CD163-binding protein

Abstract

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

Description

[Technical Field]

[0001] The present invention relates generally to the field of binding proteins, particularly antibodies, that bind to CD163 (Cluster of Differentiation 163), and in particular to binding proteins and antibodies that bind to membrane-bound porcine CD163 while not showing significant binding to soluble forms of porcine CD163. Such anti-CD163 binding proteins and antibodies, particularly when combined with other anti-CD163 binding proteins, are used for therapeutic and protective purposes, such as in the treatment or prevention of infectious diseases, such as porcine reproductive and respiratory syndrome (PRRS) virus infection, e.g., to reduce the incidence and severity of the disease. Binding protein- and antibody-based compositions, methods, and kits are also provided. [Background technology]

[0002] Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating viral swine diseases worldwide, resulting in significant economic losses to the swine industry. The causative agent is PRRSV, an enveloped RNA virus classified in the Arteriviridae family within the Nidovirales order. PRRSV has restricted host and cell tropism, with porcine alveolar macrophages (PAMs) being a key target cell. Clinical signs vary widely, but include respiratory distress and disease in young pigs and piglets, late-term abortion or stillbirth in sows and sows, early-gestation fetal resorption, reduced piglet viability in live-born piglets, and reduced growth in finishing pigs. Reduced or lost pregnancies, young piglet deaths, and reduced growth rates in all PRRSV-infected pigs are estimated to cost pork producers in the United States alone 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 the median losses to be 74,181 euros per farm (255 euros per sow).

[0003] All currently known PRRSV isolates are classified as one of two species, PRRSV-1 or PRRSV-2, which cause long-term infections and similar clinical symptoms but share only approximately 60% nucleotide identity. PRRSV-1 was first identified in Europe and tends to be found in European PRRSV isolates or lineages, whereas PRRSV-2 was first identified in North America and tends to be found in Asian or American isolates or lineages (see review by Stoian and Rowland, 2019, Vet. Sci., 6, 9).

[0004] There is great diversity within each species, and numerous strains have been identified since 2006, including new highly pathogenic strains / subtypes that have emerged particularly in China and Vietnam. Similar highly pathogenic strains have also emerged elsewhere, extending from Peninsular Malaysia to southern Russia, posing an increasing threat to pig populations (An et al., 2011, Emerging Infect Dis 17(9):1782). In China alone, over 20 million pigs were culled annually due to PRRSV infection in 2006 and 2007 (An et al., 2010, Emerging Infect Dis 16(2):365). More recently, case reports of virulent strains causing outbreaks in Europe point to the increasing emergence of PRRSV as a threat (Sinn et al., 2016, Porcine Health Management(2):28).

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

[0006] The exact mechanism of PRRSV entry is unknown, but it is thought that part of this mechanism involves PRRSV entering the endosomal compartment of the cell, where interaction of CD163 with the PRRSV GP2-GP3-GP4 heterotrimer uncoats the virus and releases the viral genome into the cytoplasm.

[0007] One proposed treatment option for PRRSV involves some kind of gene knockout or gene editing of CD163 to create pigs resistant to PRRSV infection and then breed these pigs to spread the genetic modification (Burkard et al., 2017, PLOS Pathogens 13(2):e1006206). While this has been shown to work very effectively, this treatment is complex and time-consuming, particularly in terms of being able to treat a significant percentage of a pig population. Additionally, and importantly, there is significant resistance in many markets to techniques involving genetic modification of animals, including the desirability of animal products produced from such animals.

[0008] The most common medical intervention used to limit the economic impact of PRRS is vaccination. Vaccines are used in all areas where the disease is endemic. However, due to safety concerns, they are only used under defined scenarios. Two types of vaccines are in use: either killed virus vaccines or (most often) modified live vaccines (MLVs). However, current vaccines are only partially effective and would be most valuable if deployed within an integrated approach to disease control, in which biosecurity and livestock management decisions are closely coordinated. The reasons behind the lack of vaccine efficacy are complex, but the high genetic diversity of PRRSV populations, coupled with the virus's biology (alveolar macrophage tropism and high mutability), are such that best results are seen when vaccine strains and circulating strains are tightly matched in terms of immunogenicity (reviewed by Nan et al., 2017, Front. Immunol. 8:1635). Furthermore, live vaccine strains can recombine with field strains to produce new field strains that may be pathogenic. Thus, MLVs can only be used in specific circumstances, further limiting their use. Summary of the Invention

[0009] Currently, there are no antiviral treatment options for PRRSV infection.

[0010] More recently, such alternative therapeutic or prophylactic options have been developed in the form of binding proteins and antibodies directed against porcine CD163, which can act to reduce or prevent PRRSV infection.

[0011] However, there is a clear need for alternative, preferably improved, treatment and prevention options for PRRSV infection (or other CD163-mediated infections) that can be easily used to treat or prevent infection in significant numbers of animals.

[0012] The present invention provides a means for such alternative therapeutic or preventative options by means of antibodies that have the ability to bind to the membrane-bound form of porcine CD163 on cells, but advantageously do not significantly bind to the soluble form of porcine CD163.

