without wishing to be bound by theory, it is believed that the antibodies of the present invention (such as H17B11) can bind to conformational epitopes of the membrane-bound form of CD163 generated by being tethered to the membrane through the PST2 domain, and this epitope is lost or altered once the CD163 molecule is shed or released. However, the preferred antibodies of the present invention do not bind to the PST2 region (for example, do not directly bind to the PST2 region itself).

[0083] Therefore, although the corresponding (or identical) primary amino acid sequence (linear amino acid sequence) may be present in both the membrane-bound form and the soluble form of CD163, the preferred antibodies of the present invention are able to advantageously distinguish the different forms, for example, by recognizing conformational epitopes rather than linear epitopes, such as recognizing new epitopes or new conformational epitopes present in the membrane-bound form of CD163 but not in the soluble form of CD163.

[0084] Examples provide suitable methods for obtaining the membrane-specific anti-CD163 antibodies of the present invention. However, the preferred protocol would involve immunizing a suitable animal (such as a camelid, such as a llama) with membrane-expressed CD163 (such as porcine CD163). For example, in the form of cells recombinantly expressing CD163 (such as HEK or CHO cells), for example, recombinantly expressing a construct containing a large extracellular domain of CD163, such as a construct of porcine (p)CD163-SRCR1-PST2 or pCD163-SRCR1-9, and / or in combination with PAM (or other cells naturally expressing CD163). Antibody clones prepared from such immunizations can then be screened to obtain membrane-specific anti-CD163 antibodies or subjected to appropriate rounds of selection.

[0085] The preferred rounds of selection may involve preparing a library of antibody clones, typically a phage display library, such as from the blood (PBMC) of an immunized animal, and then subjecting these clones to one or more rounds of appropriate selection. The preferred selection methods used herein involve selection against CD163 expressed natively in the membrane (here isolated pPAM cells) and CD163 expressed recombinantly in cells (here HEK cells expressing pCD163-SRCR1-PST2). One or more rounds of counter-selection (or negative selection) can also be used, for example, in the methods herein, using empty HEK wild-type cells and pPAMΔ5 cells (cells lacking SRCR domain 5) for counter-selection. Counter-selection in the presence of an excess of soluble form of CD163 is also a suitable additional or alternative selection step for obtaining membrane-specific clones.

[0086] Once the rounds of selection are completed, membrane-specific anti-CD163 antibodies can be screened.

[0087] A convenient and preferred method for performing this screening is to use flow cytometry (FACS) analysis with cells expressing cd163 that are known to be positive for the membrane-bound form of CD163 (such as pam, or cells expressing a recombinant membrane-bound form of CD163, such as the full-length membrane-bound form of CD163 described elsewhere herein). Cells with the native form of CD163, such as PAM, are preferably used. Candidate antibodies that are able to bind to the cell-expressed (membrane-bound) form of CD163 can then be identified.

[0088] The positive clones can then be further screened to assess whether they also have the ability to bind (or bind significantly) to the soluble form of CD163, e.g., the ability to distinguish between the cell membrane-bound form of CD163 and the soluble form of CD163. Similarly, any suitable method can be used, such as BiaCore or ELISA, but for accuracy and reliability, the final confirmation is preferably performed by SPR (e.g., BiaCore or an equivalent method). Suitable soluble forms of CD163, such as recombinant soluble forms, are described elsewhere in this document, including pCD163-SRCR1-PST2, pCD163-SRCR1-9, or pCD163-SRCR4-7.

[0089] Binding to the soluble form of CD163 that is not significant (insignificant) generally means that the binding to these forms of CD163 is reproducible (i.e., consistently observed) at a low or negligible level. In some cases, insignificant binding can be considered to be at background levels, e.g., compared to the levels observed in negative control experiments or without significant difference, or at undetectable or very low affinity in, for example, an SPR assay (as determined). Suitable test methods for determining whether an antibody (or binding protein) does not bind or does not significantly bind to the soluble form of CD163 are described elsewhere in this document.

[0090] In certain embodiments, the antibodies of the present invention do not bind (e.g., bind immeasurably) to the soluble form of CD163.

[0091] Another convenient method for identifying (screening) antibodies that can bind to the membrane-bound form of CD163 on cells but not to (or not significantly to) the soluble form of CD163 is to use a competitive assay, such as as part of a flow cytometry (FACS) analysis. Thus, an assay can be employed: by adding a sample of the soluble form of CD163 (e.g., the recombinant soluble form of CD163 described herein), to assess whether the soluble form of CD163 has the ability to compete with the candidate antibody for binding to the membrane-bound form of CD163 on cells. If the soluble form of CD163 can compete to a large extent, it indicates that the candidate antibody is not specific for the membrane-bound form of CD163 (since it also binds the soluble form). If the soluble form of CD163 cannot compete to a significant extent, it indicates that the candidate antibody has the ability to distinguish between the membrane-bound form of CD163 and the soluble form of CD163.

[0092] In such FACS assays, when soluble CD163 is added, a significant decrease in the signal indicates that the candidate antibody can bind to both the cell membrane-bound form and the soluble form of CD163, i.e., it does not distinguish; while when soluble CD163 is added, the signal is basically maintained or only slightly changed, indicating that the candidate antibody does not bind to (or does not significantly bind to) the soluble form of CD163, but binds to the membrane-bound form of CD163 (otherwise no positive signal would appear), i.e., the antibody can distinguish between the cell membrane-bound form and the soluble form of CD163.

[0093] The binding protein of the present invention can bind to CD163 (e.g., specifically bind), preferably to porcine CD163.

[0094] The preferred binding protein of the present invention is or comprises an antibody, particularly a VHH antibody or a single-domain antibody. However, the embodiments described herein involving antibodies (e.g., VHH antibodies or single-domain antibodies), with appropriate modifications, are equally applicable to other types of binding proteins, and vice versa.

[0095] The preferred binding protein is any single polypeptide chain that can bind to (e.g., specifically bind to) CD163 (preferably porcine CD163).

[0096] Suitable types of binding proteins that can be used in the present invention are known in the art. For example, in certain embodiments, immunoglobulin-based polypeptides are used, which typically contain CDR regions (and optionally FR regions or immunoglobulin-based scaffolds), such that the CDR regions (and optionally FR regions) of the antibodies of the present invention can be grafted onto a suitable scaffold or framework, such as an immunoglobulin scaffold.

[0097] Therefore, binding proteins containing an antigen-binding domain are also preferred, especially when the antigen-binding domain is or comprises or is derived from an antibody (e.g., contains the CDR regions of an antibody and optionally FR regions).

[0098] The binding protein of the present invention preferably may comprise multiple antibodies or antigen-binding domains that bind to CD163, such as two or three different antibodies discussed in other parts of this document.

[0099] As described elsewhere in this text, the antibodies of the present invention have the advantage that, due to their ability to distinguish between the membrane (cell surface) - bound form and the soluble form of CD163, they are not interfered with, or are not significantly interfered with, or are significantly less interfered with by the soluble form (shed) of the CD163 antigen, but rather target cells expressing CD163, which are also the cells targeted by PRRSV during infection. In fact, the antibodies of the present invention have been shown to be resistant to such soluble forms of CD163, for example, compositions have been shown to be able to very effectively inhibit PRRSV infection in the presence of the soluble form of CD163.

[0100] In addition to advantageously having the property of membrane - specific binding, the antibodies of the present invention have also been shown to have the ability to inhibit PRRSV - 2 infection.

[0101] Furthermore, when the antibodies of the present invention are combined with other CD163 antibodies, they have been shown to have excellent activity in inhibiting PRRSV infection. Without being bound by theory, it is believed that the antibodies of the present invention can help other CD163 antibodies target cells expressing CD163, thereby being able to reduce or block PRRSV infection in an efficient manner.

[0102] When two anti - CD163 antibodies are paired together (for example, one antibody of the present invention is paired with a second different anti - CD163 antibody), preferably on the same construct, excellent effects can be observed. Such bispecific molecules are preferably bis - diabody constructs, where each different antibody recognizes different epitopes (here, two different epitopes) on the same antigen (here, CD163). Therefore, such antibodies are sometimes referred to herein as bis - diabody anti - CD163 antibodies. These bis - diabody constructs exhibit good inhibition of PRRSV - 1 infection, although the effect is reduced in the presence of high concentrations of the soluble form of CD163, and also have some inhibitory effect on PRRSV - 2 infection.

[0103] When three anti - CD163 antibodies are paired together (i.e., one antibody of the present invention is paired with a second and a third different anti - CD163 antibody), preferably on the same construct, even better effects can be observed. Such trispecific molecules are preferably tris - diabody constructs, where each different antibody recognizes different epitopes (here, three different epitopes) on the same antigen (here, CD163). Therefore, such antibodies are sometimes referred to herein as tris - diabody anti - CD163 antibodies. Advantageously, the tris - diabody constructs of the present invention are able to extremely effectively inhibit PRRSV - 1 and PRRSV - 2 infection, even in the presence of high concentrations of the soluble form of CD163, such as may be found in pigs suffering from an infection (or a complex (multi - pathogen) disease), as described elsewhere in this text.

[0104] Accordingly, a preferred embodiment of the present invention provides a combination of the membrane - specific antibodies (or binding proteins) of the present invention, such as the H17B11 antibody described in Table A, or an antibody comprising three CDRs of SEQ ID NO: 2, 3, and 4 or sequences substantially homologous thereto, in combination with one or more other anti - CD163 antibodies or binding proteins. Preferably, one, two, or more other anti - CD163 antibodies or binding proteins are used in such combinations. In other words, a total of two, three, or more different anti - CD163 antibodies (or binding proteins) will be used in combination, wherein one antibody (or binding protein) is the membrane - specific antibody of the present invention.

[0105] When more than one anti - CD163 antibody (or binding protein) is used, preferably each antibody (or binding protein) binds to a different epitope on CD163, such that all antibodies (or binding proteins) in the combination can each bind to the CD163 target molecule. Preferably, these combinations are present together on the same construct, for example, linked by a suitable linker. This form is particularly suitable for the single - domain antibodies (or binding proteins) of the present invention, such as the VHH antibodies described herein.

[0106] Preferably, such combinations are provided in the form of a single construct. For example, in a bispecific construct, two (e.g., only two) such antibodies or binding proteins are present together in a single construct; or in a trispecific construct, three (e.g., only three) such antibodies or binding proteins are present together in a single construct.

[0107] A preferred anti - CD163 antibody (or binding protein) for use in combination with the membrane - specific antibody of the present invention, particularly a membrane - specific antibody based on the CDR sequences disclosed in Table A or sequences substantially homologous thereto, comprises at least one antigen - binding domain that binds to CD163 (e.g., porcine CD163), wherein the antigen - binding domain comprises a heavy - chain variable region, and the heavy - chain variable region comprises three complementarity - determining regions (CDRs), and the heavy - chain variable region comprises:

[0108] (i) a variable heavy - chain (VH) CDR1 comprising the amino acid sequence RYVMG (SEQ ID NO: 10) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1 or 2 amino acid substitutions compared to the given CDR sequence,

[0109] (ii) a variable heavy - chain (VH) CDR2 comprising the amino acid sequence AISWSGRAPYADSVKG (SEQ ID NO: 11) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and

[0110] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence GEGAIKWTTLDAYDY (SEQ ID NO:12) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence.

[0111] In a preferred embodiment, the other anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, such as porcine CD163, and the antigen-binding domain comprises a heavy chain variable region that comprises three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0112] (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence RYVMG (SEQ ID NO:10),

[0113] (ii) A variable heavy chain

[0114] (VH) CDR2 comprising the amino acid sequence AISWSGRAPYADSVKG (SEQ ID NO:11), and

[0115] (iii) A variable heavy chain

[0116] (VH) CDR3 comprising the amino acid sequence GEGAIKWTTLDAYDY (SEQ ID NO:12).

[0117] In other words, a preferred anti-CD163 antibody (or binding protein) for use in combination with the membrane-specific antibody of the present invention, particularly a membrane-specific antibody based on the CDR sequences disclosed in Table A or sequences substantially homologous thereto, comprises at least one antigen-binding domain that binds to CD163 (such as porcine CD163), and the antigen-binding domain comprises a heavy chain variable region that comprises three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0118] (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence GRTPSRYV (SEQ ID NO:26) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2 or 3 amino acid substitutions compared to the given CDR sequence,

[0119] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence ISWSGRA (SEQ ID NO:27) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2 or 3 amino acid substitutions compared to the given CDR sequence; and

[0120] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence AGGEGAIKWTTLDAYDY (SEQ ID NO:28) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence.

[0121] In a preferred embodiment, the other anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, such as porcine CD163, and the antigen-binding domain comprises a heavy chain variable region that comprises three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0122] (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence GRTPSRYV (SEQ ID NO:26),

[0123] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence ISWSGRA (SEQ ID NO:27), and

[0124] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence AGGEGAIKWTTLDAYDY (SEQ ID NO:28).

[0125] In certain preferred embodiments of the present invention, the other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises up to three light chain CDRs, preferably three light chain CDRs.

[0126] In a preferred embodiment, the other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:9 or a sequence having at least 80% sequence identity thereto (such as at least 85%, 90%, 95%, or 98% identity). In certain embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises up to three light chain CDRs, preferably three light chain CDRs.

[0127] In a preferred embodiment, the other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:9. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises up to three light chain CDRs, preferably three light chain CDRs.

[0128] An exemplary and preferred such antibody is the H03E11 antibody shown in Table B or D.

[0129] Another preferred anti - CD163 antibody (or binding protein) for use in combination with the membrane - specific antibody of the present invention, in particular a membrane - specific antibody based on the CDR sequences disclosed in Table A or sequences substantially homologous thereto, comprises at least one antigen - binding domain that binds to CD163 (such as porcine CD163), said antigen - binding domain comprising a heavy - chain variable region, said heavy - chain variable region comprising three complementarity - determining regions (CDRs), wherein the heavy - chain variable region comprises:

[0130] (i) A variable heavy - chain (VH) CDR1 comprising the amino acid sequence DYTIG (SEQ ID NO:18) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence,

[0131] wherein the substantially homologous sequence is a sequence containing 1 or 2 amino acid substitutions compared to the given CDR sequence,

[0132] (ii) A variable heavy - chain (VH) CDR2 comprising the amino acid sequence CINSITSNTYYADSVKG (SEQ ID NO:19) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and

[0133] (iii) A variable heavy - chain (VH) CDR3 comprising the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:20) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2, 3, or 4 amino acid substitutions compared to the given CDR

[0134] sequence.

[0135] In a preferred embodiment, said other anti - CD163 antibody or binding protein comprises at least one antigen - binding domain that binds to CD163, such as porcine CD163, said antigen - binding domain comprising a heavy - chain variable region, said heavy - chain variable region comprising three complementarity - determining regions (CDRs), wherein the heavy - chain variable region comprises:

[0136] (i) A variable heavy - chain (VH) CDR1 comprising the amino acid sequence DYTIG (SEQ ID NO:18),

[0137] (ii) A variable heavy - chain

[0138] (VH) CDR2 comprising the amino acid sequence CINSITSNTYYADSVKG (SEQ ID NO:19), and

[0139] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:20).

[0140] In other words, another preferred anti-CD163 antibody (or binding protein) for use in combination with the membrane-specific antibodies of the present invention, in particular a membrane-specific antibody based on the CDR sequences disclosed in Table A or sequences substantially homologous thereto, comprises at least one antigen-binding domain that binds to CD163 (such as porcine CD163), said antigen-binding domain comprising a heavy chain variable region, said heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein said heavy chain variable region comprises:

[0141] (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence GFTLDDYT (SEQ ID NO:34) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1, 2, or 3 amino acid substitutions compared to the given CDR sequence,

[0142] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence INSITSNT (SEQ ID NO:35) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1, 2, or 3 amino acid substitutions compared to the given CDR sequence; and

[0143] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence AADSGLFSGSSCLKYRAMRFGS (SEQ ID NO:36) or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence containing 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence.

[0144] In a preferred embodiment, said other anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, such as porcine CD163, said antigen-binding domain comprising a heavy chain variable region, said heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein said heavy chain variable region comprises:

[0145] (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence GFTLDDYT (SEQ ID NO:34),

[0146] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence INSITSNT (SEQ ID NO:35), and

[0147] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence AADSGLFSGSSCLKYRAMRFGS (SEQ ID NO:36).

[0148] In certain preferred embodiments of the present invention, said other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:17 or a sequence substantially homologous thereto. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain, said VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0149] In a preferred embodiment, said other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:17 or a sequence having at least 80% sequence identity thereto (such as at least 85%, 90%, 95% or 98% identity). In certain embodiments, such an antibody (or binding protein) further comprises a VL domain, said VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0150] In a preferred embodiment, said other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:17. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain, said VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0151] An exemplary and preferred such antibody is the H03D03 antibody shown in Table C or E.

[0152] In a combination using three anti-CD163 antibodies (or binding proteins), the above three specific antibodies (or binding proteins) defined based on the CDR sequences (or sequences substantially homologous thereto) disclosed in Table A, Table B (or D) and Table C (or E) respectively are preferred combinations, such as in the triple complementarity determining region constructs of the present invention.

[0153] In such an embodiment, a first preferred anti-CD163 antibody (or binding protein), for use in combination with the membrane-specific antibody of the present invention, in particular a membrane-specific antibody based on the CDR sequences disclosed in Table A or sequences substantially homologous thereto, comprises at least one antigen-binding domain that binds to CD163 (such as porcine CD163), said antigen-binding domain comprising a heavy chain variable region, said heavy chain variable region comprising three complementarity determining regions (CDRs), wherein said heavy chain variable region comprises:

[0154] (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of RYVMG (SEQ ID NO:10) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1 or 2 amino acid substitutions compared to the given CDR sequence,

[0155] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of AISWSGRAPYADSVKG (SEQ ID NO:11) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence; and

[0156] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GEGAIKWTTLDAYDY (SEQ ID NO:12) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3 or 4 amino acid substitutions compared to the given CDR sequence.

[0157] In a preferred embodiment, the first other anti - CD163 antibody or binding protein comprises at least one antigen - binding domain that binds to CD163, such as porcine CD163, and the antigen - binding domain comprises a heavy - chain variable region that comprises three complementarity - determining regions (CDRs), wherein the heavy - chain variable region comprises:

[0158] (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of RYVMG (SEQ ID NO:10),

[0159] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of AISWSGRAPYADSVKG (SEQ ID NO:11), and

[0160] (iii) A variable heavy chain

[0161] (VH) CDR3.

[0162] An alternative first other anti - CD163 antibody (or binding protein) for such an embodiment comprises at least one antigen - binding domain that binds to CD163, such as porcine CD163, and the antigen - binding domain comprises a heavy - chain variable region that comprises three complementarity - determining regions (CDRs), wherein the heavy - chain variable region comprises:

[0163] (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of GRTPSRYV (SEQ ID NO:26) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2, or 3 amino acid substitutions compared to the given CDR sequence,

[0164] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of ISWSGRA (SEQ ID NO:27) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2, or 3 amino acid substitutions compared to the given CDR sequence; and

[0165] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of AGGEGAIKWTTLDAYDY (SEQ ID NO:28) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence.

[0166] In certain preferred embodiments of the present invention, the first other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain, and the VL domain comprises at most three light chain CDRs, preferably three light chain CDRs.

[0167] In a preferred embodiment, the first other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:9 or a sequence having at least 80% sequence identity thereto (e.g., at least 85%, 90%, 95%, or 98% identity). In certain embodiments, such an antibody (or binding protein) further comprises a VL domain, and the VL domain comprises at most three light chain CDRs, preferably three light chain CDRs.

[0168] In a preferred embodiment, the first other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:9. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain, and the VL domain comprises at most three light chain CDRs, preferably three light chain CDRs.

[0169] An exemplary and preferred such first other antibody is the H03E11 antibody shown in Table B or D.

[0170] In such embodiments, the second preferred anti-CD163 antibody (or binding protein) used in combination with the two antibodies (or binding proteins) described above comprises at least one antigen-binding domain that binds to CD163, such as porcine CD163, and the antigen-binding domain comprises a heavy-chain variable region that comprises three complementarity-determining regions (CDRs), wherein the heavy-chain variable region comprises:

[0171] (i) a variable heavy-chain (VH) CDR1 comprising the amino acid sequence DYTIG (SEQ ID NO:18) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1 or 2 amino acid substitutions compared to the given CDR sequence,

[0172] (ii) a variable heavy-chain (VH) CDR2 comprising the amino acid sequence CINSITSNTYYADSVKG (SEQ ID NO:19) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and

[0173] (iii) a variable heavy-chain (VH) CDR3 comprising the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:20) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence.

[0174] In a preferred embodiment, the second other anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, such as porcine CD163, and the antigen-binding domain comprises a heavy-chain variable region that comprises three complementarity-determining regions (CDRs), wherein the heavy-chain variable region comprises:

[0175] (i) a variable heavy-chain (VH) CDR1 comprising the amino acid sequence DYTIG (SEQ ID NO:18),

[0176] (ii) a variable heavy-chain

[0177] (VH) CDR2 comprising the amino acid sequence CINSITSNTYYADSVKG (SEQ ID NO:19), and

[0178] (iii) a variable heavy-chain (VH) CDR3 comprising the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:20).

[0179] A second alternative anti-CD163 antibody (or binding protein) for such embodiments comprises at least one antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises a heavy-chain variable region that comprises three complementarity-determining regions (CDRs), and wherein the heavy-chain variable region comprises:

[0180] (i) a variable heavy-chain (VH) CDR1 comprising the amino acid sequence GFTLDDYT (SEQ ID NO:34) or a sequence that is substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, or 3 amino acid substitutions as compared to the given CDR sequence,

[0181] (ii) a variable heavy-chain (VH) CDR2 comprising the amino acid sequence INSITSNT (SEQ ID NO:35) or a sequence that is substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, or 3 amino acid substitutions as compared to the given CDR sequence; and

[0182] (iii) a variable heavy-chain (VH) CDR3 comprising the amino acid sequence AADSGLFSGSSCLKYRAMRFGS (SEQ ID NO:36) or a sequence that is substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions as compared to the given CDR sequence.

[0183] In certain preferred embodiments of the invention, the second alternative anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:17 or a sequence that is substantially homologous thereto. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises up to three light-chain CDRs, preferably three light-chain CDRs.

[0184] In a preferred embodiment, the second alternative anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:17 or a sequence having at least 80% sequence identity thereto (such as at least 85%, 90%, 95%, or 98% identity). In certain embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises up to three light-chain CDRs, preferably three light-chain CDRs.

[0185] In a preferred embodiment, the second alternative anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:17. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises up to three light-chain CDRs, preferably three light-chain CDRs.

[0186] An exemplary and preferred such second other antibody is the H03D03 antibody shown in Table C or E.

[0187] Preferred binding proteins or constructs or combinations, such as binding proteins or constructs or combinations comprising multiple antibodies, which are based on the antibody sequences listed in Table A, optionally in combination with the antibody sequences listed in Table B (or D) and / or Table C (or E). The present invention takes monoclonal antibodies of VHH antibodies (single domain antibodies) as examples, and their sequences are shown in Tables A, B, C, D and E herein. The VH CDR domains and VH domains of each of these VHH antibodies are shown in Tables A to E herein. Antibodies (or binding proteins) comprising these VH CDR domains or groups of VH domains, in particular multi-antibody constructs or binding proteins comprising such domains (or sequences substantially homologous thereto) are preferred embodiments of the present invention.

[0188] In embodiments where multiple antibodies (such as the multiple antibodies described above) are combined and coexist in a single construct, the antibodies (or binding proteins) can be provided in any order. Therefore, any reference herein to "first", "second" antibodies, etc. should not be construed as specifying the position of these antibodies in any construct. One preferred, exemplary triple complementarity-determining region construct (Tri-2) comprises (from the N-terminus to the C-terminus) an antibody based on the sequence of Table B (or D), followed by an antibody based on the sequence of Table C (or E), followed by an antibody based on the sequence of Table A. Another preferred, exemplary triple complementarity-determining region construct (Tri-10) comprises (from the N-terminus to the C-terminus) an antibody based on the sequence of Table A, followed by an antibody based on the sequence of Table B (or D), followed by an antibody based on the sequence of Table C (or E). In certain embodiments of the present invention, the membrane-specific antibodies of the present invention, such as antibodies based on the sequence of Table A, can be located at the N-terminus or C-terminus of the multiple antibodies present. Preferred antibodies are VHH antibodies, such as those shown in Tables A to E.

[0189] When present as a single construct (such as a single protein chain), the different antibodies in the construct are conveniently linked by a peptide linker, examples of which are described elsewhere herein.

[0190] In another aspect, when two anti-CD163 antibodies (or binding proteins) are used, an anti-CD163 antibody (or binding protein) based on the CDR sequences disclosed in Table B (or D) or sequences substantially homologous thereto can be combined with an anti-CD163 antibody (or binding protein) based on the CDR sequences disclosed in Table C (or E) or sequences substantially homologous thereto.

[0191] Therefore, the combination of the antibodies (or binding proteins) or double complementarity-determining region constructs in this aspect of the present invention preferably comprises:

[0192] A first antibody (or binding protein) that comprises at least one antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises a heavy-chain variable region that comprises three complementarity-determining regions (CDRs), and wherein the heavy-chain variable region comprises:

[0193] (i) a variable heavy chain (VH) CDR1 that comprises the amino acid sequence DYTIG (SEQ ID NO:18) or a sequence that is substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1 or 2 amino acid substitutions compared to the given CDR sequence,

[0194] (ii) a variable heavy chain (VH) CDR2 that comprises the amino acid sequence CINSITSNTYYADSVKG (SEQ ID NO:19) or a sequence that is substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and

[0195] (iii) a variable heavy chain (VH) CDR3 that comprises the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:20) or a sequence that is substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and

[0196] (iv) a variable heavy chain (VH) CDR4 that comprises the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:20) or a sequence that is substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and

[0197] A second antibody (or binding protein) that comprises at least one antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises a heavy-chain variable region that comprises three complementarity-determining regions (CDRs), and wherein the heavy-chain variable region comprises:

[0198] (i) a variable heavy chain (VH) CDR1 that comprises the amino acid sequence RYVMG (SEQ ID NO:10) or a sequence that is substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1 or 2 amino acid substitutions compared to the given CDR sequence,

[0199] (ii) a variable heavy chain (VH) CDR2 that comprises the amino acid sequence AISWSGRAPYADSVKG (SEQ ID NO:11) or a sequence that is substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and

[0200] (iii) a variable heavy chain (VH) CDR3 that comprises the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:20) or a sequence that is substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and

[0201] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GEGAIKWTTLDAYDY (SEQ ID NO:12) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence.

[0202] In an alternative, the first anti - CD163 antibody or binding protein for such an embodiment comprises at least one antigen - binding domain that binds to CD163, such as porcine CD163, and the antigen - binding domain comprises a heavy - chain variable region that comprises three complementarity - determining regions (CDRs), wherein the heavy - chain variable region comprises:

[0203] (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of GFTLDDYT (SEQ ID NO:34) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, or 3 amino acid substitutions compared to the given CDR sequence,

[0204] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of INSITSNT (SEQ ID NO:35) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, or 3 amino acid substitutions compared to the given CDR sequence; and

[0205] (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of AADSGLFSGSSCLKYRAMRFGS (SEQ ID NO:36) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence.

[0206] In certain further preferred embodiments, the first anti - CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:17 or a sequence substantially homologous thereto. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises at most three light - chain CDRs, preferably three light - chain CDRs; or

[0207] The first anti - CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:17 or a sequence having at least 80% sequence identity thereto (such as at least 85%, 90%, 95%, or 98% identity). In certain embodiments, such an antibody (or binding protein) further comprises a VL domain that comprises at most three light - chain CDRs, preferably three light - chain CDRs; or

[0208] The first anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:17. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain, which VL domain comprises up to three light chain CDRs, preferably three light chain CDRs.

[0209] An exemplary and preferred such antibody is the H03D03 antibody shown in Table C or E.

[0210] In an alternative such embodiment, the second anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, such as porcine CD163, and the antigen-binding domain comprises a heavy chain variable region that comprises three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0211] (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence GRTPSRYV (SEQ ID NO:26) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, or 3 amino acid substitutions as compared to the given CDR sequence,

[0212] (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence ISWSGRA (SEQ ID NO:27) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, or 3 amino acid substitutions as compared to the given CDR sequence; and

[0213] (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence AGGEGAIKWTTLDAYDY (SEQ ID NO:28) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions as compared to the given CDR sequence.

[0214] In certain preferred embodiments, the second other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:9 or a sequence substantially homologous thereto. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain, which VL domain comprises up to three light chain CDRs, preferably three light chain CDRs; or

[0215] The second other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:9 or a sequence having at least 80% sequence identity thereto (such as at least 85%, 90%, 95% or 98% identity). In certain embodiments, such an antibody (or binding protein) further comprises a VL domain, which VL domain comprises at most three light chain CDRs, preferably three light chain CDRs; or

[0216] The second other anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO:9. In certain embodiments, such an antibody (or binding protein) further comprises a VL domain, which VL domain comprises at most three light chain CDRs, preferably three light chain CDRs.

[0217] An exemplary and preferred such second other antibody is the H03E11 antibody shown in Table B or D.

[0218] The various antibodies (or binding proteins) or combinations thereof described above, or constructs comprising the antibodies (or binding proteins) or combinations thereof, all have the ability to inhibit PRRSV-1 and / or PRRSV-2 infection, and thus can be used for treating or preventing PRRSV-1 and / or PRRSV-2 infection.

[0219] Preferred uses of antibodies (or binding proteins) containing the constructs or combinations of the invention are for the treatment or prevention of pathogen infections involving CD163, most notably PRRSV infection. Generally, antibodies (or binding proteins) containing the constructs and combinations of the invention inhibit (or block or reduce) pathogen (e.g., PRRSV) infection, e.g., inhibit (or block or reduce) the ability of the pathogen (e.g., PRRSV) to cause infection (e.g., infect a suitable host cell). Preferably, said inhibition or reduction is a measurable inhibition or reduction, more preferably a significant inhibition or reduction, e.g., a statistically significant inhibition or reduction, e.g., a p-value ≤ 0.05 or < 0.05. In certain embodiments, antibodies (or binding proteins) containing the constructs or combinations of the invention can inhibit (or block or reduce) the ability of a pathogen (e.g., PRRSV) to infect a host cell, with an inhibition rate of at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95% or at least 98%, e.g., complete inhibition or 100% inhibition. Generally, such % inhibition (and other percentage inhibition levels described herein) is compared to (or relative to) an appropriate control assay or control level, e.g., a control assay or control level (e.g., a negative control or background level or assay) in the absence of the binding protein or antibody (anti-CD163 antibody). Thus, a 0% inhibition (control) level (or conversely, 100% or maximum infection level) generally refers to the level in the absence of the binding protein or antibody (anti-CD163 antibody).

[0220] This ability to inhibit infection can be determined or tested by any suitable assay method, examples of which are readily available to those skilled in the art. Suitable assays can be, for example, in vitro or ex vivo assays, e.g., involving the use of host cells expressing CD163, such as PAM or host cells expressing recombinant CD163, as described elsewhere herein. Such cells can be contacted with PRRSV or other suitable pathogen at a level sufficient to cause cell infection. Suitable assays can generally be performed in the presence of serum, e.g., porcine serum (from healthy or infected pigs, e.g., see below, or serum containing soluble form of CD163) or fetal bovine serum (FBS), or in the presence of soluble form of CD163. Those skilled in the art can readily determine the appropriate serum usage ratio, e.g., a level of 10% FBS and 80% porcine serum was used in the assays described in the Examples section.

[0221] FBS usually does not contain any soluble form of CD163. However, porcine serum usually contains at least some soluble form of CD163. Therefore, in order to test the ability of an antibody (or binding protein) containing the construct or combination of the present invention to inhibit PRRSV infection in the presence of soluble CD163, porcine serum, usually 80% porcine serum, was used. In pigs with an infection (e.g., pigs experiencing an inflammatory response), the level of soluble CD163 is usually significantly elevated. Therefore, in order to test the ability of an antibody (or binding protein) containing the construct or combination of the present invention to inhibit PRRSV infection in the presence of a high (but physiological) level of soluble CD163, the serum of infected pigs (e.g., pigs infected with Lawsonia intracellularis), usually 80% porcine serum, was used. Similarly, the serum from pigs infected with other bacteria or viruses can be used such that the level of soluble CD163 is elevated. The method for determining the level of soluble CD163 in the relevant serum samples can be carried out routinely to determine the level of soluble CD163 present. In the porcine serum assays described herein, the level of soluble CD163 measured using healthy porcine serum was 0.40 mg / l, while in infected pigs it was 4.50 mg / l. Therefore, such levels of soluble CD163 are exemplary and preferred, and in certain embodiments of the present invention, the inhibition levels described herein are the levels observed in the presence of these concentrations of soluble CD163.

[0222] Then the ability of an antibody (or binding protein) containing the construct or combination of the present invention to inhibit or reduce such infection can be easily analyzed, for example, by comparison (or relative to) the 100% infection level set in a control assay. Suitable and exemplary infection assays are described in the Examples section.

[0223] Antibodies (or binding proteins) containing the constructs or combinations of the present invention at any suitable concentration can be used to inhibit or reduce infection. The exemplary constructs or combinations of the present invention have the ability to cause inhibition, for example, at the inhibition levels described herein, when used at a concentration of at least 1, 2, 4, 5, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300 or 400 μg / ml, for example, up to a concentration of 200, 300 or 400 μg / ml, for example, between 50 or 100 and 200, 300 or 400 μg / ml, the inhibition level of the antibody (especially VHH). The preferred constructs or combinations of the present invention have the ability to cause inhibition, for example, at the inhibition levels described herein, when used at a concentration of or at least 50, 60, 70, 80, 90, 100, 120, 140, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 450, 500, 600, 700, 800 or 900 ng / ml, for example, up to a concentration of 100, 200, 300, 400, 450, 500, 600, 700, 800 or 900 ng / ml, for example, between 50 or 100 and 200, 300, 400 or 500 ng / ml, the inhibition level of the antibody (especially VHH). If an antibody combination (such as a VHH antibody) is used, then in certain embodiments, these levels can refer to the total amount of antibody (such as VHH) present, i.e., the sum of the concentrations of each antibody present.

[0224] Exemplary constructs or combinations of the present invention have the ability to inhibit antibodies (especially VHHs) at a concentration of or at least 5, 10, 15, 20, 25, or 30 μM, such as the inhibition levels described herein. Preferred constructs or combinations of the present invention have the ability to cause inhibition, such as the inhibition levels described herein, when used at a concentration of or at least 50, 60, 70, 80, 90, 100, 120, 140, 160, 170, 180, 190, 200, 250, 275, 300, 325, 350, 375, 400, 500, 600, 700, 800, or 900 nM, such as a concentration up to 100, 200, 300, 400, 500, 600, 700, 800, or 900 nM, such as between 50 or 100 and 200, 300, 400, or 500 nM, of the inhibition level of an antibody (especially a VHH). Even more preferred constructs of the present invention have the ability to cause inhibition, such as the inhibition levels described herein, when used at a concentration of or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nM, such as a concentration up to 10, 20, 30, 40, or 50 nM, such as between 5 or 10 and 30, 40, or 50 nM, of the inhibition level of an antibody (especially a VHH). If an antibody combination (such as a VHH antibody) is used, then in certain embodiments, these levels may refer to the total amount of antibody (such as a VHH) present, i.e., the sum of the concentrations of each antibody present.

[0225] In certain embodiments, an antibody (or binding protein) comprising the construct or combination of the present invention can inhibit (or block or reduce) the ability of PRRSV-1 or PRRSV-2 to cause infection (e.g., infect host cells expressing CD163). In certain embodiments, the binding protein or antibody of the present invention can inhibit (or block or reduce) the ability of PRRSV-1 and PRRSV-2 to cause infection (e.g., infect host cells expressing CD163). Notably, the antibody (or binding protein) comprising the construct or combination of the present invention targets host cell CD163 rather than PRRSV (or other pathogenic entity) itself. This provides an important advantage of being able to inhibit any viral infection, such as PRRSV using the same binding region on CD163 for infection or pathogenesis. Thus, the antibody etc. of the present invention can provide a method to block multiple PRRSV strains or isolates, including highly pathogenic strains or isolates, provided that they use CD163 to infect cells. It is believed that CD163 is common in the infection of multiple PRRSV strains. Therefore, the constructs and combinations of the present invention have a wide range of uses. This is in contrast to some known PRRSV methods (e.g., vaccination), which may be strain-specific and whose effectiveness (or whether it is effective) may vary depending on the strain. Thus, the construct or combination of the present invention provides important advantages and flexibility over such existing methods.