[0013] Such antibodies (or binding proteins constituting such antibodies) are sometimes referred to herein as membrane-specific CD163 antibodies (or binding proteins). Such antibodies (or binding proteins) have the ability to target only membrane-bound CD163. In other words, they do not target, or do not significantly target, soluble CD163. This is advantageous because soluble CD163 is cleaved from the cell surface and can be detected at high concentrations in serum in some swine infections. Therefore, soluble CD163 may effectively act as a sink for anti-CD163 antibodies used in therapy, potentially interfering with their action. However, due to their ability to distinguish between membrane (cell surface) and soluble forms of CD163, the antibodies of the present invention should not be attracted to such soluble (shed) antigens but instead target cells expressing CD163, which are also cells targeted by PRRSV during infection. Indeed, the antibodies of the present invention have been shown to provide tolerance to such soluble forms of CD163, resulting in compositions that function very effectively to inhibit PRRSV infection even in the presence of, for example, soluble CD163.

[0014] 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 wishing to be bound by theory, it is believed that the antibodies of the present invention are useful for targeting other CD163 antibodies to cells that express CD163, thereby reducing or preventing PRRSV infection with high efficiency.

[0015] When two anti-CD163 antibodies are paired (e.g., an antibody of the present invention and a second, different anti-CD163 antibody), very good results are observed. Such antibodies are sometimes referred to herein as biparatopic anti-CD163 antibodies. These biparatopic constructs effectively inhibit PRRSV-1 infection and, to some extent, PRRSV-2 infection, although their effectiveness is reduced in the presence of high concentrations of soluble CD163. Even better results are observed when three anti-CD163 antibodies are combined (i.e., an antibody of the present invention and second and third, different anti-CD163 antibodies). Such antibodies are sometimes referred to herein as triparatopic anti-CD163 antibodies. Advantageously, the triparatopic constructs of the present invention are highly effective in inhibiting both PRRSV-1 and PRRSV-2 infection, even in the presence of high concentrations of soluble CD163, such as those found in infected pigs, e.g., pigs suffering from viral or bacterial infections, as described elsewhere herein. For example, non-PRRSV infections such as Lawsonia intracellularis infection, pigs with complex (multiple pathogen) disease, pigs with multiple (or mixed) infections, and pigs with severe infections are frequently encountered in the field. [Effects of the Invention]

[0016] Thus, the antibodies and constructs of the present invention (e.g., other binding proteins containing the CD163 antigen-binding domain described herein) may provide a new type of therapeutic molecule that can preferentially target the cell membrane form of CD163, and thus may provide a highly effective therapeutic option for the treatment of both PRRSV-1 and PRRSV-2 infections. Because such antibodies do not bind significantly to the soluble form of CD163, they may be effective at lower doses, which is an additional advantage for the treated animal and from a cost perspective. DETAILED DESCRIPTION OF THE INVENTION

[0017] In one embodiment, the invention provides a binding protein, e.g., an antibody, that binds to CD163, e.g., porcine CD163, wherein said binding protein or antibody: (i) binds to the membrane-bound form of CD163 on cells, e.g., porcine CD163; and (ii) it does not significantly bind to soluble forms of CD163, such as porcine CD163;

[0018] In one embodiment, the invention provides a binding protein, e.g., an antibody, that binds to porcine CD163, wherein said binding protein or antibody comprises: (i) binds to the membrane-bound form of porcine CD163 on cells; and (ii) it does not significantly bind to the soluble form of porcine CD163;

[0019] As discussed elsewhere herein, preferred antibodies (or binding proteins) of the invention that are suitable for use in the therapeutic methods described herein are capable of binding to the SRCR5 domain of CD163 (e.g., porcine CD163), e.g., have an epitope (or a portion of an epitope) in the SRCR5 domain of CD163. Additionally, preferred antibodies (or binding proteins) are capable of inhibiting PRRSV-2 infection.

[0020] In a further embodiment, the invention provides a binding protein, e.g., an antibody, comprising at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, wherein said antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 2) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, or three amino acid substitutions compared to the CDR sequence in question; (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of IGWTGGTT (SEQ ID NO: 3) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, or three amino acid substitutions compared to the CDR sequence; (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of AADQAGWRTAGVRNTYEYDY (SEQ ID NO: 4), 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 CDR sequence in question.

[0021] In a further embodiment, the invention provides a binding protein, e.g., an antibody, comprising at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, wherein said antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of GRTFSSYA (SEQ ID NO: 2); (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of IGWTGGTT (SEQ ID NO: 3), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence AADQAGWRTAGVRNTYEYDY (SEQ ID NO: 4).

[0022] As described elsewhere herein, the antibody (or binding protein) preferably has the ability to bind to a membrane-bound form of CD163 on a cell, e.g., porcine CD163, but does not significantly bind to a soluble form of CD163, e.g., porcine CD163.

[0023] Certain preferred embodiments of the invention provide antibodies (or binding proteins) that bind to CD163, e.g., porcine CD163, and which comprise a VH domain having or comprising the amino acid sequence of SEQ ID NO: 1, or a sequence substantially homologous thereto. In some embodiments, such antibodies (or binding proteins) also comprise a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0024] In preferred embodiments, the invention provides antibodies (or binding proteins) that bind to CD163, e.g., 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 thereto (e.g., at least 85%, 90%, 95%, or 98% identity). In some embodiments, such antibodies (or binding proteins) also comprise a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0025] In preferred embodiments, the invention provides antibodies (or binding proteins) that bind to CD163, e.g., porcine CD163, and which comprise a VH domain having or comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, such antibodies (or binding proteins) also comprise a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0026] As described elsewhere herein, the antibody (or binding protein) preferably has the ability to bind to a membrane-bound form of CD163 on a cell, e.g., porcine CD163, but does not significantly bind to a soluble form of CD163, e.g., porcine CD163.

[0027] As described above, the present invention provides binding proteins, such as antibodies or binding proteins comprising an antigen-binding domain, that bind to (or specifically recognize or specifically bind to) CD163, preferably porcine CD163. CD163 is also known as M130, MM130, SCAR1, macrophage-associated antigen, hemoglobulin scavenger receptor, and scavenger receptor cysteine-rich type 1 protein M130.

[0028] CD163 is a 130 kDa type I transmembrane protein containing a signal peptide followed by nine scavenger receptor cysteine-rich (SRCR) domains, each approximately 100 amino acids in length. A 35-amino acid proline-serine-threonine (PST)-rich region (PST-1) separates SRCR domain 6 (SRCR6) from SRCR7. A second PST-rich region (PST-2) connects SRCR9 to the transmembrane domain and a short cytoplasmic tail containing a functional internalization motif. Surface expression of CD163 is restricted to cells of the monocyte-macrophage lineage.

[0029] Thus, the binding proteins or antibodies of the present invention bind to or are capable of binding to CD163. Accordingly, the binding proteins or antibodies of the present invention are sometimes referred to herein as anti-CD163 binding proteins or antibodies. The CD163 used in the present invention may be derived from any species, particularly mammals (e.g., pig (porcine), human, bovine (bovine), canine (canine), feline (feline), ovine (obine), equine (equine), mouse, or monkey). In a preferred embodiment, the CD163 is pig CD163, and the antibody (or binding protein) binds to or is capable of binding to (or specifically recognizes or specifically binds to) pig CD163. For example, it may be referred to as an anti-pig CD163 antibody.

[0030] Of particular relevance to the present invention, CD163 is expressed on the surface of porcine alveolar macrophages (PAMs) and is thought to play an important role in the ability of various pathogens, including viral pathogens, particularly PRRSV, to cause disease in pigs.

[0031] As described above, the present invention also provides an antibody or binding protein, preferably an isolated antibody or binding protein, that binds to CD163 (preferably porcine CD163), wherein the antibody (or binding protein) binds to the membrane-bound form of CD163 on cells and does not significantly bind to the soluble form of CD163. Such an antibody (or binding protein) of the present invention is sometimes referred to herein as a membrane-specific antibody (or binding protein).

[0032] The term "membrane-bound form of CD163" or "membrane-bound form of CD163 on a cell," or other equivalent terms, refers to CD163 that is attached, bound, embedded, or otherwise associated with, or is a component of, the plasma membrane of a cell. Thus, a membrane-bound form of CD163 can be referred to, for example, as a cell surface form of CD163, or a cell surface CD163 molecule, or a cell surface-bound CD163 molecule, or a cell-expressed CD163 molecule, or full-length CD163 (e.g., including the transmembrane domain and, optionally, the cytoplasmic tail). Such membrane-bound forms often represent (or correspond to) the native or natural form of CD163, e.g., the form found on cells that naturally express or overexpress CD163.

[0033] Unless otherwise specified, in the context of the present invention, the term "cell" is used to refer to a CD163-positive (CD163-expressing) cell. In the context of the present invention, the term "cell" is used to refer to a cell that includes a nucleus.

[0034] Suitable cell types that naturally express CD163 are well known to those of skill in the art and include monocytes and macrophages. A preferred cell type is porcine alveolar macrophages (PAMs). Alternatively, CD163 can be expressed or overexpressed in a membrane-bound form, e.g., by recombinant means (or by other engineering means), using a cell type that does not normally express CD163, i.e., a recombinant membrane-bound form of CD163, e.g., full-length CD163.

[0035] Thus, in some embodiments, the membrane-bound form of CD163 on a cell is the membrane-bound form of CD163 on a PAM, and the antibody (or binding protein) of the present invention has the ability to bind to PAM, also referred to herein as porcine PAM (or pPAM). In other words, in certain embodiments, the membrane-bound form of CD163 on a cell is PAM-associated CD163. In some embodiments, the membrane-bound form of CD163 on a cell is the membrane-bound form of CD163 on a cell transfected with (and thus expressing or overexpressing) 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 and described in the art, and several examples, such as HEK293 cells and CHO cells, are provided elsewhere herein. Typically, CD163-negative cells, i.e., cells that do not express CD163 before transfection, are used.