[0226] The preferred construct or combination of the present invention can almost completely inhibit or completely inhibit PRRSV-1 infection. For example, at least 90%, 95% or 98% inhibition can be observed, or 100% inhibition can be observed. Alternatively, at least 50%, 60%, 70%, 75% or 80% inhibition can be observed. In certain embodiments, the preferred antibody can show at least 80% inhibition of PRRSV-1 infection, more preferably at least 85%, 90%, 95% or 98% inhibition.

[0227] The preferred construct or combination of the present invention can almost completely inhibit or completely inhibit PRRSV-2 infection. For example, at least 90%, 95% or 98% inhibition can be observed, or 100% inhibition can be observed. Alternatively, at least 50%, 60%, 70%, 75% or 80% inhibition can be observed. In certain embodiments, the preferred antibody can show at least 80% inhibition of PRRSV-2 infection, more preferably at least 85%, 90%, 95% or 98% inhibition.

[0228] PRRSV-2

[0229] Other preferred constructs or combinations of the present invention are capable of inhibiting PRRSV-1 and / or PRRSV-2 infection by at least 40%, at least 45%, at least 50%, at least 55%, at least 60% or at least 65%, more preferably at least 70%, 80%, 85%, 90%, 95% or 98% in the presence of soluble CD163 (e.g., in the presence of relatively low levels of soluble CD163, such as up to or about 0.40 mg / L).

[0230] Other preferred constructs or combinations of the present invention are capable of inhibiting PRRSV-1 and / or PRRSV-2 infection by at least 40%, at least 45%, at least 50%, at least 55%, at least 60% or at least 65%, more preferably at least 70%, 80%, 85%, 90%, 95% or 98% in the presence of soluble CD163 (e.g., in the presence of relatively high levels of soluble CD163, such as up to or about 4.50 mg / L).

[0231] In certain embodiments, the constructs or combinations of the present invention can inhibit (or block or reduce) the ability of PRRSV-2 to infect host cells. In certain embodiments, the binding proteins or antibodies of the present invention are capable of specifically inhibiting (or blocking or reducing) the ability of PRRSV-2 to cause infection (e.g., infecting host cells expressing CD163 or specifically inhibiting PRRSV-2 infection). Thus, exemplary antibodies can be capable of inhibiting at least 25%, 30%, 35%, 40%, 45% or 50% of PRRSV-2 infection (e.g., inhibiting the ability of PRRSV-2 to infect host cells by at least 25%, 30%, 35%, 40%, 45% or 50%).

[0232] Exemplary constructs or combinations comprise three VHH antibodies shown in Tables A, B (or D) and C (or E).

[0233] In certain embodiments, the constructs or combinations of the present invention have an IC 50 (e.g., for inhibiting PRRSV-1 and / or PRRSV-2 infection of host cells, such as PAM). In certain embodiments, the IC 50 is 2.0, 2.5, 3.0 or 3.5 to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0 nM. Specific ICs are also shown in the examples.50 Value examples.

[0234] In certain embodiments, the constructs or combinations of the present invention have an IC of 30.0 nM or lower, 25.0 nM or lower, 20.0 nM or lower, 15.0 nM or lower, 12 nM or lower, 10.0 nM or lower, 9.5 nM or lower, 9.0 nM or lower, 8.5 nM or lower, 8.0 nM or lower, 7.5 nM or lower, 7.0 nM or lower, 6.5 nM or lower, 6.0 nM or lower, 5.5 nM or lower, 5 nM or lower, 4.5 nM or lower, 4.0 nM or lower, 3.5 nM or lower or 3.0 nM or lower 90 (e.g., for inhibiting PRRSV-1 and / or PRRSV-2 from infecting host cells, such as PAM). In certain embodiments, the IC 90 is 2.0, 2.5, 3.0 or 3.5 to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0, 20.0, 25.0 or 30.0 nM. Specific IC value examples are also shown in the examples. 90 Value examples.

[0235] The preferred IC as described above 50 or IC 90 values are preferably determined by appropriate viral infectivity assays, such as those described above or in the Examples section.

[0236] In alternative embodiments of the present invention, the constructs or combinations of the present invention can be used to reduce the risk of PRRSV infection or prevent PRRSV infection.

[0237] Preferably, compared to an appropriate control level, the above-mentioned capabilities and characteristics are observed at a measurable or significant level, more preferably at a statistically significant level. Appropriate significance levels are discussed in other parts of this document. More preferably, when compared to the capabilities observed with prior art antibodies, the above-mentioned one or more capabilities and characteristics are manifested at a measurably better, or more preferably significantly better (preferably statistically significantly better) level.

[0238] In any statistical analysis mentioned herein, the probability value of a statistically significant difference from a relevant control or other comparison entity or measurement is preferably ≤0.1 or <0.1, preferably ≤0.05 or <0.05. Appropriate methods for determining statistical significance are well-known and documented in the art, and any of them can be used.

[0239] In certain embodiments, the constructs or combinations of the present invention have one or more, preferably two or more, or three or more, most preferably all of the functional characteristics described herein, particularly the preferred functional characteristics.

[0240] Throughout the application, the term "a" means "at least one", "at least the first", "one or more", or "multiple" of the recited component or step, except where an upper limit is specifically stated hereinafter. Thus, for example, an "antibody" as used herein means "at least the first antibody".

[0241] Furthermore, when the terms "comprising", "containing", "having", "include" or other equivalent terms are used herein, in some more specific embodiments, such as in the definitions of the CDR or FR sequences herein, these terms include the terms "consisting of" or "consisting essentially of" or other equivalent terms.

[0242] A nucleic acid molecule encoding a binding protein or antibody of the present invention (or a binding protein or antibody for use in the present invention), or encoding a construct or combination defined herein, or a part or fragment thereof, or a nucleic acid molecule substantially homologous thereto constitutes a further aspect of the present invention.

[0243] Preferred nucleic acid molecules are those encoding a VHH antibody or VH region or domain of the present invention (such as a nucleic acid molecule encoding SEQ ID NO: 1, 9 or 17). Other preferred nucleic acid molecules are those encoding any of the three sets of CDR sequences defined in Tables A, B, C, D or E, or sequences substantially homologous thereto. Preferred such nucleic acid molecules also encode appropriate framework regions, such as FR1, FR2, FR3 and FR4 regions, preferably a set of FR sequences defined in any of Tables A, B, C, D or E, or sequences substantially homologous thereto.

[0244] The nucleic acid molecules of the present invention can be, for example, DNA or RNA molecules.

[0245] The term "substantially homologous" as related to an amino acid or nucleic acid sequence herein includes sequences having at least 55%, 60%, 65%, 70% or 75% sequence identity with the disclosed amino acid or nucleic acid sequence, preferably at least 80%, even more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In certain embodiments, an antibody (or binding protein) of the present invention comprises one or at least one heavy chain variable region (or VH domain) that comprises an amino acid sequence region having at least about 55%, 60%, 65%, 70% or 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90% or 95%, most preferably at least about 97%, 98% or 99% amino acid sequence identity with the amino acid sequences of SEQ ID NO: 1, 9 and 17.

[0246] Thus, the substantially homologous sequences of the present invention include alterations (additions, substitutions, insertions or deletions) of one or more bases or amino acids of the sequences of the present invention. At the amino acid level, the preferred substantially homologous sequences contain at most 5, such as only 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably 1 or 2 altered amino acids in one or more framework regions and / or one or more CDRs constituting the sequences of the present invention. The alterations can be conservative amino acid alterations or non-conservative amino acid alterations. Preferably, the alterations are substitutions, preferably conservative amino acid substitutions.

[0247] In certain embodiments, if a given starting sequence is relatively short (e.g., five amino acids in length), then the number of amino acid substitutions that can occur in a sequence substantially homologous to it is less than the number of amino acid substitutions that can be optionally made in a sequence substantially homologous to a longer starting sequence. For example, in certain embodiments, a sequence substantially homologous to a starting VH CDR1 sequence according to the present invention, e.g., a starting VH CDR1 sequence that may be 5 amino acid residues in length in certain examples, preferably has 1 or 2 (more preferably 1) altered amino acids compared to the starting sequence. Thus, in certain embodiments, the number of altered amino acids in a substantially homologous sequence (e.g., a substantially homologous CDR sequence) can be adjusted according to the length of a given starting CDR sequence. For example, depending on the length of a given starting CDR sequence, there can be different numbers of altered amino acids in order to achieve a particular % sequence identity, e.g., at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, in each CDR. Thus, by way of example, in a CDR sequence 20 or 22 amino acids in length herein, there can be at most 8, such as only 1, 2, 3, 4, 5, 6, 7 or 8, or 1, 2, 3, 4, 5, 6 or 7, or 1, 2, 3, 4, 5 or 6, or 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably 1 or 2 altered amino acids.

[0248] Other preferred examples of substantially homologous sequences are sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% amino acid sequence identity to the amino acid sequence of one or more CDR regions or one or more FR regions disclosed in Table A or B or C or D or E. Thus, in certain embodiments, a substantially homologous CDR sequence can be a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to a given CDR sequence described herein.

[0249] In certain embodiments, in an antibody having a substantially homologous sequence or a certain degree of sequence identity to a given sequence, the altered amino acid residues are not in the CDR regions. For example, in certain embodiments, in an antibody having a VH domain with a certain degree of sequence identity to the given VH domain sequence of a specific antibody of the present invention (or for use in the present invention) (e.g., as disclosed in Table A, B or C, D or E), the altered (or variant) residues are not in the CDR regions. Thus, in certain embodiments, in an antibody having a substantially homologous sequence or a certain degree of sequence identity to a given sequence, the altered amino acid residues are in one or more framework regions.

[0250] As will be apparent from other parts of this document, in other embodiments, in an antibody having a substantially homologous sequence or a certain degree of sequence identity to a given sequence, the altered amino acid residues can be in the CDR regions.

[0251] In certain embodiments, for an antibody (or binding protein) having a substantially homologous sequence or a certain degree of sequence identity to a given sequence, the three VH CDR amino acid sequences (i.e., all three VH CDR sequences together) are considered the complete CDR complementary region of the antibody, and the amino acid sequence of the complete CDR complementary region of the antibody has at least 60%, 65% or 70% identity, preferably at least 75% or 80%, or at least 85% or 90%, or at least 95% identity, compared to the corresponding complete CDR complementary region of a given starting (or reference) antibody. The starting (or reference) antibody can have the CDR sequences of the antibodies disclosed in Table A, B or C, D or E.

[0252] The altered residues can be conservative or non-conservative amino acid substitutions, or a mixture thereof. In such embodiments, the preferred alterations are conservative amino acid substitutions.

[0253] In all embodiments, the binding protein (e.g., antibody) containing the substantially homologous sequence retains the ability to bind to CD163 (e.g., porcine CD163). Preferably, the binding protein (e.g., antibody) containing the substantially homologous sequence retains one or more (preferably all) of the other properties associated with the H17B11 (Table A), H03E11 (Table B), or H03D03 (Table C) antibodies, as appropriate, as described herein.

[0254] The CDRs of the antibodies (or binding proteins) of the present invention are preferably separated by appropriate frameworks, such as the framework regions found in naturally occurring antibodies and / or effectively engineered antibodies. Thus, the V H (e.g., VHH), V L and each CDR sequence are preferably located within or incorporated into an appropriate framework or scaffold to effect antigen (here, CD163) binding. Such framework sequences or regions may correspond to the naturally occurring framework regions FR1, FR2, FR3, and / or FR4 to form a suitable scaffold as needed, or may correspond to consensus framework regions, such as those determined by comparing various naturally occurring framework regions. Alternatively, non-antibody scaffolds or frameworks, such as T cell receptor frameworks, may be used.

[0255] Suitable sequences for the framework regions are well known and documented in the art, and any of them may be used. Preferred sequences for the framework regions are one or more of the framework regions that constitute the VHH antibodies of the present invention (or used in the present invention), preferably one or more of the framework regions of the H17B11, H03E11, or H03D03 VHH antibodies disclosed in Tables A, B (or D), and C (or E), respectively, or framework regions that are substantially homologous thereto, particularly framework regions capable of maintaining antigen specificity, such as framework regions that result in an antibody having a substantially identical or completely identical 3D structure.

[0256] In certain preferred embodiments, the antibody (or binding protein) of the present invention contains all four variable heavy chain (SEQ ID NOs: 5, 6, 7, and 8) framework regions (FRs), as appropriate, or FR regions that are substantially homologous thereto, particularly antibodies (or binding proteins) based on the CDRs of SEQ ID NOs: 2, 3, and 4.

[0257] In other preferred embodiments, the antibody (or binding protein) of the present invention contains all four variable heavy chain (SEQ ID NOs: 13, 14, 15, and 16) framework regions (FRs), as appropriate, or FR regions that are substantially homologous thereto, particularly antibodies (or binding proteins) based on the CDRs of SEQ ID NOs: 10, 11, and 12.

[0258] In other preferred embodiments, the antibodies (or binding proteins) of the invention comprise all four variable heavy chain (SEQ ID NOs: 21, 22, 23, and 24) framework regions (FRs), as appropriate, or FR regions that are substantially homologous thereto, particularly antibodies (or binding proteins) based on the CDRs of SEQ ID NOs: 18, 19, and 20.

[0259] In other preferred embodiments, the antibodies (or binding proteins) of the invention comprise all four variable heavy chain (SEQ ID NOs: 29, 30, 31, and 32) framework regions (FRs), as appropriate, or FR regions that are substantially homologous thereto, particularly antibodies (or binding proteins) based on the CDRs of SEQ ID NOs: 26, 27, and 28.

[0260] In other preferred embodiments, the antibodies (or binding proteins) of the invention comprise all four variable heavy chain (SEQ ID NOs: 37, 38, 39, and 40) framework regions (FRs), as appropriate, or FR regions that are substantially homologous thereto, particularly antibodies (or binding proteins) based on the CDRs of SEQ ID NOs: 34, 35, and 36.

[0261] The CDR sequences of certain antibodies of the invention are listed in Tables A, B, C, D, and E. In certain embodiments, the CDR sequences of the antibodies of the invention can be the CDR sequences in the VH (VHH) domains of the antibodies of the invention identified using any suitable method (or tool), such as the sequences identified according to Kabat (e.g., Kabat et al., “Sequences of Proteins of Immunological Interest”, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 647 - 669, 1991) or Chothia (e.g., Chothia C et al., (1989) Nature, 342:877–883, or Al - Lazikani et al., (1997) JMB 273, 927 - 948), or the sequences identified using the IMGT numbering scheme (e.g., Lefranc, M.-P., The Immunologist, 7, 132 - 136 (1999); www.imgt.org) )。

[0262] Conventional methods in the art, such as alanine - scanning mutagenesis and / or crystal structure analysis of antigen - antibody complexes, can be used to determine which amino acid residues in the CDRs do not contribute or contribute insignificantly to antigen binding. Thus, in embodiments of the invention involving substantially homologous sequences, these residues are good candidates for alteration or replacement.

[0263] Once identified, one or more amino acids in the amino acid sequence of a parental antibody are added, deleted, substituted, or inserted to form a new antibody, where the parental antibody is one of the antibodies of the invention as defined elsewhere herein, and the resulting new antibody is tested to identify an antibody that binds CD163 according to the invention, such as a membrane-specific antibody as described elsewhere herein. Such methods can be carried out using conventional techniques in the art. Such methods can be used to construct a variety of new antibodies, all of which can be tested for their ability to bind CD163, such as the membrane-specific binding ability as described elsewhere herein. Preferably, the addition, deletion, substitution, or insertion of one or more amino acids occurs in one or more CDR domains.

[0264] For example, the manipulation can be conveniently carried out at the nucleic acid level by genetic engineering, where the nucleic acid molecule encoding the appropriate binding protein and its domains is modified such that the amino acid sequence of the resulting expressed protein is also modified in an appropriate manner. Testing the ability of one or more modified antibodies to bind CD163, and their ability to exhibit membrane-specific binding as described elsewhere herein, can be carried out by any suitable method known and described in the art. Suitable methods are also described elsewhere herein and in the Examples section.

[0265] New antibodies generated, obtained, or obtainable by these methods constitute another aspect of the invention.

[0266] The term "substantially homologous" also includes modifications or chemical equivalents of the amino acid and nucleotide sequences of the invention that perform substantially the same function as the protein or nucleic acid molecule of the invention in substantially the same manner. For example, any substantially homologous antibody should retain the ability to bind CD163 as described above. Preferably, any substantially homologous antibody should retain one or more (or all) of the functional properties of the starting antibody, for example, in appropriate cases, retain the membrane-specific binding ability as described elsewhere herein.

[0267] Preferably, any substantially homologous antibody should retain the ability to bind specifically to the same epitope on CD163 recognized by the starting antibody involved, such as the CDR domains of one or more antibodies of the invention or the same epitope recognized by the VH (VHH) domain of the invention as described herein, for example, bind to the same epitope as one or more of the various antibodies of the invention (such as one or more of the VHH antibodies shown in Tables A, B, C, D, or E). Thus, preferably, any substantially homologous antibody should retain the ability to compete with one or more different antibodies of the invention (such as the VHH antibodies shown in Tables A, B, C, D, or E, as appropriate) for binding to CD163 in a suitable assay.

[0268] Binding to the same epitope / antigen can be readily tested by methods well-known and described in the art, such as using binding assays (e.g., competitive assays) or by analyzing the crystal structure of the antigen-antibody complex. Retention of other functional properties can also be readily tested by methods well-known and described in the art or herein.

[0269] Thus, those skilled in the art will understand that binding assays can be used to test whether any antibody (e.g., a "substantially homologous" antibody) has the same binding specificity, e.g., binds the same epitope or has the same / equivalent affinity as the antibodies and antibody fragments of the present invention, such as binding assays like the competitive assays or ELISA assays described elsewhere herein. For example, assays that preferably measure the binding of an antibody to cells expressing CD163 (e.g., FACS assays) can be used to determine whether the antibody (or binding protein) binds to the membrane-bound form of CD163 (e.g., porcine CD163). SPR, such as a BIAcore assay, can also be readily used to determine whether an antibody (e.g., a "substantially homologous" antibody) is able to bind to CD163, e.g., whether it is able to bind (or not bind) to the soluble form of CD163. In fact, the SPR (BIAcore) assay is a preferred option for determining whether an antibody (or binding protein) does not bind or does not significantly bind to the soluble form of CD163 (e.g., porcine CD163). Those skilled in the art will be aware of other suitable methods and variations.