[0036] The preferred membrane-bound form of CD163 on cells is the native 130 kDa form or the corresponding recombinant form, which consists of the entire CD163 domain, including the nine SRCR domains, PST-rich region, transmembrane region, and cytoplasmic tail, except for the signal sequence (which is removed during intracellular processing and subsequent cell surface expression).

[0037] A preferred membrane-bound form of CD163 on a cell 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 a cell. The antibodies of the present invention may bind to a membrane-bound form of CD163 on a cell that corresponds to this membrane-bound form of CD163 on a cell (e.g., a different CD163 isoform or CD163 from a different species).

[0038] Antibodies of the invention preferably bind to the SRCR5 domain of CD163, e.g., porcine CD163 (or an epitope comprising one or more residues of said SRCR5 domain). Thus, preferred membrane-specific anti-CD163 binding proteins or antibodies of the invention have the ability to bind to the SRCR5 domain of CD163 or an epitope (or part of an epitope) of the SRCR5 domain, preferably the porcine SRCR5 domain.

[0039] Thus, in some embodiments, membrane-specific anti-CD163 antibodies of the invention do not bind (or do not significantly bind) to CD163 molecules containing deletions of or mutations in the SRCR5 domain. Thus, in some embodiments, membrane-specific antibodies of the invention do not bind (or do not significantly bind) to the PST-2 domain or an epitope (or a portion of an epitope) in the PST-2 domain of CD163, e.g., porcine CD163.

[0040] Preferably, porcine forms of CD163 are used to assess the binding capacity of antibodies of the invention, although equivalent forms from other species, e.g., other mammalian species, can also be used, e.g., to assess cross-reactivity.

[0041] The sequences of CD163 in various species are well known and described in the art and can be obtained, for example, from various sequence databases, such as Uniprot. For ease of reference, porcine CD163 has Uniprot number Q2VL90, which is reproduced below for reference.

[0042] The sequence of the porcine SRCR5 domain is shown below, corresponding to residues 477-577 of Uniprot Q2VL90: PRLVGGDIPCSGRVEVQHGDTWGTVCDSDFSLEAASVLCRELQCGTVVSLLGGAHFGEGSGQIWAEEFQCEGHESHLSLCPVAPRPDGTCSHSRDVGVVCS (SEQ ID NO: 41).

[0043] The sequence of porcine CD163 is shown below, corresponding to the entire sequence of Uniprot Q2VL90:

[0044] The ability of an antibody (or binding protein) to bind to membrane-bound CD163 (or CD163 expressed on the cell surface) on cells can be easily tested using methods well known and routinely performed in the art, and any appropriate method can be used. For example, flow cytometry (e.g., FACS) can be used. In an exemplary flow cytometry method, CD163-expressing cells (e.g., PAM cells, e.g., pPAM cells, or cells expressing a recombinant form of CD163, e.g., cells transfected with CD163, e.g., cells transfected with a construct containing full-length CD163, e.g., the entire region of CD163, e.g., cells transfected with a construct containing the signal sequence, all nine SRCR domains and both PST-rich regions, the transmembrane region, and the cytoplasmic tail) are incubated with the antibody (or binding protein) to be investigated, and the antibody (or binding protein) bound to CD163 on the cells is detected by fluorescence. For example, the antibody is fluorescently labeled. Such labeling can be achieved, for example, by incubating the cell-antibody mixture with a secondary antibody that recognizes the antibody under investigation (e.g., an anti-myc antibody if the antibody under investigation has a myc tag) and a fluorescently labeled third antibody (such third antibody recognizes the second antibody). Alternatively, the second antibody can also be fluorescently labeled. Thus, when the antibody (or binding protein) under investigation binds to the membrane-bound form of CD163 on cells, the cells become fluorescently labeled, and such cells, and therefore antibodies (or binding proteins) capable of binding to the membrane-bound form of CD163 on cells, can be easily identified using a flow cytometer. A particularly preferred flow cytometry assay for testing the ability of an antibody (or binding protein) to bind to the membrane-bound form of CD163 on cells, is described in the Examples.

[0045] In some embodiments, PAM cells with deleted SRCR5 domains can be used in these binding assays to assess whether an antibody (or binding protein) has the ability to bind to the SRCR5 domain (e.g., as described in Burkard et al., 2017, PLoS Pathogens, 13(2):e1006206).

[0046] Another method for testing the ability of an antibody (or binding protein) to bind to the membrane-bound form of CD163 is immunohistochemistry.Another method for testing the ability of an antibody (or binding protein) to bind to the membrane-bound form of CD163 is microscopic observation (e.g., confocal microscopy) of cells that have been fluorescently labeled as a result of antibody (or binding protein) binding to membrane-bound CD163.

[0047] As discussed elsewhere herein, the antibodies (or binding proteins) of the invention do not significantly bind (or do not bind) to soluble forms of CD163, eg, porcine CD163.

[0048] A soluble form of CD163 refers to a form of CD163 that exists in solution or in the soluble phase. Therefore, this form of CD163 is not membrane-bound, particulate, or in the form of insoluble aggregates or precipitates. A preferred form of soluble CD163 is (or corresponds to) CD163 that has been bound to the surface of a cell, e.g., a PAM, or another macrophage or monocyte, and then shed or lost from the cell membrane by cleavage, e.g., proteolytic cleavage, to form a soluble form of CD163 (such forms of CD163 are also referred to as "shed" or "shed" forms of CD163). Thus, a soluble form of CD163 can be derived from a membrane-bound form by cleavage, e.g., by native cleavage within the PST2 domain (the likely cleavage site is between residue HATG (residue 1041) and RSS).

[0049] A preferred soluble form of CD163 to which antibodies of the present invention and the like do not significantly bind is a soluble form of membrane-bound CD163 that has been shed (cleaved) from cells. Thus, a soluble form of CD163 comprises (or is comprised of) the same primary amino acid sequence as the membrane-bound form of CD163 on cells, or comprises (or consists of) a substantial portion (fragment) of the primary amino acid sequence of the membrane-bound form of CD163 on cells. For example, a soluble form of CD163 can comprise (or consist of) an amino acid 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 corresponding to the amino acid sequence of the membrane-bound form of CD163 on cells. Such forms generally comprise or consist of fragments from the extracellular domain of CD163. For example, a fragment comprising or consisting of, in order from N-terminus to C-terminus of the CD163 molecule, amino acid sequences from SRCR1-6, PST-1, SRCR7-9, and PST-2, or a fragment taken from amino acid residues 51 to 1044 or 51 to 1041 of SEQ ID NO: 42. In some embodiments, the soluble form of CD163 has a corresponding sequence (e.g., in a different CD163 isoform or CD163 from a different species).

[0050] In a preferred embodiment of the present invention, soluble CD163 maintains the tertiary structure naturally formed under physiological conditions, e.g., physiological conditions present in mammals, e.g., pigs. Thus, the soluble form of CD163 has its native tertiary structure, i.e., for example, the soluble 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 soluble CD163 can be a folded structure. Suitable soluble forms can also be recombinantly prepared, e.g., recombinant or synthetic molecules.

[0051] 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 thereto (e.g., in a different CD163 isoform or CD163 from a different species).

[0052] 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 includes the PST-1 domain. In some embodiments, the soluble form of CD163 has a corresponding sequence thereto (e.g., in a different CD163 isoform or CD163 from a different species).

[0053] 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., in a different CD163 isoform or CD163 from a different species).

[0054] 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., in a different CD163 isoform or CD163 from a different species).

[0055] Soluble (or shed) CD163 is found in the blood of subjects, such as pigs (serum CD163), as well as in the interstitium of tissues. Thus, the soluble form of CD163 is naturally occurring or corresponds to the naturally occurring or native form of soluble CD163.

[0056] Soluble CD163 can be obtained from any suitable source, ie, any sample or source in which CD163 is present in a soluble form.

[0057] One suitable and preferred source is recombinant CD163 (e.g., recombinant porcine CD163). Related forms of soluble recombinant CD163, such as porcine CD163, can be readily produced or synthesized using standard techniques, such as the sequence information provided herein and elsewhere in the art. For example, a recombinant CD163 construct encoding a desired soluble form of CD163, as described elsewhere herein, can be readily constructed and expressed in a suitable cell line, allowing the soluble CD163 protein to be isolated / purified.

[0058] For CD163 shed from cells into the circulation, a suitable source would be the blood or serum of a relevant subject (e.g., a pig or porcine subject). For example, relatively low levels of soluble CD163 (e.g., around or up to 0.5 mg / ml) are found in the serum of healthy pigs. Furthermore, higher levels of soluble CD163 (e.g., around or up to 4.5 mg / ml) are found in the serum of infected pigs (pigs suffering from an infection) or pigs undergoing an inflammatory response. Such high levels of soluble CD163 are found, for example, in any infectious disease that induces an inflammatory response or inflammation in animals, such as any bacterial or viral infection, including non-PRRSV-infected pigs. However, typical and paradigmatic infections frequently occurring in the field are those caused by Lawsonia intracellularis, as well as infections caused by M hyo, P. multocida, and S. suis. Additionally, because multiple simultaneous infections (complex / multipathogen diseases) are common in this field, such subjects (e.g., pigs or porcine subjects), or pigs with more severe infections, are likely to have higher levels of soluble CD163 than those found in healthy subjects (e.g., pigs or porcine subjects). Therefore, whole blood or serum from such healthy or infected pigs (e.g., serum from pigs infected with Lawsonia intracellularis or pigs suffering from other infectious diseases) can be a source of soluble CD163.

[0059] The soluble CD163 (or soluble CD163-related peptide) to which the antibodies (or binding proteins) of the present invention preferably do not significantly bind may therefore be CD163 in a physiological fluid (e.g., serum), for example, a physiological fluid (e.g., serum) from a healthy pig or an infected pig.