[0270] As described below, a competitive binding assay can be employed to detect whether an antibody (e.g., a "substantially homologous" antibody) retains the ability to specifically bind to an epitope that is substantially the same as that recognized by the antibodies shown in the various sequence listings of the present invention, or whether it has the ability to compete with one or more of the various antibodies of the present invention shown in the sequence listings herein. The method described below is only one example of a suitable competitive assay. Those skilled in the art will be aware of other suitable methods and variations.

[0271] Exemplary competitive assays involve: evaluating the binding of various effective concentrations of the antibodies of the present invention to CD163 (e.g., the membrane-bound form of CD163) in the presence of different concentrations of a test antibody (e.g., a substantially homologous antibody). The amount of binding inhibition induced by the test antibody can then be evaluated. If the test antibody shows increased competition with the antibodies of the present invention as its concentration increases (i.e., an increase in the concentration of the test antibody results in a corresponding decrease in the amount of binding of the antibodies of the present invention to CD163, e.g., binding to the membrane-bound form of CD163), it demonstrates binding to substantially the same epitope. Preferably, the test antibody significantly reduces the amount of binding of the antibodies of the present invention to CD163 (e.g., the membrane-bound form of CD163). Preferably, the test antibody reduces the amount of binding of the antibodies of the present invention to CD163 (e.g., the membrane-bound form of CD163) by at least about 95%. ELISA and flow cytometry can be used to evaluate binding inhibition in such competitive assays, but other suitable techniques are also well known to those skilled in the art. When membrane-specific antibodies of the present invention are involved, flow cytometry assays are particularly preferred.

[0272] Another embodiment of the present invention is an antibody (monoclonal antibody) that has the ability to specifically bind to an epitope that is substantially the same (or identical) or overlapping with the epitope of CD163 recognized by the antibodies of the present invention (e.g., the membrane-specific antibody of the present invention, such as the VHH antibody H17B11 shown in Table A), or is capable of competing with the antibodies of the present invention (e.g., the membrane-specific antibody of the present invention, such as the VHH antibody H17B11 shown in Table A).

[0273] As used herein, the term "competing antibody" refers to an antibody that binds to an epitope that is substantially the same, essentially the same, or identical to the epitope of a "reference antibody". "Competing antibodies" include antibodies with overlapping epitope specificities. Thus, a competing antibody can effectively compete with a reference antibody for binding to CD163, e.g., the membrane-bound form of CD163. Preferably, the competing antibody can bind to the same epitope as the reference antibody. In other words, the competing antibody preferably has the same epitope specificity as the reference antibody.

[0274] As used herein, a "reference antibody" refers to an antibody that can bind to CD163 in the present invention, which preferably has a VH domain as defined herein, more preferably has a VH domain or is a VHH antibody comprising SEQ ID NO: 1, 9, or 17 (or the relevant three CDR sequences of said sequences) listed in Tables A, B, C, D, or E. A preferred reference antibody has a VH domain or is a VHH antibody comprising SEQ ID NO: 1 (or the relevant three CDR sequences of said sequence) listed in Table A. In other words, the preferred reference antibody is the membrane-specific antibody defined in Table A.

[0275] Since reference antibodies (such as the antibodies listed in the sequence listing herein) have been provided, it is a routine technical matter to identify one or more competing antibodies or antibodies that bind to the same epitope. Particularly preferred is the identification of one or more competing antibodies that bind to the same epitope as the VHH antibodies in Table A and exhibit membrane-specific binding activity. Since competing antibodies or antibodies that bind to the same epitope can be determined by comparison with a reference antibody, it should be understood that it is not actually necessary to determine the epitope to which either or both antibodies bind in order to identify competing antibodies or antibodies that bind to the same epitope. However, if desired, standard techniques can be used for epitope mapping.

[0276] Accordingly, on the other hand, there is provided an antibody (or binding protein) comprising an antigen-binding domain that binds to or specifically binds to CD163 (such as porcine CD163), wherein the antibody (antigen-binding domain) binds to the same epitope as the VHH antibodies in Table A (or an antibody having the CDRs defined in Table A, or CDRs that are substantially homologous thereto), and exhibits membrane-specific binding activity as described elsewhere herein.

[0277] To the inventors' knowledge, antibodies (such as monoclonal antibodies) that are capable of binding to (or specifically binding to) porcine CD163, wherein the antibody binds to membrane-bound porcine CD163 but does not significantly bind to the soluble form of porcine CD163 as described herein, have not been described in the art but are described herein. Preferably, the antibody binds within the SRCR5 domain of CD163.

[0278] Thus, the individual membrane-specific monoclonal antibodies described herein (such as in Table A) are both uncommon and advantageous. In addition, the epitopes to which such antibodies bind, as well as antibodies that bind to the same epitope, are also of significance. Accordingly, it is believed that the antibodies of the present invention can bind to a novel epitope, such as a conformational epitope, in the SRCR5 region of cell surface porcine CD163, thereby conferring the ability for membrane-specific binding.

[0279] Substantially homologous sequences of the proteins of the present invention include, but are not limited to, conservative amino acid substitutions, or alterations such as those that do not affect the VH, VL, or CDR domains of the antibody (such as an antibody to which a tag sequence, toxin, or other component that does not participate in antigen binding has been added), or alterations made to convert one type or form of binding protein, antibody molecule, or fragment to another type or form of binding protein, antibody molecule, or fragment (such as converting from a VHH to a Fab or scFv or full antibody, and vice versa), or converting an antibody molecule to an antibody molecule of a specific class or subclass (such as converting an antibody molecule to an IgG or its subclass, such as IgG2).

[0280] As used herein, "conservative amino acid substitution" refers to the replacement of an amino acid residue with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In other examples, families of amino acid residues can be grouped according to hydrophobic or hydrophilic side groups.

[0281] Homology can be evaluated by any convenient method. However, in order to determine the degree of homology between sequences, computer programs capable of performing multiple sequence alignments can be used, such as Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal W algorithm can be used in conjunction with the BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915–10919, 1992), with a gap opening penalty of 10 and a gap extension penalty of 0.1, to obtain an alignment result with the highest degree of match between two sequences, where at least 50% of the total length of at least one of the sequences is involved in the alignment. Other methods that can be used to align sequences are the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970), as revised by Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482, 1981), in order to obtain the highest degree of match between two sequences and to determine the number of identical amino acids between the two sequences. Other methods for calculating the percent identity between two amino acid sequences are generally recognized in the art, such as the method described by Carillo and Lipton in (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988), and the methods described in “Computational Molecular Biology”, Lesk, e.d. Oxford University Press, New York, 1988, “Biocomputing: Informatics and Genomics Projects”.

[0282] Typically, such calculations are performed using computer programs. Programs for comparing and aligning sequence pairs, such as ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990), and gapped BLAST (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984) are also useful for this purpose. In addition, the Dali server at the European Bioinformatics Institute provides a structure-based protein sequence alignment service (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, J. Mol. Biol., 233:123-38, 1993; Holm, Nucleic Acid Res., 26:316-9, 1998).

[0283] As a reference benchmark, sequences having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology, sequence identity, etc. in the present invention can be determined using the ALIGN program with default parameters (e.g., available online through the GENESTREAM network server at the IGH, Montpellier, France).

[0284] As used herein, the terms "antibody" and "immunoglobulin" broadly refer to any immunoconjugate that contains an antigen-binding domain, including polyclonal antibodies and monoclonal antibodies. Monoclonal antibodies are preferred, however. In other words, in certain embodiments, the antibodies of the invention are not polyclonal antibodies. Depending on the type of constant region in the heavy chain, whole antibodies are classified into one of five major classes: IgA, IgD, IgE, IgG, and IgM, and the antibodies of the invention can belong to any of these classes. Several of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc., such that camel antibodies are typically IgG antibodies with IgG2 or IgG3 constant domains. The heavy chain constant domains corresponding to the different immunoglobulin classes are designated α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of the different classes of immunoglobulins are well known.

[0285] Generally, when whole antibodies rather than antigen-binding regions are used in the invention, IgG-class antibodies are preferred because such antibodies are the most common in physiological environments and are the easiest to prepare under laboratory conditions.

[0286] The "light chain" of mammalian antibodies is classified into one of two distinct types: κ and λ, based on the amino acid sequence of its constant domain and some amino acids in its variable domain framework regions.

[0287] As used herein, the term "heavy chain complementarity-determining region" ("heavy chain CDR") refers to the hypervariable regions within the variable domain (V H domain) of an antibody molecule heavy chain or within a VHH antibody molecule. The heavy chain variable domain has three CDRs from the amino terminus to the carboxyl terminus, designated heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, respectively. The heavy chain variable domain also has four framework regions (FR1, FR2, FR3, and FR4 from the amino terminus to the carboxyl terminus). These framework regions separate the CDRs.

[0288] As used herein, the term "heavy chain variable domain" (V H domain) refers to the variable domain of an antibody molecule heavy chain.

[0289] As used herein, the term "light chain complementarity-determining region" ("light chain CDR") refers to the hypervariable regions within the variable domain (V L domain) of an antibody molecule light chain. The light chain variable domain has three CDRs from the amino terminus to the carboxyl terminus, designated light chain CDR1, light chain CDR2, and light chain CDR3, respectively. The light chain variable domain also has four framework regions (FR1, FR2, FR3, and FR4 from the amino terminus to the carboxyl terminus). These framework regions separate the CDRs.

[0290] As used herein, the term "light chain variable domain" (V L domain) refers to the variable domain of an antibody molecule light chain.

[0291] Those skilled in the art will understand that the immunoconjugate reagents covered by the term "antibody" include or extend to all antibodies and their antigen-binding fragments, including intact antibodies, dimeric, trimeric, and multimeric antibodies; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies and their fragments.

[0292] Accordingly, the term "antibody" is used to refer to any antibody-like molecule having an antigen-binding region (e.g., an antigen-binding region containing CDRs derived from or corresponding to an antibody molecule and optionally FRs), and the term includes antibody fragments containing an antigen-binding domain, such as Fab', Fab, F(ab')2, single-domain antibodies (DABs), TandAbs dimers, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bisomes, trisomes (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabodies; κ(λ) bodies (scFv-CL fusions); BiTE (bispecific T cell engager, scFv-scFv tandem for recruiting T cells); DVD-Ig (dual variable domain antibody, bispecific form); SIP (small immunoprotein, a type of minibody); SMIP ("small modular immunopharmaceutical", scFv-Fc dimer); DART (ds-stabilized diabody "dual affinity retargeting"); and small antibody mimetics containing one or more CDRs, etc.

[0293] Techniques for preparing and using various antibody-based constructs and fragments are well known in the art.

[0294] Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragments can be treated by reducing disulfide bonds to generate Fab' fragments. Papain digestion can result in the formation of Fab fragments. Fab, Fab', F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized recombinantly or chemically synthesized. Techniques for generating antibody fragments are well known in the art and have been described.

[0295] In all embodiments of the present invention, single-domain antibodies (also known as VHH antibodies, sdAbs, DABs, dAbs, nanobodies, camelid antibodies, vNAR (shark) antibodies, VH antibodies or VL antibodies) are preferred, particularly VHH antibodies, nanobodies, camelid antibodies and vNAR (shark) antibodies. Such antibodies comprise a single monomeric variable antibody domain, typically a VH domain, which is capable of binding to an antigen (although single VL domains with antigen-binding capabilities have been described and can be used). Thus, in certain such preferred embodiments, the antibody (or antigen-binding domain) of the present invention comprises (or consists of) one (or a single or only a single or one or only one) heavy-chain variable region (VH or VHH), although in certain embodiments, multiple such individual heavy-chain variable regions having the same or different sequences may co-exist in the same construct or molecule.

[0296] Such antibodies can be obtained or prepared using standard techniques well known and described in the art. For example, such antibodies can be obtained by immunizing a suitable animal (e.g., a camelid such as a llama) or a shark with the desired antigen, and then cloning the VH domain of the resulting antibody into a suitable expression vector and selecting for binders. VH domain libraries (e.g., human VH domain phage display libraries) are also available, or can be generated and screened.

[0297] Due to the relatively small molecular weight of single-domain antibodies, their half-life may be relatively short, for example, the plasma half-life is relatively short. Therefore, such antibodies are sometimes modified to extend or maintain their half-life, and such modified antibodies (binding proteins) form part of the present invention. The techniques for achieving this goal are well-known and documented in the art, and any of these methods can be employed. Examples include linking, conjugating, or fusing the antibody (binding protein) to albumin / serum albumin (or another protein or entity that itself has a long (or longer) half-life, such as a protein or entity with a longer half-life than the fused antibody, or an alternative entity or protein that can extend the half-life of the protein (such as an antibody) to which it is linked), or linking, conjugating, or fusing the antibody (binding protein) to another protein or entity that itself can interact with a protein or entity having a long (or longer) half-life (such as an antibody, e.g., a VHH antibody) (e.g., linking to an antibody that can bind to IgG (such as porcine IgG), e.g., a VHH antibody), or linking or conjugating the antibody (binding protein) to PEG (or other polymers, such as hydrophilic polymers), or linking, conjugating, or fusing the antibody (binding protein) to an antibody or other protein or entity that binds to FcRn. In this regard, fusion with the IgG Fc region is a well-established strategy for extending the half-life of therapeutic proteins. Therefore, preferably the antibody (binding protein) comprises an Fc region or domain, for example, is fused with an Fc region or domain (in other words, is an Fc fusion). Such Fc regions or domains are known in the art and generally comprise the CH2 and CH3 domains of the antibody heavy chain, which combine to form a homodimer. These regions can be derived from any suitable source or species, for example, a source or species different from the host species used to produce the antibody (such as by immunization), or a source or species different from the antibody source, but preferably correspond to or are derived from a porcine Fc region or domain.

[0298] Thus, in certain embodiments of the present invention, the antibody (or binding protein) comprises, is linked to, or otherwise fused to an entity that can extend the half-life, preferably albumin or the IgG Fc region.

[0299] In certain embodiments of the present invention, the antibody (or binding protein) comprises, is linked to, or otherwise fused to an entity that can extend the half-life, for example, another antibody that can extend the half-life, such as a VHH antibody.

[0300] In certain embodiments, the antibodies or antibody fragments of the invention comprise all or part of a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG2 heavy chain constant region or a portion thereof. Additionally, the antibody or antibody fragment may comprise all or part of a κ light chain constant region or a λ light chain constant region, or a portion thereof. These constant regions may be naturally occurring in whole or in part, or may be obtained in whole or in part by synthetic means. Suitable sequences for such constant regions are well known and documented in the art. When the antibodies of the invention comprise the complete complement of heavy and light chain constant regions, such antibodies are commonly referred to as "full-length" antibodies or "complete" antibodies. In some embodiments, IgG2 antibodies are preferred.

[0301] In other embodiments, it is preferred that no constant regions are present, such as no heavy or light chain constant regions, such that the variable domain or the heavy chain variable domain (VH) is the only part present in the antibody.

[0302] The antibody or antibody fragment may occur naturally or may be prepared in whole or in part by synthetic means.

[0303] Many antibodies or antibody fragments comprise an antibody light chain variable region (V L ) containing three CDR domains and an antibody heavy chain variable region (V H ) containing three CDR domains. The VL and VH typically form the antigen binding site.

[0304] However, the prior art literature has amply demonstrated that the presence of the three CDRs of the light chain variable region and the three CDRs of the heavy chain variable region of an antibody is not always necessary for antigen binding. Thus, constructs smaller than the above-described classical antibody fragments are known to be equally effective.

[0305] For example, camel antibodies have a broad antigen binding spectrum but lack a light chain. Additionally, results with single domain antibodies comprising only the VH domain or the VL domain have shown that these domains are capable of binding antigen with acceptably high affinity and have other advantages, such as their small size and ease of production. Thus, the three CDRs can effectively bind antigen, and the single domain antibodies (such as VHH antibodies) described and exemplified herein are preferred.

[0306] The antibodies, binding proteins, and nucleic acid molecules of the present invention are generally "isolated" or "purified" molecules, as they are distinct from any such components that may be present in a human or animal body (e.g., a camelid) or in a tissue sample derived from a human or animal body (e.g., a camelid). However, these sequences may correspond to or be substantially homologous to sequences found in a human or animal body (e.g., a camelid). Thus, the terms "isolated" or "purified" as used herein in reference to nucleic acid molecules or sequences and proteins or polypeptides (e.g., antibodies) refer to molecules that have been separated, purified, or substantially removed from their natural environment, such as being isolated or purified from a human or animal body (if they are indeed naturally occurring), or to molecules produced by technical means, i.e., including recombinantly and synthetically produced molecules.

[0307] It is noted that the antibodies and the like of the present invention do not exist in nature and are thus artificial constructs in this regard, as they do not correspond to naturally occurring molecules. For example, preferred antibodies are single-domain antibodies that can be produced by engineering or recombination. Even in species that naturally produce such antibodies (e.g., camelids), such species do not produce antibodies against CD163 (especially porcine CD163) unless induced experimentally, such as by immunization. In other words, the antibodies and the like of the present invention are non-natural.

[0308] As used herein, the term "fragment" refers to a biologically relevant fragment, such as a fragment involved in antigen binding (e.g., forming part of an antigen-binding site), and / or a fragment that contributes to the functional properties of a CD163 antibody. Certain preferred fragments comprise or consist of the variable region of the heavy chain (V H domain or three VH CDRs) of the antibody of the present invention.

[0309] Those skilled in the art will understand that the proteins and polypeptides of the present invention, such as heavy and light chain CDRs, heavy and light chain variable regions, antibodies, and antibody fragments, can be prepared by a variety of methods known and described in the art, but are most preferably prepared using recombinant methods.

[0310] The nucleic acid fragments encoding the variable regions of the heavy and light chains of the antibodies of the present invention can be derived or produced by any suitable method (e.g., cloning or synthesis) as the case may be.

[0311] Once nucleic acid fragments encoding the variable regions of the heavy and / or light chains of the antibodies of the present invention are obtained, these fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region fragments into full-length antibody molecules with appropriate constant region structures, or into antibody fragments in specific forms discussed elsewhere herein, such as single-domain antibodies (e.g., VHH, Fab fragments, scFv fragments, etc.), or forms in which multiple antibodies (e.g., single-domain antibody / VHH antibody) are present, such as the bispecific and trispecific constructs described herein. Generally, or as part of further manipulation procedures, the nucleic acid fragments encoding the antibody molecules of the present invention are typically integrated into one or more suitable expression vectors to facilitate the production of the antibodies of the present invention, or, for example, by integration into a phage display vector to facilitate selection or screening.