[0060] To assess the ability of binding proteins and antibodies to bind to soluble CD163, exemplary soluble forms of CD163, such as recombinant CD163, as described above and elsewhere herein, can be used. Exemplary are constructs containing the appropriate extracellular portion of CD163, such as constructs containing different subsets of CD163 SRCR domains, such as CD163-SRCR1-PST2, CD163-SRCR1-9, CD163-SRCR4-7, or CD163-SRCR5-6. Other combinations of CD163 SRCR domains, or fragments containing subsets of different CD163 SRCR domains, PST-1, and PST-2 domains, can also be used, provided only the extracellular portion is present. While porcine forms are preferably used to assess the antibodies of the invention, equivalent forms from other species, such as other mammalian species, can also be used, e.g., to assess cross-reactivity.

[0061] Methods for assessing binding (or ability to bind) to a suitable soluble form of CD163 are well known to those skilled in the art, and any suitable method may be used.

[0062] Convenient and suitable methods for assessing binding include in vitro binding assays, such as ELISA assays, which assess the binding of an antibody or binding protein to an immobilized antigen, such as an immobilized form of soluble CD163 as described herein. Those skilled in the art are familiar with ELISA assays and will readily be able to establish appropriate conditions for assessing the ability of a binding protein or antibody to bind to CD163 in such an assay. A particularly preferred ELISA assay is described in the Examples section.

[0063] A preferred antibody (or binding protein) of the present invention does not bind, does not significantly bind, or does not measurably bind to a soluble form of CD163, preferably porcine CD163, as assessed by ELISA. Exemplary soluble forms are described elsewhere herein. Preferred soluble forms are pCD163 SRCR1-PST2 and pCD163-SRCR1-9. See the Examples section for further details.

[0064] A preferred method for assessing binding (or ability to bind) to a suitable soluble form of CD163 (e.g., porcine CD163) is a surface plasmon resonance (SPR) assay (e.g., a BIACore assay). Suitable SPR assays are known in the art and are preferred because they allow for easier and more consistent quantification of binding. In certain preferred SPR assays, a suitable form of soluble CD163 is captured (or immobilized) on a solid support (e.g., a sensor chip) via, for example, amine coupling (e.g., 2000-2500 or 2500-3500 RU of CD163 is immobilized), and then various concentrations (e.g., a dilution series, e.g., a 2-fold or 3-fold dilution series) of the binding protein or antibody to be tested are injected. Preferred concentrations and flow rates for injection are described in the Examples section. A preferred pH for evaluation is pH 7.4. Exemplary soluble forms of CD163 for use in such SPR assays are described elsewhere herein. Preferred soluble forms are CD163-SRCR1-PST2 or CD163-SRCR1-9 or CD163-SRCR4-7.

[0065] Such SPR assays can also be useful for measuring the binding dynamics of antibody-antigen interactions, e.g., kinetic parameters such as association rate (k), dissociation rate (k), and affinity (K). In certain embodiments, measurements can be performed in a suitable buffer, e.g., a standard HEPES-EDTA buffer such as HBS-EP (available from GE Healthcare Life Sciences, 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.0005% surfactant P20), at pH 7.4 and 25°C. Kinetic parameters can be determined or calculated using any suitable model or software by fitting sensogram experimental data assuming a 1:1 interaction, e.g., using BIAevaluation software. Particularly preferred SPR assays are described in the Examples section of this specification.

[0066] Thus, in particularly preferred embodiments, the binding proteins or antibodies of the invention are deemed not to bind to soluble forms or not to bind significantly to soluble forms of CD163 (e.g., porcine CD163) in a surface plasmon resonance (SPR) assay (e.g., a BIACore assay).

[0067] In certain preferred embodiments, antibodies of the invention that do not bind or do not significantly bind soluble CD163, e.g., in the case of a VHH or VH format, have undetectable or essentially undetectable binding affinity for soluble CD163 (e.g., porcine CD163), e.g., a K in the range of 100 μM or higher. D (equilibrium dissociation constant affinity) has (poorer / stronger binding).

[0068] Thus, preferably, the antibodies of the invention have a K (as determined, assessed) in a surface plasmon resonance (SPR) assay of greater than 20 μM, 50 μM, 100 μM, 150 μM, 200 μM or 250 μM, for example in the case of a VHH or VH format. Dor is undetectable by SPR, or is so low that it is essentially unmeasurable, or is unreliable to measure, e.g., cannot be fitted or adequately fitted assuming a 1:1 interaction. Particularly exemplary binding affinities are disclosed in the Examples. Thus, for example, the H17B11 VHH antibody of the invention (shown in Table A) has a binding affinity of at least 100 μM, e.g., at least 200 or 250 μM, as assessed by surface plasmon resonance (SPR) assay / BIACore assay.

[0069] An exemplary form of soluble CD163 that can be used to assess such binding affinity is recombinant soluble CD163 as described herein, such as porcine CD163, for example, a form of recombinant porcine CD163 comprising SRCR4-7 or SRCR1-9. Suitable exemplary forms are described in the Examples section, such as the constructs pCD163-SRCR4-7 (optionally with human (hu)Fc) or pCD163-SRCR1-9 (optionally with huFc) or pCD163-SRCR1-PST2 (optionally with a 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 are used interchangeably to refer to porcine CD163 constructs comprising the full-length CD163 sequence from SRCR1 to PST2. Thus, the above-described binding affinities may be observed when antibodies of the invention, e.g., in VHH or VH format, are assayed with these constructs, e.g., in an SPR assay.