[0312] Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), provided that the vector is compatible with the host cell used. Such an expression vector "is suitable for transformation of a host cell" means that the expression vector contains the nucleic acid molecule of the present invention and regulatory sequences selected according to the host cell to be used for expression, and these regulatory sequences are operably linked to the nucleic acid molecule. "Operably linked" means that the nucleic acid is linked to the regulatory sequences in a manner that permits its expression.

[0313] Accordingly, the present invention contemplates an expression vector, such as a recombinant expression vector, that contains or comprises the nucleic acid molecule of the present invention or a fragment thereof, and regulatory sequences required for transcription and translation of the protein sequence encoded by the nucleic acid molecule of the present invention.

[0314] The expression vector can be introduced into a host cell to produce a transformed host cell. The terms "transformation", "transfection", "transformation process", and "transfection process" are intended to encompass the introduction of a nucleic acid (e.g., a vector) into a cell by one of many possible techniques known in the art. Methods suitable for transformation and transfection of host cells can be referred to the works of Sambrook et al. in 1989 (Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and other laboratory textbooks.

[0315] Suitable host cells include a variety of eukaryotic host cells and prokaryotic cells. For example, the proteins of the present invention can be expressed in yeast cells or mammalian cells. In addition, the proteins of the present invention can be expressed in prokaryotic cells, such as Escherichia coli.

[0316] Other expression vectors will include RNA or mRNA expression vectors, such as self - amplifying RNA expression vectors, which can be used, for example, to express the antibodies or constructs or combinations of the present invention in a subject to be treated.

[0317] The proteins of the present invention can also be prepared by chemical synthesis using techniques well - known in protein chemistry, such as solid - phase synthesis.

[0318] Another aspect provides an expression construct or expression vector or expression system (such as a virus or bacterium or other expression construct, vector or system) that contains one or more nucleic acid fragments or segments or molecules of the present invention. Preferably, the expression construct, vector or system is recombinant. Preferably, the construct, vector or system also contains regulatory sequences required for transcribing and / or translating the protein sequences encoded by the nucleic acid molecules of the present invention. Preferably, constructs etc. should be able to achieve persistent or continuous expression of the antibodies (or binding proteins) of the present invention in the host target species (such as in pigs). Such expression can be transient, such as episomal, or more persistent, such as through genomic integration, as long as sufficient expression levels and durations are achieved to observe therapeutic or biological effects. Similarly, self - amplifying RNA expression vectors are a suitable example.

[0319] On the other hand, a host cell (such as a mammalian, bacterial or yeast host cell) or virus, or other delivery vector (such as a lipid - based delivery vector, such as liposomes or lipid nanoparticles) is provided, which contains one or more expression constructs or expression vectors of the present invention. Host cells, viruses or delivery vectors containing one or more nucleic acid molecules of the present invention are also provided. Host cells (such as mammalian host cells, bacterial host cells or yeast host cells) or viruses, or delivery vectors that express the antibodies (or binding proteins) or constructs or combinations of the present invention, or that contain the expression constructs or nucleic acid molecules of the present invention, constitute another aspect.

[0320] Such expression constructs or vectors or systems, or host cells or viruses or delivery vectors, or other nucleic acid products or fragments encoding the antibodies (or binding proteins) or constructs or combinations of the present invention, can be administered as therapeutic agents to a subject to produce in situ in the subject the antibodies (or binding proteins) etc. of the present invention, thereby exerting their therapeutic effects.

[0321] Another aspect of the present invention provides a method for producing (or manufacturing) the antibody, binding protein, protein construct or combination of the present invention, which comprises the step of culturing the host cell of the present invention. The preferred method comprises the following steps: (i) culturing a host cell (e.g., in a culture medium) containing one or more recombinant expression vectors or one or more nucleic acid sequences of the present invention under conditions suitable for expressing an antibody or binding protein; and optionally (ii) isolating or obtaining the antibody or binding protein from the host cell or the growth medium / culture supernatant. Such production (or manufacturing) methods may also include the steps of purifying the antibody or binding protein product and / or formulating the antibody or product into a composition containing at least one other component (e.g., a pharmaceutically acceptable carrier or excipient).

[0322] In embodiments where the antibody or binding protein of the present invention consists of more than one polypeptide chain (e.g., certain fragments, such as Fab fragments or full antibodies), all polypeptides are preferably expressed in the host cell, from the same or different expression vectors, such that the complete protein (e.g., the antibody protein of the present invention) can be assembled within the host cell and isolated or purified therefrom.

[0323] On the other hand, the present invention provides a method for binding to CD163, which comprises contacting a composition containing CD163 with the antibody of the present invention.

[0324] In yet another aspect, the present invention provides a method for detecting CD163, which comprises contacting a composition suspected of containing CD163 with the antibody of the present invention under conditions effective to permit the formation of a CD163 / antibody complex and detecting the formed complex.

[0325] A composition containing at least one first antibody (or binding protein) or construct or combination of the present invention, or a nucleic acid molecule, expression vector or host cell of the present invention constitutes another aspect of the present invention. A preparation (composition) containing one or more antibodies or constructs or combinations of the present invention, or a nucleic acid molecule, expression vector or host cell of the present invention and mixed with a suitable diluent, carrier or excipient constitutes a preferred embodiment of the present invention. Such preparations can be used for medical purposes, for example, for animal health applications or veterinary uses (e.g., in the farming industry), and thus the compositions of the present invention are preferably pharmaceutically acceptable or suitable for administration to non-human animals (e.g., mammals, preferably pigs). Suitable diluents, excipients and carriers are known to those skilled in the art.

[0326] The compositions of the present invention can be in a form suitable for oral, nasal, parenteral (e.g., intramuscular, subcutaneous or intradermal), intravenous, topical or rectal administration, for example. Intramuscular administration is particularly convenient.

[0327] The active compounds defined herein (e.g., the antibodies of the present invention) can exist in conventional pharmacological administration forms, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules or sustained-release forms. These dosage forms can be prepared using conventional pharmaceutical excipients and common production methods.

[0328] Injection solutions can be prepared, for example, in a conventional manner, such as by adding preservatives (e.g., parabens) or stabilizers (e.g., EDTA). The solution can then be filled into injection bottles or ampoules.

[0329] Those skilled in the art can determine appropriate dosage units.

[0330] In the context of a combination therapy or co - administration regimen, the pharmaceutical composition can also contain other active ingredients (e.g., as described elsewhere herein).

[0331] Another aspect of the present invention provides an anti - CD163 antibody (or binding protein) or construct or combination as defined herein, or a nucleic acid molecule, expression vector or host cell of the present invention, for use in therapy, particularly for treating or preventing any disease or disorder associated with CD163, or in which CD163 plays a role, e.g., a pathogenic role (e.g., a total or pathogenic role) or a key role. For example, the anti - CD163 antibody of the present invention can be used to treat or prevent any infection caused by a virus or other pathogen, wherein the infection is associated with CD163, or in which CD163 plays a role, e.g., a pathogenic role (e.g., a total or pathogenic role) or a key role. Exemplary diseases include PRRSV infection or simian hemorrhagic fever virus (SHFV). In other words, according to the present invention, an anti - CD163 antibody (or binding protein) or construct or combination, etc. can target and inhibit or reduce the function of CD163, particularly CD163 expressed on or within PAM or other CD163 - positive cells. Thus, an anti - CD163 antibody (or binding protein) or construct or combination as defined herein can be used to treat or prevent any disease or disorder that requires inhibition of CD163 or blocking or reduction of the function of CD163.

[0332] Preferred embodiments provide an anti - CD163 antibody (or binding protein) or construct or combination, etc. of the present invention for treating or preventing swine infections, preferably swine viral infections. Particularly preferably for treating or preventing PRRSV infection. In embodiments for treating swine, the anti - CD163 antibody (or binding protein) or construct or combination, etc. of the present invention is typically an anti - swine CD163 antibody (or binding protein), or comprises or encodes such an anti - swine CD163 antibody (or binding protein).

[0333] CD163 is considered a possible receptor for all PRRS virus strains. However, as described elsewhere herein, there are two types of PRRSV: PRRSV-1 and PRRSV-2. Although the PRRSV-1 and PRRSV-2 viruses are phenotypically similar at multiple levels, there are differences between the virus species. The antibodies (or binding proteins) or constructs or combinations, etc. of the present invention can be used to treat or prevent PRRSV-1 and / or PRRSV-2 infections, such as PRRSV-1 and PRRSV-2 infections, or to treat or prevent (e.g., specifically treat or prevent) PRRSV-2 infections.

[0334] In the treatment methods and uses of the present invention, the binding proteins, antibodies, constructs or combinations, etc. are administered to a subject in need of treatment (an animal or mammal, such as a pig) in a pharmaceutically, therapeutically or physiologically effective amount. Therefore, the said methods and uses may involve additional steps of identifying the subject in need of treatment.

[0335] According to the present invention, the treatment of a disease or disorder (e.g., treating an existing disease) includes curing the disease or disorder, or any alleviation or remission of a disease (e.g., reducing the severity of the disease) or disorder.

[0336] The treatment methods and uses of the present invention are applicable to the prevention of diseases as well as the active treatment of diseases (e.g., treating an existing disease). Therefore, the present invention also encompasses prophylactic treatment and post-prophylactic treatment (treating a group of subjects when a disease breaks out, e.g., treating a group of subjects after a part of the group is diagnosed with infection and / or clinical disease, with the aim of preventing the spread of the infectious disease to animals in close contact and / or at high risk). Therefore, in the methods and uses of the present invention, treatment also includes prophylactic treatment, post-prophylactic treatment or preventive treatment, as appropriate.

[0337] Such prophylactic (or protective) measures can be conveniently carried out on healthy, normal or at-risk subjects, and can include complete prevention and significant prevention. Similarly, significant prevention can include cases where the severity of the disease or the symptoms of the disease are reduced (e.g., measurably or significantly reduced) compared to the severity or symptoms expected without treatment.

[0338] The subjects preferably to be treated are those who have been infected or are at risk of infection with PRRSV. However, as described elsewhere herein, the treatment methods of the present invention can also be used for subjects suffering from other (non-PRRSV) infections (e.g., suffering from other bacterial or viral infections, or even complex (multi-pathogen) infections). In particular, such treatment methods are applicable to subjects with a measurable level of soluble CD163 in the serum, or a level of soluble CD163 that is elevated (e.g., significantly elevated) compared to healthy subjects.

[0339] For example, the clinical symptoms of PRRSV infection include fetal resorption, stillbirth, and late-term abortions in pregnant or gilts, as well as respiratory diseases and syndromes in all pigs (especially piglets and suckling pigs), such as respiratory distress. Other symptoms include anorexia (which typically results in reduced growth rate or weight loss, or decreased weight gain, such as reduced average daily weight gain), fever, lethargy, respiratory distress (such as pneumonia or pneumonia / lung lesions), reproductive failure, and diarrhea (especially in piglets), central nervous system (CNS) symptoms, and death. Individuals infected with PRRSV are also susceptible to endemic diseases, such as meningitis, Glasser's disease, exudative dermatitis, sarcoptic mange, and bacterial bronchopneumonia, which have been reported to have a generally increased incidence (Diseases of Swine, Eleventh Edition, Editor(s): Jeffrey J. Zimmerman, Locke A. Karriker, Alejandro Ramirez, Kent J. Schwartz, Gregory W. Stevenson, Jianqiang Zhang, First published: 29 March 2019) or to PCV2 infection. Such diseases are typically controlled with antibacterial products such as antibiotics. Thus, the present invention helps reduce the use of antibacterial products on farms.

[0340] Thus, the antibody or binding protein or construct or combination of the present invention can be used to treat or prevent clinical diseases or symptoms, such as those associated with PRRSV infection or downstream endemic diseases (such as the downstream endemic diseases described above), or to reduce the circulation of a virus (such as PRRSV) (such as the viral load in serum) or to prevent infection (such as a first infection) or a new infection (such as a second or subsequent infection), such as a PRRSV infection (such as a first PRRSV infection) or a new PRRSV infection (such as a second or subsequent PRRSV infection).

[0341] Accordingly, preferred subjects for treatment according to the present invention include all types of pigs (sometimes also referred to as swine breeds), such as any pig, swine breed or species of the family Suidae, including pigs of all ages and species, provided they are susceptible to or capable of being infected by the pathogens (in particular PRRSV) defined herein. Piglets, especially those born to infected sows or live-born piglets (up to 80% of which may die), are particularly preferred subjects for treatment, as are nursery pigs (e.g., pigs up to 12 weeks of age after weaning) and growing or finishing pigs (e.g., pigs up to slaughter age), especially growing pigs. Piglets before weaning, e.g., piglets up to 4 weeks of age (especially piglets of infected sows, where infection may be transmitted through the mammary secretions of the infected sow), as well as gilts, sows and pregnant sows are also preferred subjects for treatment. Since the preferred subjects include all types of pigs, it should be understood that in all embodiments and aspects, the subjects include groups of subjects, e.g., herds and litters of pigs, but treatment of individual animals is not excluded.

[0342] In certain embodiments, e.g., when it comes to prevention, the subject is a subject at risk of being affected by the disease or disorder, e.g., a subject at risk of being infected by the above-mentioned pathogen or virus (e.g., PRRSV) and developing the disease. Such subjects can be healthy subjects, or subjects that do not exhibit any symptoms of the disease, or any other suitable "at-risk" subject. In another embodiment, the subject is a subject suffering from, suspected of suffering from (or developing), or likely to suffer from (or develop) the above-mentioned disease or disorder.

[0343] In other words, the present invention provides a method for treating or preventing a disease or disorder associated with or in which CD163 plays a role, e.g., a disease with a pathogenic effect (e.g., a total or pathogenic effect) or a key role, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-CD163 antibody (or binding protein) or construct or combination of the present invention as defined herein. Suitable diseases, disorders or subjects are described elsewhere herein.

[0344] Preferably for the treatment or prevention of pig infections, especially for pig viral infections. Particularly preferred for the treatment or prevention of PRRSV infection, e.g., the treatment or prevention of PRRSV-1 and / or PRRSV-2 infection (e.g., PRRSV-1 and PRRSV-2 infections), or the treatment or prevention (e.g., specific treatment or prevention) of PRRSV-2 infection.

[0345] Accordingly, another embodiment provides a method of treating or preventing porcine PRRSV infection, e.g., a method of treating or preventing porcine PRRSV-1 and / or PRRSV-2 infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of a monoclonal antibody that binds to porcine CD163. CD163 antibodies (or binding proteins) or constructs or combinations suitable for such methods are described herein.

[0346] Accordingly, in another aspect, provided is a method of treating or preventing PRRSV infection in a subject (preferably a pig), e.g., a method of treating or preventing PRRSV-1 and / or PRRSV-2 infection in said subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody, binding protein, or combination thereof of the invention. Such methods of treatment may alternatively involve administering one or more nucleic acid molecules, expression vectors, or host cells of the invention.

[0347] The therapeutically effective amount will be determined based on clinical assessment and is readily monitored.

[0348] The embodiments of the therapeutic uses of the invention described herein, mutatis mutandis, apply to this aspect of the invention.

[0349] In further other words, the invention provides the use of an anti-CD163 antibody (or binding protein) or construct or combination of the invention (e.g., a monoclonal antibody of the invention as defined herein) in the preparation of a medicament for treatment. Preferred therapeutic uses are described elsewhere herein, particularly for treating or preventing any disease or disorder in which CD163 is involved or plays a role, e.g., a pathogenic role (e.g., a full or pathogenic role) or a key role. For example, the anti-CD163 antibody (or binding protein) of the invention can be used to treat or prevent any infection caused by a virus or other pathogen, wherein the infection is associated with CD163 or CD163 plays a role therein, e.g., a pathogenic role (e.g., a full or pathogenic role) or a key role. In other words, according to the invention, the anti-CD163 antibody (or binding protein) can target and inhibit or reduce the function of CD163, particularly CD163 expressed on or within PAM or other CD163-positive cells. Accordingly, the anti-CD163 antibody (or binding protein) or construct or combination as defined herein can be used to treat or prevent any disease or disorder in which it is desired to inhibit CD163 or block or reduce the function of CD163.

[0350] Preferred embodiments provide for the use of the anti-CD163 antibody (or binding protein) of the invention in the preparation of a medicament for treating or preventing a swine infection, preferably a swine viral infection. Particularly preferred is for treating or preventing PRRSV infection, such as treating or preventing PRRSV-1 and / or PRRSV-2 infection (e.g., PRRSV-1 and PRRSV-2 infection), or treating or preventing (e.g., specifically treating or preventing) PRRSV-2 infection.

[0351] Accordingly, in another aspect, there is provided the use of a monoclonal antibody that binds to porcine CD163 in the preparation of a medicament for treating or preventing a porcine PRRS virus infection (preferably PRRSV-1 and / or PRRSV-2 infection). CD163 antibodies (or binding proteins) or constructs or combinations suitable for such use are described herein.

[0352] Accordingly, in a further aspect, there is provided the use of the antibody, binding protein or combination of the invention in the preparation of a medicament for treating a subject, preferably for treating or preventing a PRRSV infection in a subject, such as for treating or preventing a PRRSV-1 and / or PRRSV-2 infection in a subject (preferably a swine). Such use may alternatively involve the use of one or more nucleic acid molecules, expression vectors or host cells of the invention.

[0353] The embodiments of the therapeutic uses of the invention described herein, mutatis mutandis, are applicable to this aspect of the invention.

[0354] In certain embodiments, the antibodies (or binding proteins) of the invention can be used in combination. For example, the membrane-specific antibodies of the invention (such as the antibodies defined in Table A) can be used in combination with one, two or more alternative anti-CD163 antibodies (such as the antibodies defined in Table B (or D) and / or the antibodies defined in Table C (or E)). Preferred anti-CD163 antibody combinations are combinations in which each anti-CD163 antibody in the combination binds to a different epitope on the CD163 molecule, such as the bispecific (two different epitopes) or trispecific (three different epitopes) constructs described herein.

[0355] Any combination of the VHH antibodies shown in Table A, Table B (or D) and Table C (or E) can be used.

[0356] Preferred combinations include:

[0357] The VHH antibody of Table A and the VHH antibody of Table B (or D).

[0358] The VHH antibody of Table A and the VHH antibody of Table C (or E).

[0359] The VHH antibody of Table B (or D) and the VHH antibody of Table C (or E).

[0360] The VHH antibodies of Table A, the VHH antibodies of Table B (or D), and the VHH antibodies of Table C (or E).

[0361] In all of the above combinations, antibodies having the 3 CDRs shown in Tables A to E, or substantially homologous sequences thereof, may also be used.

[0362] Preferred combinations are those that can improve or enhance (preferably significantly improve or enhance) the therapeutic effect as compared to any antibody of the invention (e.g., VHH) administered as a single active agent (monotherapy), single antibody, or single anti-CD163 agent. Other preferred combinations are those in which the individual anti-CD163 antibodies in the combination bind to different epitopes on the CD163 molecule. As described elsewhere herein, in certain embodiments of the invention, the antibodies (or binding proteins) of the invention may also be used in combination with entities that can extend the half-life, such as another antibody (e.g., a VHH antibody) that can extend the half-life, or any other suitable entity for extending the half-life.

[0363] For such combination therapies using two or more antibodies (or binding proteins) of the invention, the second (or subsequent, e.g., third) anti-CD163 antibody of the invention can be administered to a subject substantially simultaneously with the first anti-CD163 antibody of the invention, e.g., by a single pharmaceutical composition or by administering two pharmaceutical compositions closely together (simultaneously or at similar times). Alternatively, the second (or subsequent, e.g., third) anti-CD163 antibody of the invention can be administered to the subject at a time before or after the administration of the first anti-CD163 antibody of the invention. As used herein, "before or after" means "staggered", i.e., the second antibody is administered to the subject at a time different from the administration of the first anti-CD163 antibody component. Generally, the two (or more) components can be administered at effectively spaced times or simultaneously such that each component exerts its respective therapeutic effect, i.e., they are administered in a "biologically effective amount" over a "biologically effective time interval" and as part of the same treatment regimen.

[0364] As described elsewhere herein, preferably, a combination of the anti-CD163 antibodies (or binding proteins) of the invention (and optionally an entity for extending the half-life), if appropriate, can be conveniently administered as part of the same molecule or construct, e.g., can be conjugated or linked together, e.g., by an artificial linker. This mode of administration can be particularly suitable for VHH antibodies (or other types of antibody molecules consisting of a single polypeptide chain), in which the individual antibodies can be conveniently linked in a single polypeptide chain containing multiple VHH (or other) antibodies by an appropriate peptide (or other) linker (e.g., a non-natural peptide or an artificial linker), and these antibodies can be of the invention or can be combined with other VHHs or other antibodies.

[0365] Suitable linkers are well known in the art and have been described. Exemplary linkers can include GS linkers, such as one or more repeats of the G4S linker (GGGGS, SEQ ID NO:43). The linker sequence used in the constructs employed in the appended examples was GGGGSGGGGS (SEQ ID NO:44), i.e., 2 repeats of GGGGS. Linkers with 5 repeats were also used. However, it should be understood that linkers (spacers) with other sequences and lengths can also be used, such as other GS linkers, or other suitable linkers.

[0366] In such embodiments, the antibodies are typically linked together using appropriate techniques (such as spacing) such that each component can perform its respective function, such as binding to CD163. For example, in embodiments where the anti-CD163 antibodies of the invention bind to different epitopes on CD163, combinations of such antibodies are preferred and the constructs are designed appropriately such that each antibody can bind to CD163, such as to its CD163 epitope.

[0367] Thus, in certain embodiments, the anti-CD163 antibodies (or binding proteins) of the invention can be used as the sole active drug in a treatment regimen (monotherapy), or multiple anti-CD163 antibodies of the invention can be used in combination, such as as described above. In certain embodiments, the anti-CD163 antibodies (or binding proteins) (or appropriate combinations) of the invention can be used as the sole active anti-CD163 agent or the sole active anti-CD163 antibody in a treatment regimen, or they can be the sole active anti-PRRSV drug in a treatment regimen. However, in certain embodiments, other anti-CD163 agents or anti-PRRSV agents can be used.

[0368] Thus, the anti-CD163 binding proteins or antibodies (or appropriate combinations) of the invention can be combined with one or more other (other CD163-targeted or non-CD163-targeted) active agents, such as in combination with at least one therapeutic agent or biologic, where the anti-CD163 binding protein or antibody (or combination of such binding proteins or antibodies) of the invention is the first therapeutic agent or biologic.

[0369] The anti-CD163 antibodies (or binding proteins) (or appropriate combinations) of the invention can be combined, for example, with any other therapeutic agent or vaccine that can be used to treat or prevent the diseases described elsewhere herein (such as PRRSV or other diseases).

[0370] Suitable and exemplary dosing regimens for such combination therapies can be as described elsewhere herein for anti-CD163 antibody combinations.

[0371] The present invention also includes a kit, which comprises one or more antibodies, constructs or compositions of the present invention, or one or more nucleic acid molecules encoding the antibodies or constructs of the present invention, or one or more recombinant expression vectors comprising the nucleic acid sequences of the present invention, or one or more host cells or viruses comprising the recombinant expression vectors or nucleic acid sequences of the present invention. Preferably, the kit is for the methods and uses described herein, such as the therapeutic methods described herein. Preferably, the kit comprises instructions for use of the kit components. Preferably, the kit is for treating the diseases or disorders described in other parts of this document, and optionally comprises instructions for use of the kit components for treating such diseases or disorders. Equivalent embodiments of the binding proteins of the present invention are also provided.

[0372] The antibodies (or binding proteins) of the present invention as defined herein can also be used as molecular tools for in vitro or in vivo applications and assays. Since antibodies (and binding proteins) have antigen-binding sites, they can function as members of a specific binding pair, and these molecules can be used in any assay that requires a member of a specific binding pair.

[0373] Accordingly, another aspect of the present invention provides a reagent comprising the antibodies (or binding proteins) of the present invention as defined herein, and the use of such antibodies (or binding proteins) as molecular tools, such as for in vitro or in vivo assays.

[0374] As used herein, the terms "reduce" or "decrease" (or equivalent terms) include any measurable reduction or decrease compared to an appropriate control. An appropriate control can be readily identified by those skilled in the art and can include untreated or placebo-treated subjects, healthy subjects, or samples or assays that do not contain the antibodies (or binding proteins) of the present invention. Preferably, the reduction or decrease will be significant, such as clinically or statistically significant.

[0375] As used herein, the term "increase" (or equivalent terms) includes any measurable increase or elevation compared to an appropriate control. An appropriate control can be readily identified by those skilled in the art and can include untreated or placebo-treated subjects, healthy subjects, or samples or assays that do not contain the antibodies (or binding proteins) of the present invention. Preferably, the increase will be significant, such as clinically or statistically significant.

[0376] Preferably, such an increase (and other increases, improvements or positive effects mentioned elsewhere in this text) or such a decrease (and other decreases, reductions or negative effects mentioned elsewhere in this text) is a measurable increase, decrease, etc. (as appropriate), more preferably a significant increase, decrease, etc., preferably a clinically or statistically significant increase, decrease, etc., for example a probability value ≤ 0.05 or < 0.05, when compared with an appropriate control level or value (e.g., compared with untreated or placebo-treated subjects, or with healthy or normal subjects, or with the same subject before treatment, or with a sample or assay in the absence of the antibody (or binding protein) of the present invention).

[0377] Methods for determining the statistical significance of differences between test groups of subjects or differences in the levels of specific parameters are well known in the art and are documented. For example, in this text, when a statistical comparison is made using an appropriate significance test (such as the Student t-test, Mann-Whitney U Rank-Sum test, chi-square test, Fisher's exact test, one-way analysis of variance or two-way analysis of variance), if the probability value ≤ 0.05 or < 0.05, an increase / decrease in the level of a specific parameter or a difference between test groups of subjects is generally considered to be statistically significant.

[0378] The amino acid sequence listing and its sequence identifiers (SEQ ID NO) disclosed in this article

[0379] All amino acid sequences are listed herein from the N-terminus to the C-terminus in accordance with the convention of this technical field.

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387] The present invention will now be further described in the following non-limiting examples with reference to the following drawings:

[0388] Figure 1 – Dose-response FACS binding experiment performed on pPAM wild-type cells using an exemplary bispecific antibody candidate.

[0389] Figure 2 - Under the condition of increasing the concentration of the soluble form of CD163 protein, perform a FACS competition experiment on pPAM wild-type cells using the exemplary bispecific antibody candidate.

[0390] Figure 3 - Infection experiment: Monomeric VHH, PRRSV-1BOR57 infection.

[0391] Figure 4 - Infection experiment: Monomeric VHH, PRRSV-2MN184 infection.

[0392] Figure 5 - Infection experiment: Bispecific VHH, PRRSV-1BOR57 infection.

[0393] Figure 6 - Infection experiment: Bispecific 03E11 + 03D03 2(G4S) dose response, PRRSV-1LT3, Sigma RPMI, 10% FBS.

[0394] Figure 7 - Infection experiment: Bispecific 03E11 + 03D03 2(G4S) dose response, PRRSV-1LT3, Sigma RPMI 80% low CD163 porcine serum.

[0395] Figure 8 - Infection experiment: Bispecific 03E11 + 03D03 2(G4S) dose response, PRRSV-1LT3, Sigma RPMI 80% high CD163 porcine serum.

[0396] Figure 9 - Infection experiment: Bispecific 03E11 + 03D03 2(G4S) dose response, PRRSV-2MN184, Sigma RPMI 10% FBS.

[0397] Figure 10 - Infection experiment: Trispecific-10 17B11-03E11-03D03 2(G4S) dose response, PRRSV-1LT3, Sigma RPMI 80% low CD163 porcine serum.

[0398] Figure 11 - Infection experiment: Trispecific-10 17B11-03E11-03D03 2(G4S) dose response, PRRSV-1LT3, Sigma RPMI 80% high CD163 porcine serum.

[0399] Figure 12– Infection experiment: Triple complementarity - 10 17B11 - 03E11 - 03D03 2(G4S) dose response, PRRSV - 2NA174, Sigma RPMI 80% low CD163 porcine serum.

[0400] Figure 13 – Infection experiment: Triple complementarity - 10 17B11 - 03E11 - 03D03 2(G4S) dose response, PRRSV - 2NA174, Sigma RPMI 80% high CD163 porcine serum. Example

[0401] Example 1: Immunization, library construction, screening, and clone selection

[0402] Materials and methods

[0403] Immunity

[0404] Single - domain antibodies were obtained from llamas immunized with HEK293T cells expressing different porcine CD163 constructs, followed by a final boost with porcine (p) alveolar macrophage (pPAM) wild - type cells. The llamas were injected with HEK293T cells expressing pCD163 - SRCR - FL - PST2 (i.e., a full - length construct containing from SRCR1 to the end of PST2) and received three boost immunizations, and finally one boost immunization with pPAM wild - type cells. The animals were immunized every two weeks for a total of 5 times. Serum was collected 6 days after the last boost immunization, and the antibody titer against the pCD163 - SRCR - FL - PST2 - His protein was measured by ELISA. In this ELISA, 96 - well plates (Maxisorp; Nunc) were coated with the recombinant protein. After blocking and adding diluted serum samples, the presence of anti - pCD163 antibodies was demonstrated by using mouse anti - llama IgG antibody (FJB, product number FJ1203MAB01B09) and donkey anti - mouse IgG - HRP antibody (JIR, product number 715 - 035 - 150).

[0405] Library construction

[0406] RNA was extracted from the PBMC of 2 immunized llamas (400 μl each). 40 μg of RNA was used for cDNA synthesis using random primers. The cDNA was used for a first - round PCR amplification using unlabeled primers annealing to the leader sequence and hinge CH1 region, and then a second - round PCR amplification was performed to introduce restriction enzyme sites for cloning the VHH gene into the pDCL1 phagemid vector. The library was electroporated into TG1 Escherichia coli cells, and the bacterial glycerol stock of the immune library was stored at - 80 °C.

[0407] Screening

[0408] Using the pCD163 recombinant protein, HEK293T cells expressing different porcine CD163 constructs or pPAM wild-type cells, phage products were obtained from the llama VHH library pool through two consecutive rounds of phage display screening. The first round of screening was carried out on pPAM wild-type cells, and negative selection was preformed using empty vector HEK293T wild-type cells. The second round of screening was conducted on HEK293T cells expressing pCD163-SRCR-FL-PST2, and negative selection was preformed using pPAMΔ5 domain cells (cells with deletion of SRCR domain 5, Burkhard et al., 2017).

[0409] When screening HEK293T cells expressing the pCD163-SRCR-FL-PST2 construct, non-specific phages were washed with PBS buffer at pH 7.4, and then specific phages were eluted with trypsin (complete elution). For the screening round on pPAM, 5E106 cells were used in PBS buffer at pH 7.4, non-specific phages were washed, and then specific phages were eluted with trypsin (complete elution).

[0410] The eluted phages were serially diluted and used to infect exponentially growing TG1. The infected TG1 was plated on LBCarb100Glu 2% plates, and enrichment values were calculated on the background (no antigen was used for screening).

[0411] ELISA screening

[0412] Single clones from the output of the second round of screening conditions were picked into a 96-well master plate and tested for binding as periplasmic extracts (P.E.) to pCD163-SRCR-FL-PST2-His or pCD163-SRCR1-9-huFc (i.e., the construct from the start of SRCR-1 to the end of SRCR-9 but without PST2) or huCD6-pPST2-His protein by binding ELISA at pH 7.0.

[0413] For the P.E. binding ELISA, MaxiSorp TMThe 96-well ELISA plates with high protein binding capacity were coated with 1 μg / ml pCD163-SRCR-FL-PST2-His or huCD6-pPST2-His or pCD163-SRCR1-9-huFc protein diluted in PBS overnight at 4 °C. The next day, the plates were washed 3 times with PBS Tween 0.05% (pH 7.4) and blocked with 250 μl / well of 4% Marvel / PBS for 1 hour at room temperature. After blocking, the plates were washed 3 times with PBS Tween 0.05% (pH 7.4), and each well was incubated with 20 μl of P.E + 80 μl in 1% Marvel / PBS (pH 7.4) with shaking at room temperature for 1 hour. The plates were washed 3 times with PBS Tween 0.05% (pH 7.4), and 100 μl of anti-c-Myc antibody (Roche; product number 11667203001) was added, followed by incubation with the secondary antibody DAM-HRP (JIR; product number 715-035-150) in 1% Marvel / PBS (pH 7.4) with shaking at room temperature for 1 hour. The plates were washed 3 times with PBS Tween 0.05% (pH 7.4), and the substrate solution (TMB solution) was added to the plates. The reaction was terminated with H2SO4, and the plates were read at 450 nm in a plate reader.

[0414] Cell screening (FACS):

[0415] The periplasmic extract (P.E) from the selected clones was incubated with an anti-c-Myc antibody (Roche; product number 11667203001) specific for the c-myc tag present in the soluble form of the VHH with stirring at room temperature (RT) for 30 minutes. The mixture (P.E + anti-c-Myc antibody) was added to pPAM wild type or pPAMΔ5 domain (cells lacking SRCR domain 5), and incubated with gentle shaking at 4 °C for 60 minutes.

[0416] The cells were washed 3 times with 150 μl / well of FACS buffer and incubated with 50 μl / well of the secondary antibody GAM-APC at 4 °C for 30 minutes with shaking in the dark.

[0417] The cells were washed 3 times with 150 μl / well of FACS buffer and resuspended in 75 μl / well of FACS buffer for measurement in the RL-1 channel (APC channel) in a FACS machine (Attune TM NxT), and a total of 10,000 cells were acquired for each sample.

[0418] Sequencing

[0419] Send the positive conjugates for sequencing. Classify the clones into families based on different HCDR3 sequences.

[0420] Expression and purification of VHH candidate antibodies

[0421] A synthetic gene encoding the VHH variable domain with FLAG and His tags was obtained. Each DNA construct was digested with restriction enzymes, the inserts were gel purified, and each variable domain insert was ligated to the mammalian expression vector pcDNA3.1. ExpiCHO-S cells were transfected with the VHH sequences using 40 μg of the total DNA plasmid construct. A total volume of 25 mL of cells was used for 8-day protein production (32 °C, 5% CO2). The produced VHH antibodies were captured from the clarified supernatant using a HisTrap HP 5 mL IMAC column (GE Healthcare, product number 17-5248-02) on a Pure25 FPLC system. The buffer of the eluted antibody peak fractions was changed to 1x PBS pH 7.4 and concentrated using a 3 kDa MCO spin concentrator (Amicon, product number UFC900324). The purified protein was analyzed for the presence of the correct chain by analytical size exclusion chromatography (aSEC) and SDS-PAGE.

[0422] Performing dose-response ELISA using purified VHH

[0423] MaxiSorp TM 96-well ELISA plates with high protein-binding capacity were coated overnight at 4 °C with 1 μg / ml of pCD163-SRCR1-9-huFc (diluted in PBS). The next day, the plates were washed 3 times with PBS Tween 0.05% (pH 7.4), and 250 μl of 4% Marvel was added to each well and incubated at room temperature for 1 hour for blocking. After blocking, the plates were washed 3 times with PBS Tween 0.05% (pH 7.4).

[0424] VHH in PBS (pH 7.4) was serially diluted 3-fold from an initial concentration of 200 nM to 0.0034 nM and added to the wells of the ELISA plates pre-coated with pCD163-SRCR1-9-huFc and blocked, and incubated at room temperature for 1 hour.

[0425] Wash the plate three times with 0.05% PBS Tween (pH 7.4), and then incubate it with a PBS solution (pH 7.4) of anti-histidine-HRP (Miltenyi Biotec, product number 130-092-783) at room temperature for 1 hour. Wash the plate three times with 0.05% PBS Tween (pH 7.4), and add the substrate solution (TMB solution) to the plate. Terminate the reaction with H2SO4 and read the plate at 450 nm in a plate reader.

[0426] Determining the affinity of purified VHH using Biacore

[0427] To evaluate the affinity of the selected purified clones for the pCD163 protein, the pCD163-SRCR1-9-huFc and pCD163-1-PST2-His (also referred to herein as pCD163-SRCR-FL-PST2-His) proteins were coated onto a CM5 sensor chip (GE Healthcare) using the amine coupling method.

[0428] The binding kinetics of the selected single-domain antibodies were measured at pH 7.4 using surface plasmon resonance technology (SPR) (Biacore 3000, GE Healthcare). The pCD163-SRCR1-9-huFc (approximately 2075 to 2423 RU) or pCD163-1-PST2-His (approximately 2859 to 3286 RU) was dissolved in acetic acid buffer at pH 5.0 or pH 5.5 at 20 or 30 μg / ml and immobilized onto the CM5 chip through a standard amine coupling procedure.

[0429] Commercial anti-pig CD163 (BioRad, product number MCA2311GA) diluted to 30 nM in HBS-EP pH 7.4 buffer was used for immobilization quality control.