[0070] Importantly, the antibodies (or binding proteins) of the present invention bind to the membrane-bound form of CD163 on cells, but do not significantly bind to the soluble form of CD163. This combination of properties may be particularly important in terms of antibody-based therapies (such as treating or preventing PRRSV infection). As noted above, without wishing to be bound by theory, antibodies that bind to the membrane-bound form of CD163 on cells (e.g., PAM, porcine PAM) but not to the soluble form will not be attracted by soluble (shed) CD163 in the circulation or interstitium of the treated subject, but instead will directly target the cell membrane of appropriate disease-related cells (e.g., CD163-expressing PAM targeted by PRRSV).

[0071] Preferred antibodies of the invention retain the ability to bind to membrane-bound CD163 on cells and do not significantly bind to soluble CD163 in the presence of any physiological concentration of soluble CD163 (e.g., any concentration found in the human or animal body), for example, when soluble CD163 is present at high concentrations, e.g., at or up to or at least 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.

[0072] Therefore, the preferred antibody of the present invention distinguishes between membrane-bound CD163 and soluble CD163 on cells. Therefore, the antibody of the present invention can positively distinguish membrane-bound CD163 on cells. Therefore, the antibody of the present invention is considered to be specific for the membrane-bound form of CD163 on cells.

[0073] Therefore, the antibodies of the present invention may bind to conformational epitopes on CD163. Such conformational epitopes of CD163 are present in membrane-bound CD163 on cells, but are absent (or significantly reduced or altered) in soluble CD163. Without wishing to be bound by theory, it is believed that the conformational epitope on membrane-bound CD163 on cells bound by the antibodies of the present invention (e.g., H17B11) may be generated by being tethered to the membrane via the PST2 domain, and that this epitope disappears or changes when the CD163 molecule is shed or released. However, preferred antibodies of the present invention do not bind to the PST2 region (e.g., do not directly bind to the PST2 region itself).

[0074] Thus, although corresponding (or identical) primary amino acid sequences (linear amino acid sequences) may be present in membrane-bound CD163 on cells and in soluble CD163, preferred antibodies of the present invention can advantageously distinguish between the different forms, for example, by recognizing a conformational epitope rather than a linear epitope, for example, by recognizing a neoepitope or conformational neoepitope that is present in membrane-bound CD163 on cells but not in soluble CD163.

[0075] Suitable methods for obtaining membrane-specific anti-CD163 antibodies of the present invention are described in the Examples. However, a preferred protocol involves immunizing a suitable animal (e.g., a camelid, e.g., a llama) with membrane-expressed CD163, e.g., porcine CD163, for example, in the form of cells (e.g., HEK cells or CHO cells). Cells (e.g., HEK cells or CHO cells) recombinantly expressing CD163, e.g., cells recombinantly expressing constructs containing a significant portion of the extracellular domain of CD163, such as porcine (p)CD163-SRCR1-PST2 or pCD163-SRCR1-9, and / or PAM (or other cells with native expression of CD163). Antibody clones prepared from such immunizations are screened for membrane-specific anti-CD163 antibodies or subjected to appropriate selection rounds.

[0076] A preferred selection round may involve preparing a library of antibody clones, typically a phage display library, e.g., from the blood (PBMCs) of an immunized animal, and then subjecting these clones to one or more appropriate selection rounds. A preferred selection method used herein involves selection against both native membrane-expressed CD163 (here, isolated pPAM cells) and recombinant cell-expressed CD163 (here, HEK cells expressing pCD163-SRCR1-PST2). For example, in the method herein, counter-selection was performed using both empty HEK wild-type cells and pPAMΔ5 cells (cells in which SRCR domain 5 has been deleted). Counter-selection in the presence of excess soluble CD163 may also be a suitable additional or alternative selection step to obtain membrane-specific clones.

[0077] Once the selection rounds have been performed, it becomes possible to screen for membrane-specific anti-CD163 antibodies.

[0078] A convenient and preferred method for conducting this screening would be to perform flow cytometry (FACS) analysis using CD163-expressing cells known to be positive for the membrane-bound form of CD163 (e.g., PAMs, or recombinant membrane-bound forms of CD163, e.g., cells expressing the full-length membrane-bound form of CD163, e.g., as described elsewhere herein). Preferably, cells with native CD163, e.g., PAMs, are used. Candidate antibodies capable of binding to the cellular (membrane-bound) form of CD163 can then be identified.

[0079] Positive clones can be further screened to assess whether they do not bind (or significantly bind) to soluble CD163, e.g., whether they have the ability to distinguish between membrane-bound and soluble CD163 on cells. Again, suitable methods such as BiaCore or ELISA can be used, but final confirmation is preferably performed using SPR (e.g., BiaCore or an equivalent method) for accuracy and reliability. Suitable soluble forms of CD163, e.g., recombinant soluble forms, are described elsewhere herein and include pCD163-SRCR1-PST2, pCD163-SRCR1-9, or pCD163-SRCR4-7.