[0430] 1x HBS-EP pH 7.4 was used as the running buffer for binding kinetics measurements. The purified VHH was serially diluted from 200 nM to 12.5 nM in HBS-EP pH 7.4 and injected at 30 μl / min for 2 minutes, followed by a dissociation rate wash for 1 minute between injections. In each cycle, a dissociation rate wash was performed 300 seconds after the last concentration injection. After regeneration by injecting 10 μl of 1M NaCl, 1 mM glycine (pH 1.5) between two samplings, the RU level was restored to the baseline level.

[0431] Apply the 1:1 binding and mass transfer fitting to the set sample curves using the simultaneous fitting option of BIAevaluation software to calculate the kinetic constants of the antibody-antigen interaction, including the association rate (ka), dissociation rate (kd), and affinity (KD). After visually inspecting the residuals and considering the Chi2 value, curves were removed from the fitting: at least 4 curves were considered for simultaneous fitting.

[0432] Results and Discussion

[0433] It has been confirmed that the clone has membrane-specific binding, as shown by clone 17B11. As can be seen from Tables 1, 3, and 4, clone 17B11 did not bind to non-cell surface-associated (soluble form) CD163 in ELISA, ELISA EC50, or Biacore experiments.

[0434] As shown in Table 2, clone 17B11 did bind to pPAM WT cells expressing CD163. In addition, clone 17B11 did not bind to pPAMΔ5WT cells, indicating that its binding specificity requires the SRCR5 domain of cell surface-expressed CD163.

[0435] Overall, these results indicate that clone 17B11 is specific for cell surface-associated CD163.

[0436] Table 1 – P.E.ELISA screening results of clone 17B11

[0437]

[0438] Table 2 – P.E.FACS screening results of clone 17B11

[0439]

[0440] Table 3 – Purified VHH dose-response ELISA of clone 17B11 binding to pCD163-SRCR1-9-huFc

[0441]

[0442] Table 4 – Affinity of clone 17B11 determined by Biacore

[0443]

[0444] N / D = Not determined due to too weak binding.

[0445] The sequence of 17B11 is shown in Table A (also sometimes referred to as clone 39 in this article).

[0446] Example 2: Functional assays using bispecific and trispecific constructs containing 17B11 VHH antibodies Anti Porcine CD163 clones 03E11 (H03E11) and 03D03 (H03D03)

[0447] Previously screened anti-pig (p)CD163 VHH clones 03D03 and 03E11 were selected from libraries generated from two llamas immunized with pCD163-SRCR4-7-huFc and finally boosted with pCD163-SRCR1-9-huFc. Two consecutive rounds of phage display screening were performed using pCD163 recombinant protein or pPAM WT cells. For the recombinant protein screening rounds, 10 μg / ml of pCD163-SRCR1-9-huFc or pCD163-SRCR4-7-huFc in pH 7.4 (PBS buffer) was used. Non-specific phages were washed first, and then specific phages were eluted with trypsin (complete elution). The screening rounds on pPAM cells were performed as described above.

[0448] These clones were identified by P.E.ELISA screening with recombinant protein (soluble form of CD163) and P.E.FACS screening with cells (membrane CD163). P.E.ELISA screening was performed on pCD163-SRCR4-7-huFc or pCD163-SRCR5-6-huFc protein at pH 7.0 (PBS). P.E.FACS screening was performed on pPAM WT and pPAMΔ5 cells. Both clones showed binding to soluble form of porcine CD163 (recombinant protein) and membrane-bound form of porcine CD163 (pPAM WT), but no significant binding to pPAMΔ5 cells. Therefore, these clones were considered to be able to bind to the SRCR5 domain of porcine CD163. The 03E11 antibody has been shown to inhibit PRRSV-1 or PRRSV-2 infection (see Figure 3 and Figure 4 ), and the sequence of 03E11 is shown in Table B (sometimes referred to as clone 19 in this article). The 03D03 antibody has been shown to inhibit PRRSV-2 infection (see Figure 4 ), and its sequence is shown in Table C (sometimes referred to as clone 17 in this article).

[0449] These antibodies were used in combination with the 17B11 antibody to construct bispecific (bis-antibody) and trispecific (tris-antibody) constructs as described below.

[0450] Materials and Methods

[0451] Construction of dual-complementary and triple-complementary site candidate antibodies

[0452] Single VHHs were assembled into bi - and tri - complementary site combinations using 2x(G4S) or 5x(G4S) flexible linkers. These linkers were placed between each individual antibody in the construct. The combination construction was semi - rational, taking into account non - competing epitopes, aiming for high affinity and high potency against both PRRSV1 and PRRSV2 subtypes, as well as the ability to bind to cell - surface CD163 and block infection in the presence of competitive soluble forms of the CD163 protein. This lack of interference was considered important because high levels of soluble forms of CD163 may be present in the sera of wild animals, especially those suffering from bacterial and / or viral infections such as Lawsonia intracellularis infection, which is common and can act as an absorption point for any therapeutic agent that does not preferentially target cell - surface CD163.

[0453] First, bi - complementary site constructs (containing two different anti - porcine CD163 antibodies that can bind to different epitopes on porcine CD163) were prepared and their binding to pPAM WT cells was evaluated. In a competitive setting, their binding to pPAM WT cells was further tested as the concentration of the soluble form of CD163 increased. Examples of the prepared bi - complementary site constructs are summarized in Table 5 and include: 03E11 + 03D03 2(G4S), 03E11 + 03D03 5(G4S), 03E11 + 17B11 2(G4S), 03E11 + 17B11 5(G4S), 17B11 + 03D03 2(G4S), and 17B11 + 03D03 5(G4S). 2(G4S) indicates that the individual VHHs are separated by two repeated G4S linkers, while 5(G4S) indicates that the individual VHHs are separated by five repeated G4S linkers.

[0454] Tri - complementary site constructs (containing three different anti - porcine CD163 antibodies that can bind to different epitopes on porcine CD163) were also constructed. The constructed tri - complementary site constructs are summarized in Table 6 and include: 03E11 + 03D03 + 17B11 2(G4S) (sometimes called Tri - 2), 03E11 + 17B11 + 03D03 2(G4S), 03D03 + 03E11 + 17B11 2(G4S), 03D03 + 17B11 + 03E11 2(G4S), 17B11 + 03E11 + 03D03 2(G4S) (sometimes called Tri - 10), and 17B11 + 03D03 + 03E11 2(G4S). 2(G4S) indicates that the individual VHHs are separated by two repeated G4S linkers.

[0455] Expression and purification of dual-complementary and triple-complementary site candidate antibodies

[0456] The synthetic gene encoding the VHH variable domain was appropriately combined with FLAG and His tags and linked into the mammalian expression vectors pcDNA3.1 or pcDNA3.4 through an appropriate linker. ExpiCHO-S or HEK293T cells were transfected with the DNA plasmid construct and cultured for 7 - 10 days at 32 °C and 5% CO2 to produce the protein. The resulting bispecific and trispecific VHH antibody constructs were captured from the clarified supernatant on an FPLC system using a HisTrap IMAC column (GE Healthcare, product number 17 - 5248 - 02). The buffer of the eluted antibody peak fractions was changed to 1x PBS pH 7.4 and concentrated using a 3 kDa MCO spin concentrator (Amicon, product number UFC900324). The purified protein was analyzed by analytical size exclusion chromatography (aSEC) and SDS - PAGE for the presence of the correct chains.

[0457] Dose - response FACS of pPAM WT cells

[0458] The bispecific and trispecific antibody constructs in PBS (pH 7.4) were diluted in 3 - fold serial dilutions from 150 nM to 0.023 nM in FACS buffer (1x PBS pH 7.4 solution with 0.5% FBS and 0.5 mM EDTA). The bispecific and trispecific candidates were incubated with anti - FLAG - biotin (Sigma, product number F9291) by shaking at iced temperature for 30 minutes. This mixture was added to pPAM WT cells and incubated by gentle shaking at iced temperature for 60 minutes. The cells were washed 3 times with 150 μL / well of FACS buffer and incubated by shaking at iced temperature and protected from light for 30 minutes with 50 μL / well of the secondary detection reagent streptavidin R - P.E conjugated antibody (Invitrogen, product number SA10044). Then the cells were washed 3 times with 150 μL / well of FACS buffer and resuspended in 50 μL / well of FACS buffer for measurement on a FACS machine (Attune TM NxT), and a total of 10,000 cells were obtained for each sample.

[0459] FACS competition experiment on pPAM WT cells: Binding with dual-complementary site antibody candidates in the presence of soluble form of CD163 protein Binding

[0460] Dilute the dual complementary bit candidates to 1 nM in FACS buffer and mix with pCD163-SRCR1-9-PST2 at final concentrations of 0, 1, 10, and 100 nM in FACS buffer. Then incubate the mixture with pPAM WT cells with shaking on ice for 60 minutes. For VHH detection, add anti-FLAG-biotin (Sigma, product number F9291) antibody to the cell mixture and shake on ice for 30 minutes. Wash the cells 3 times with 150 μL / well of FACS buffer and incubate with the secondary detection reagent anti-mouse IgG-APC (Invitrogen, product number A865) with shaking on ice and protected from light for 30 minutes. Wash the cells 3 times with 150 μL / well of FACS buffer and resuspend in 50 μL / well of FACS buffer for measurement on a FACS machine (Attune TM NxT), and a total of 10,000 cells are obtained for each sample.

[0461] Results and Discussion

[0462] As Figure 1 and Table 5 show, the dual complementary bit VHH candidates bind very well to pPAM WT cells. As Figure 2 shown, in the presence of the competitive soluble form of CD163, the binding ability of the dual complementary bit combination to pPAM WT cells will decrease. For example, 03E11+03D03 5(G4S) is significantly inhibited at 10 nM and 100 nM of the competitive soluble form of CD163. However, other dual complementary bits containing the membrane-specific anti-CD163 17B11 VHH antibody (exemplified by 03E11+17B11 5(G4S) and 17B11+03D03 5(G4S)) maintain a higher level of binding to pPAM WT cells even in the presence of a large excess of up to 100 nM of the soluble form of the CD163 protein. Therefore, the data clearly show that compared with the dual complementary bit combinations without 17B11, the dual complementary bit combinations containing 17B11 can maintain a good binding level to pPAM WT cells in the presence of 10 nM and even in the presence of up to 100 nM of the competitive soluble form of the CD163 protein.

[0463] Table 5. Dose-response FACS of pPAM WT cells using example dual complementary bit antibody candidates

[0464]

[0465]

[0466] As shown in Table 6, the triple complementary bit VHH candidates also bind very well to pPAM WT cells.

[0467] Table 6. Dose - response FACS of pPAM WT cells with Example 3 complementary - site antibody candidates

[0468] Sample ID EC50 (nM) of PAM WT cells <![CDATA[03E11+03D03+17B11 2(G4S)]]> 9.1 <![CDATA[03E11+17B11+03D03 2(G4S)]]> 18.2 <![CDATA[03D03+03E11+17B11 2(G4S)]]> 3.8 <![CDATA[03D03+17B11+03E11 2(G4S)]]> 13.6 <![CDATA[17B11+03E11+03D03 2(G4S)]]> 11.6 <![CDATA[17B11+03D03+03E11 2(G4S)]]> 8.0

[0469] Example 3: Inhibition of porcine reproductive and respiratory syndrome (PRRS) virus infection in primary porcine alveolar macrophages by dual - and triple - complementary - site constructs including the 17B11 VHH antibody

[0470] Materials and methods

[0471] PRRS virus infection protocol

[0472] Reagents

[0473] Control antibody:

[0474] Primary antibody: Anti - PRRS1AC7, Ingenasa

[0475] Secondary antibody: Goat anti - mouse IgG(H + L) Alexa Fluor Plus 488, ThermoFisher, A32723

[0476] Medium:

[0477] Complete RPMI, 10% FBS, 80% low sCD163 porcine serum or 80% high sCD163 porcine serum, Ultra Glutamine, Pen / Strep (sCD163 is the soluble form of CD163)

[0478] PAM isolation: Porcine alveolar macrophages were isolated as described by Burkard et al. in 2017.

[0479] Virus isolates:

[0480] Type 1 virus: BOR57 isolate (Roslin Institute, Edinburgh, UK)

[0481] Type 1 virus: LT3 (PRRSV - 1 subtype 2 strain, Roslin Institute, Edinburgh, UK)

[0482] Type 2 virus: MN184 US strain (Han et al., 2006)

[0483] Type 2 virus: NA174 (Roslin Institute, Edinburgh, UK)

[0484] Infection protocol

[0485] Day 1 - Seeding cells

[0486] Seed 20 million porcine alveolar macrophages per well in complete RPMI in a 48-well plate and place in a CO2 incubator overnight

[0487] Day 2 - VHH treatment and infection challenge

[0488] 1. Pretreatment (30 minutes before infection)

[0489] a. Aspirate the medium from the cells

[0490] b. Add 100 μL of medium to the untreated uninfected control and the untreated infected control

[0491] c. Add 20 μL of PBS in 100 μL of medium to the mock-treated infected control

[0492] d. Add an appropriate amount of VHH stock solution in 100 μL of medium to the treated infected samples

[0493] e. Return the plate to the CO2 incubator for 30 minutes

[0494] 2. Thaw the virus stock solution and sonicate for 15 seconds before use

[0495] 3. Infection challenge (2 hours)

[0496] a. Remove the medium from the cells and save the VHH-containing medium for the overnight incubation step

[0497] b. Add 100 μL of medium to the untreated uninfected control

[0498] c. Add 10 μL of virus in 100 μL of medium to the untreated infected control

[0499] d. Add 10 μL of virus plus 20 μL of PBS in 100 μL of medium to the mock-treated infected control

[0500] e. Add an appropriate amount of VHH stock solution and 10 μL of virus in 100 μL of medium to the treated infected samples

[0501] f. Gently swirl the plate and return to the CO2 incubator

[0502] g. Gently swirl the plate every 15 minutes for 2 hours

[0503] 4. Incubate overnight (15 hours)

[0504] a. Aspirate the medium from the cells

[0505] b. Add 100 μL of medium to the untreated uninfected control and the untreated infected control

[0506] c. Add 20 μL of PBS in 100 μL of the medium to the mock-treated infected control

[0507] d. Add the VHH-containing medium reserved in the pretreatment step to the appropriate samples

[0508] e. Place the plate back into the CO2 incubator for 15 hours

[0509] Day 3 - Assay and virus infection readings

[0510] 5a. Quantify viral RNA in the culture supernatant by direct lysis RT-qPCR protocol

[0511] a. Collect 5 μl of the culture supernatant at 24 HPI

[0512] b. Dilute the sample 1:2 with lysis buffer (20 mM Trizma hydrochloride buffer, pH 7.5, 300 mM NaCl, 2.5%

[0513] CA-630, 1:2000 Plus RNase inhibitor) and mix

[0514] c. Incubate the sample at room temperature for 20 minutes

[0515] d. Dilute the sample with nuclease-free H2O (1:5) and use for subsequent qRT-PCR protocol (primers

[0516] optimized and validated for PRRSV)

[0517] e. Readings: Relative to mock-treated TCID50 / ml (%)

[0518] 5b. In-well fixation and staining protocol

[0519] a. Aspirate the medium from the cells

[0520] b. Fix the cells in 4% formaldehyde / PBS++ (with calcium and magnesium) solution at room temperature for 30 minutes

[0521] c. Wash once with PBS++

[0522] d. Permeabilize with Triton-X (1%, in PBS++) at room temperature for 5 minutes

[0523] e. Wash once with PPBS++ or blocking solution (PBS++ / 5% FBS)

[0524] f. Block with blocking solution (PBS++ / 5% FBS) at room temperature for 20 minutes

[0525] g. Add the first antibody anti-PRRS1AC7 at a ratio of 1:5000 to all wells except the unstained control and the control with only the second antibody.

[0526] h. Incubate at room temperature for 1 hour.

[0527] i. Wash three times with PBS++.

[0528] j. Add the second antibody goat anti-mouse IgG(H+L) Alexa Fluor Plus 488 at a ratio of 1:5000 to all wells except the unstained control.

[0529] k. Incubate at room temperature for 45 minutes.

[0530] l. Wash three times with PBS++.

[0531] m. Add 300 μL of PBS++.

[0532] n. Scratch the cells with a wide-bore p200 pipette tip, then scratch around the edge of the well with a normal p200 tip, and then wash the well surface 3 times with a p1000. Collect the cells and transfer them to a FAC tube.

[0533] o. Perform measurements on a Fortessa x20.

[0534] Serum containing soluble form of CD163

[0535] Serum containing low soluble forms of CD163 was collected from healthy pigs. Serum with high soluble forms of CD163 was collected from pigs infected with Lawsonia intracellularis. Lawsonia intracellularis infection causes shedding of CD163, followed by an increase in the level of soluble CD163 in serum, and any Lawsonia intracellularis in the serum does not interfere with the detection of viral PRRS virus infection.

[0536] The serum concentration of soluble CD163 was calculated based on standard sera whose concentrations were previously detected by ELISA. The low CD163 serum from healthy pigs and the high CD163 serum from infected pigs were diluted in PBS at ratios of 1:1 and 1:9 respectively, and then the concentrations were measured by dot blot using the first antibody anti-PRRS1AC7 (Ingenasa) and the second antibody goat anti-mouse IgG(H+L) Alexa Fluor Plus 488 (ThermoFisher, A3272).

[0537] Upon detection, the content of soluble CD163 in the serum of healthy pigs was 0.4 mg / l (±0.015 STDEV), and the content of soluble CD163 in the serum of infected pigs was 4.5 mg / l (±0.45 STDEV). The medium containing 10% FBS did not contain soluble CD163.

[0538] Results and Discussion

[0539] The ability of single VHH, dual complementarity-determining region (CDR) and triple CDR VHH constructs to inhibit the infection of pPAM host cells by members of the PRRS virus family is described below. As described above, this assay was used to measure the degree of virus infection, which was quantified by the proliferative ability of the virus and measured by FACS or RT-qPCR after a 17-hour infection cycle.

[0540] The data clearly show that VHHs are able to inhibit the efficient infection of porcine alveolar macrophages by PRRSV-1 (see Figure 3 ) and PRRSV-2 (see Figure 4 ). Single VHHs that showed activity in the infection assay could be divided into VHHs that were effective against both PRRSV-1 and PRRSV-2 infections (exemplified by 03E11) and VHHs that had no inhibitory activity against PRRSV-1 infection but had inhibitory activity against PRRSV-2 infection (exemplified by 17B11 and 03D03).

[0541] In the infection assay using BOR57 PRRSV-1, dual CDR combinations of VHH 03D03 (clone 17), 03E11 (clone 19) and 17B11 (clone 39) were used ( Figure 5 ). Then, in the presence of medium containing 10% FBS, low soluble CD163 porcine serum or high soluble CD163-containing serum, the best-performing dual CDR construct (19-17, 03E11-03D03 2(G4S)) was used in the infection assay using LT3 PRRSV-1 ( Figures 6 to 8 ) or MN184 PRRSV-2 ( Figure 9 ).

[0542] Dual CDRs without membrane-specific anti-CD163 VHHs, such as those shown by 03E11+03D03 (19-17), were able to reduce the infection potential of PRRSV-1 virus by approximately 65% to 75% at most in medium containing only 10% FBS (see Figure 6 ). In medium containing 80% low CD163 porcine serum, such dual CDRs were able to reduce the infection potential of PRRSV-1 virus by approximately 75% (see Figure 7 ).

[0543] The data also show that in a medium containing 80% highly soluble form of CD163 porcine serum, the infectivity potential of PRRSV-1 virus was only reduced by about 50% (see Figure 8 ). In the above FACS competition assay, the dual complementarity 03E11+03D03 was also vulnerable to competition by the soluble form of CD163 when binding to pPAM WT cells ( Figure 2 ). This clearly shows that the competitive soluble form of CD163 can reduce the effectiveness of potential therapeutic agents that are not specific to membrane-bound CD163 and are sensitive to blockade by the competitive soluble form of CD163.

[0544] Taking the dual complementarity combination of 03D03+03E11 as an example, it can reduce the infectivity potential of PRRSV-2 virus by about 40% in a medium containing 10% FBS (see Figure 9 ). Although effective, its effect on PRRSV-2 infection is lower than that on PRRSV-1 infection even in the absence of the soluble form of CD163. It would be beneficial to improve this effect.

[0545] Although dual complementarity combinations such as 03D03+03E11 may show reduced efficacy in the presence of the highly soluble form of CD163, the data also show that in the same assay, triple complementarity combinations such as triple complementarity Tri-2 and Tri-10 containing membrane-specific VHH such as 17B11 can reduce the infectivity potential of PRRSV-1 and PRRSV-2 by 100%, even in the presence of the highly soluble form of CD163. In addition, triple complementarity combinations that combine a membrane-specific anti-CD163 VHH (such as 17B11) with other VHHs that can individually inhibit PRRSV-2 (such as 03D03 or 03E11) and / or PRRSV-1 (such as 03E11) infection can combine these properties in an advantageous way.

[0546] Triple complementarity combinations assembled from VHHs that can individually block PRRSV-2 or PRRSV-1 and PRRSV-2 infection and membrane-specific anti-CD163 VHHs, examples of which are triple complementarity Tri-2 (03E11-03D03-17B11 2(G4S)) and Tri-10 (17B11-03E11-03D03 2(G4S)).

[0547] Such triple complementarity combinations can completely inhibit the infectivity potential of PRRSV-1, and the IC Figure 10 values in low soluble form of CD163 serum (see Figure 11 and Table 7) and high soluble form of CD163 serum (see 50 ) are very low, in the range of 2.81 - 4.02 nM.

[0548] In addition, such three-complementary-site combinations are capable of completely inhibiting the infectivity potential of PRRSV-2 with very low IC Figure 12 values in the range of 2.13 - 7.60 nM in both low-soluble forms of CD163 serum (see Figure 13 and Table 8) and high-soluble forms of CD163 serum (see 50 and Table 8).

[0549] The data clearly show that the VHHs that can individually block PRRSV-1 and PRRSV-2 or PRRSV-2, or the combination of the VHHs of PRRSV-2 with membrane-specific VHHs showing reduced sensitivity to the competitive soluble form of CD163, can lead to excellent blocking of the infection of PRRSV-1 or PRRSV-2 family members. The advantage is that such combinations can enhance the blocking of the infection of PRRS virus family members in the case of elevated levels of soluble CD163, for example, soluble CD163 is present at significant levels in serum in persistent infections and can serve as an absorption point. Such multi-complementary-site VHHs will have obvious and significant advantages as therapeutic agents for the prevention and treatment of PRRS virus infections.

[0550] Table 7. Summary of the three-complementary-site PRRSV1 LT3 infection assay

[0551]

[0552] Table 8. Summary of the three-complementary-site PRRSV2 infection assay

[0553]

[0554] References

[0555] Burkard C et al., "Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function PLoS Pathogens", 13(2) 2017: e1006206 Han et al., 2006, "Complete genome analysis of RFLP184 isolates of porcine reproductive and respiratory syndrome virus", Virus Res., 122: 175 - 182.

[0556] Example 4: Efficacy of repeated administration of a triple - complementarity construct (including 17B11 antibody) against experimental infection of piglets with PRRSV - 1 (LT3 strain)

[0557] Materials and methods

[0558] Animals

[0559] Twenty - six piglets (Sus scrofa domesticus), healthy and approximately 5 - 6 weeks old at the time of challenge.

[0560] Breed: Large White pig x Pietrain pig,

[0561] Both male and female are acceptable,

[0562] PCR - negative and serologically negative for both PRRSV - 1 and PRRSV - 2 before challenge

[0563] Challenge

[0564] Challenge strain: LT3 strain of PRRSV - 1, at a concentration of approximately 10 6 TCID 50 / mL (Roslin Institute, Edinburgh, UK)

[0565] Inoculum: 5 mL was administered intranasally to each piglet once at 0:00 on day 0 (D0). Treatment group: VHH (Tri2) - 12 pigs received LT3 challenge at T0 on day 0

[0566] VHH: Three complementarity-determining regions 03E11 + 03D03 + 17B112 (G4S), designated as "Tri2".

[0567] Formulation: Injectable solution, Tri-2 protein sample, recovered from Pichia pastoris, 111 mg / mL, dissolved in 15 mM purified sodium phosphate, 100 mM arginine hydrochloride, pH 7.00, 0.2 μm filter

[0568] Dose: 10 mg / kg, intramuscular injection (neck), starting from D0: approximately 5 hours before T0 (challenge), then approximately 3 hours after T0. From day 1 (D1) to day 10 (D10), repeat the injection every morning and approximately 8 hours later.

[0569] Control group: Buffer – 12 piglets received LT3 challenge at T0 on day 0

[0570] VHH: None

[0571] Formulation: Injectable solution, 15 mM purified sodium phosphate, 100 mM arginine hydrochloride, pH 7.00, 0.2 μm filter

[0572] Dose: The same volume as Tri2; intramuscular injection (neck), starting from D0: approximately 5 hours before T0 (challenge), then approximately 3 hours after T0. From D1 to D10, repeat the injection every morning and approximately 8 hours later.

[0573] Clinical observation and autopsy observation

[0574] Animals were observed from D-2 (2 days before challenge) to D11. Rectal temperature, general health status, clinical observation of respiratory signs, local tolerance at the injection site, and general tolerance were measured daily.

[0575] Body weights at D-7, D-2, D5, and D11 were used to determine weight gain and average daily weight gain; euthanasia was performed at D11 and necropsy was carried out to score gross pneumonic lesions (Halbur et al., 1995) and collect samples.

[0576] Sample collection and analysis

[0577] · Blood samples were collected before challenge, at D2, D5, D7, D9, and D11.

[0578] o PRRSV qPCR and serology

[0579] o Determination of Tri2 concentration

[0580] o Biochemical analysis was performed only on pre-challenge and D11 samples to evaluate general tolerance

[0581] · Collect fecal samples at the same time points for PRRSV qPCR detection.

[0582] · Collect nasal secretions by nasal swab at D11, only for PRRSV qPCR.

[0583] · Samples collected at necropsy:

[0584] o Bronchoalveolar fluid for PRRSV qPCR and potential Tri2 determination; processed to collect alveolar macrophages (PAM) for potential additional studies

[0585] o Lungs for histopathology to score pneumonia and PRRSV qPCR

[0586] o Tonsils, inguinal lymph nodes, and spleen for PRRSV qPCR

[0587] o Injection sites on both sides of the neck for histopathological evaluation of local tolerance

[0588] o Additional analyses can be performed on these tissues

[0589] The sampling schedule for piglets in the safety group is the same. Collect additional blood samples at D-2 and D-1 to evaluate the Tri2 concentration within hours after dosing.

[0590] Laboratory tests (including serum analysis) are performed by personnel unaware of the treatment assignment.

[0591] Summary of results

[0592] Based on the observations of the injection sites, no abnormalities were reported during the study period, indicating good local tolerance to Tri2.

[0593] In addition, no systemic adverse reactions were observed in any clinical observation evaluations throughout the study, indicating good systemic tolerance to Tri2.

[0594] Abnormal clinical signs related to the challenge were more common in the control group. Cough was only sporadically observed in the control group, and the incidence of high fever (rectal temperature ≥ 40°C) was significantly higher: there were 45 observations in the control group, while only 15 in the Tri2 group (P < 0.001) (Table 9).

[0595] Body weight and weight gain: The groups were consistent and homogeneous at the start of the study. On day 11 before necropsy, the average body weight difference between the control group (9.7 ± 1.01 kg) and the Tri2 group (10.7 ± 1.67 kg) was approximately 1 kg. Compared with the control group (2.1 ± 0.71 kg), the average daily weight gain of the Tri2 group (3.0 ± 0.52 kg) between D-2 and D11 was significantly higher (p < 0.05) (Table 9).

[0596] The primary endpoint was that at each observation time point from D2 to D9, the PRRSV viral load in the serum of Tri2 piglets was significantly lower than that of control piglets (Table 9). The viral load was evaluated by Ct count, and the Ct count was inversely proportional to the viral load: a Ct of 37 to 40 was considered a negative result, and the viral load increased as the Ct decreased. A difference of approximately 3.3 Ct represented a 1 log10 difference in virus titer.

[0597] PRRSV serological data are shown in Table 10, indicating that the development of the humoral immune response may be slightly delayed under Tri2 treatment.

[0598] Compared with the control group (11.1 ± 16.91), the gross lung lesions (measured by Halbur score) in Tri2 piglets (5.3 ± 5.11) were generally less severe, but the difference in scores was not statistically significant, which may be due to data variability and sample size. Notably, severely affected piglets, i.e., pigs with a lung lesion score equal to or higher than 15 (4 out of 12 pigs, Table 11), were only observed in the control group.

[0599] Table 9. Summary of clinical and virological observations in pigs receiving LT3 challenge, Tri2 treatment, or no treatment

[0600]

[0601]

[0602] *Ct = 40 was considered a negative result. Detection kits: PCR SDRP EU / M-VBIM / M / 008 Biosellal and PCR SDRP US / M-VBIM / M / 008 Biosellal

[0603] Table 10. PRRSV serology in piglets at D0 after infection with LT3 strain

[0604] D0 to D7 D9 D11 Treatment group All negative 12 cases negative 5 cases positive / 7 cases negative Control group All negative 3 cases positive / 9 cases negative 10 cases positive / 2 cases negative

[0605] Neg: Negative; pos: Positive. Detection kit: IDVET Elisa indirect ID PRRS Indirect-IDvet (id-vet.com)

[0606] Table 11. Distribution of lung lesion scores (scoring system from Halbur et al., 1995)

[0607]

[0608] Conclusion

[0609] Treatment of piglets with 10 mg / kg of Tri2 twice daily for 10 days effectively controlled the clinical and virological effects of PRRSV infection induced by PRRSV-1 (LT3) challenge administered after the first Tri2 treatment.

[0610] When compared to the untreated control group, Tri2 treatment significantly reduced PRRSV viremia at all time points of observation from D2 to D9 post-challenge. Seroconversion was observed in both groups, but was slightly delayed in the treated group.

[0611] In the Tri2-treated piglets, the frequency of high fever was significantly reduced. Treated piglets gained on average 1 kg more than control piglets over a 12-day period, and the difference was statistically significant. Gross pneumonic lesions observed and scored at necropsy 11 days post-challenge were generally less severe in the treated group; severe lesions defined as score >15 were observed in 4 untreated piglets (33%), but not in any of the treated piglets.

[0612] The preparation was well tolerated after multiple intramuscular injections.

[0613] Reference: Halbur PG et al., “Comparison of the pathogenicity of two US Porcine Reproductive and Respiratory Syndrome Virus isolates with that of the Lelystad virus”. Vet. Pathol. 32:648-660 (1995).

Claims

1. An antibody that binds to porcine CD163, wherein the antibody: (i) binds to membrane-bound porcine CD163; and (ii) does not significantly bind to soluble porcine CD163.

2. The antibody according to claim 1, wherein the antibody has the ability to bind to the SRCR5 domain of porcine CD163.

3. The antibody or binding protein according to claim 1 or 2, which comprises at least one antigen-binding domain that binds to porcine CD163, the antigen-binding domain comprising a heavy-chain variable region, the heavy-chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy-chain variable region comprises: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence GRTFSSYA (SEQ ID NO:2) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, or 3 amino acid substitutions compared to the given CDR sequence, (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence IGWTGGTT (SEQ ID NO:3) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, or 3 amino acid substitutions compared to the given CDR sequence; and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence AADQAGWRTAGVRNTYEYDY (SEQ ID NO:4) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence that contains 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence.

4. The antibody or binding protein according to claim 3, wherein the heavy-chain variable region comprises: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence GRTFSSYA (SEQ ID NO:2), (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence IGWTGGTT (SEQ ID NO:3), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence AADQAGWRTAGVRNTYEYDY (SEQ ID NO:4). (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence AADQAGWRTAGVRNTYEYDY (SEQ ID NO:4). (VH) CDR3.

5. The antibody or binding protein according to any one of claims 1 to 4, which comprises a VH domain having the amino acid sequence of SEQ ID NO:1 or a sequence having at least 70%, 75%, or 80% sequence identity thereto.

6. An antibody that binds to the same epitope on porcine CD163 as the antibody according to claim 3 or 4.

7. A combination of an antibody or binding protein according to any one of claims 1 to 6 and one or more other anti-porcine CD163 antibodies or binding proteins, preferably a combination with one or two other anti-porcine CD163 antibodies or binding proteins.

8. The combination according to claim 7, wherein each antibody or binding protein binds to a different epitope on porcine CD163.

9. The combination according to claim 7 or 8, wherein the other anti - porcine CD163 antibody or binding protein comprises at least one antigen - binding domain that binds to porcine CD163, the antigen - binding domain comprises a heavy - chain variable region, and the heavy - chain variable region comprises three complementarity - determining regions (CDRs), wherein the heavy - chain variable region comprises: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence RYVMG (SEQ ID NO:10) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1 or 2 amino acid substitutions compared to the given CDR sequence, (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence AISWSGRAPYADSVKG (SEQ ID NO:11) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence GEGAIKWTTLDAYDY (SEQ ID NO:12) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and / or (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence GEGAIKWTTLDAYDY (SEQ ID NO:12) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and / or wherein the other anti - porcine CD163 antibody or binding protein comprises at least one antigen - binding domain that binds to porcine CD163, the antigen - binding domain comprises a heavy - chain variable region, and the heavy - chain variable region comprises three complementarity - determining regions (CDRs), wherein the heavy - chain variable region comprises: wherein the other anti - porcine CD163 antibody or binding protein comprises at least one antigen - binding domain that binds to porcine CD163, the antigen - binding domain comprises a heavy - chain variable region, and the heavy - chain variable region comprises three complementarity - determining regions (CDRs), wherein the heavy - chain variable region comprises: wherein the other anti - porcine CD163 antibody or binding protein comprises at least one antigen - binding domain that binds to porcine CD163, the antigen - binding domain comprises a heavy - chain variable region, and the heavy - chain variable region comprises three complementarity - determining regions (CDRs), wherein the heavy - chain variable region comprises: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence DYTIG (SEQ ID NO:18) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1 or 2 amino acid substitutions compared to the given CDR sequence, (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence CINSITSNTYYADSVKG (SEQ ID NO:19) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence; and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:20) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence. (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:20) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence having 1, 2, 3, or 4 amino acid substitutions compared to the given CDR sequence.

10. The combination according to any one of claims 7 to 9, wherein the combination of the anti - porcine CD163 antibody or binding protein is present in a single construct, preferably, wherein the combination comprises two or three anti - porcine CD163 antibodies or binding proteins.

11. The antibody or antibody combination according to any one of claims 1 to 10, wherein one or more or all of the antibodies are single - domain antibodies.

12. The antibody, binding protein or combination according to any one of claims 1 to 11, further comprising an entity capable of extending the half-life of the antibody, binding protein or combination, preferably albumin or the IgG Fc region.

13. One or more nucleic acid molecules comprising a nucleotide sequence encoding the antibody, binding protein or combination according to any one of claims 1 to 12.

14. One or more expression vectors comprising the one or more nucleic acid molecules according to claim 13.

15. One or more host cells comprising the expression vector according to claim 14 or the nucleic acid molecule according to claim 13, or expressing the antibody, binding protein or combination according to any one of claims 1 to 12.

16. A method for preparing an antibody, binding protein or combination according to any one of claims 1 to 12, the method comprising the following steps: (i) Culturing a host cell comprising the expression vector according to claim 14 or the nucleic acid molecule according to claim 13 under conditions suitable for expressing an encoded antibody, binding protein or combination; and optionally (ii) isolating or obtaining the antibody, binding protein or combination from the host cell or the culture medium / supernatant.

17. A composition comprising the antibody, binding protein or combination according to any one of claims 1 to 12, the one or more nucleic acid molecules according to claim 13, the one or more expression vectors according to claim 14 or the one or more host cells according to claim 15.

18. Use of the antibody, binding protein or combination according to any one of claims 1 to 12, the one or more nucleic acid molecules according to claim 13, the one or more expression vectors according to claim 14 or the one or more host cells according to claim 15 for therapy in a subject, preferably for treating or preventing PRRSV infection in a subject.

19. Use of the antibody, binding protein, composition, nucleic acid molecule, expression vector or host cell according to claim 18, wherein the subject is a pig.

20. A method of treating or preventing a subject from being infected with PRRSV, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody, binding protein or combination according to any one of claims 1 to 12, the one or more nucleic acid molecules according to claim 13, the one or more expression vectors according to claim 14 or the one or more host cells according to claim 15.

21. Use of the antibody, binding protein or combination according to any one of claims 1 to 12, the one or more nucleic acid molecules according to claim 13, the one or more expression vectors according to claim 14 or the one or more host cells according to claim 15 in the preparation of a medicament for therapy, preferably for treating or preventing PRRSV infection in a subject.

22. The method or use according to claim 20 or 21, wherein the subject is a pig.

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