[0080] Insignificant (insignificant) binding to soluble forms of CD163 generally means that binding to these forms of CD163 is reproducibly (i.e., consistently observed) low or negligible. In some cases, insignificant binding can be considered background levels, e.g., levels observed in negative control experiments, or levels that are not significantly different, or undetectable or with very low affinity, e.g., in an SPR assay. Suitable tests for determining whether an antibody (or binding protein) does not bind or does not significantly bind to soluble forms of CD163 are described elsewhere herein.

[0081] In some embodiments, the antibodies of the invention do not bind (eg, do not measurably bind) to soluble forms of CD163.

[0082] Another convenient way to identify (screen) antibodies that bind to the membrane-bound form of CD163 on cells but do not bind (or do not significantly bind) to the soluble form of CD163 is to use a type of competition assay, for example, as part of flow cytometry (FACS) analysis. Thus, an assay can be used in which a sample of soluble CD163 (e.g., a recombinant form of soluble CD163 as described elsewhere herein) is introduced to assess whether soluble CD163 (e.g., a recombinant form of soluble CD163 as described elsewhere herein) has the ability to compete with the binding of a candidate antibody to a source of membrane-bound CD163 on cells. If soluble CD163 can compete to a significant extent, this indicates that the candidate antibody is not specific for membrane-bound CD163 on cells (because it also binds to the soluble form). If soluble CD163 cannot compete significantly, this indicates that the candidate antibody has the ability to distinguish between membrane-bound and soluble CD163 on cells.

[0083] In such a FACS assay, a significant decrease in signal upon addition of soluble CD163 indicates that the candidate antibody binds to, and is unable to distinguish between, both membrane-bound and soluble forms on cells, whereas a large or significant retention of signal upon addition of soluble CD163 indicates that the candidate antibody does not bind (or does not significantly bind) to soluble CD163 but binds to membrane-bound CD163 (or has no positive signal), indicating that the antibody can distinguish between membrane-bound and soluble forms on cells.

[0084] The binding proteins of the invention are capable of binding to (eg, specifically binding to) CD163, preferably porcine CD163.

[0085] Preferred binding proteins of the present invention are antibodies, in particular VHH antibodies or single domain antibodies, however, the embodiments described herein relating to antibodies, e.g. VHH antibodies or single domain antibodies, apply mutatis mutandis to other types of binding proteins as well, and vice versa.

[0086] A preferred binding protein is any single polypeptide chain that is capable of binding (eg, specifically binding) to CD163, preferably porcine CD163.

[0087] Suitable types of binding proteins that can be used in the present invention are known in the art. For example, in some embodiments, immunoglobulin-based polypeptides are used, which generally include CDR regions (and optionally FR regions or immunoglobulin-based scaffolds), and 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.

[0088] Therefore, binding proteins comprising an antigen-binding domain are also preferred, particularly where the antigen-binding domain is, comprises or is derived from an antibody (e.g., comprises the CDR and optionally FR regions of an antibody).

[0089] The binding proteins of the invention may preferably comprise multiple antibody or antigen binding domains that bind to CD163, for example, from two or three different antibodies as discussed elsewhere herein.

[0090] As explained elsewhere herein, the antibodies of the invention, due to their ability to distinguish between membrane (cell surface) and soluble forms of CD163, have the advantage of not being attracted, or being significantly less attracted, by soluble (shed) CD163 antigen, but instead targeting cells expressing CD163, which are also cells targeted by PRRSV during infection. Indeed, antibodies of the invention have been shown to provide tolerance to such soluble forms of CD163, resulting in compositions that function very effectively to inhibit PRRSV infection, even in the presence of soluble CD163.

[0091] In addition to possessing the advantageous property of membrane-specific binding, the antibodies of the present invention have also been shown to have the ability to inhibit PRRSV-2 infection.

[0092] Furthermore, the antibodies of the present invention have been shown to have excellent activity in inhibiting PRRSV infection when combined with other CD163 antibodies. Without wishing to be bound by theory, it is believed that the antibodies of the present invention are useful for targeting other CD163 antibodies to cells that express CD163, thereby reducing or preventing PRRSV infection with high efficiency.

[0093] When two anti-CD163 antibodies are paired (e.g., an antibody of the present invention and a second, different anti-CD163 antibody), preferably on the same construct, extremely good results are observed. Such bispecific molecules are preferably biparatopic constructs in which each different antibody recognizes a different epitope (here, two different epitopes) on the same antigen (here, CD163). Such antibodies are sometimes referred to herein as biparatopic anti-CD163 antibodies. These biparatopic constructs effectively inhibit PRRSV-1 infection and also inhibit PRRSV-2 infection to some extent, although the effect is reduced in the presence of high concentrations of soluble CD163.

[0094] Even better results are observed when three anti-CD163 antibodies are combined (i.e., an antibody of the present invention and a second and third different anti-CD163 antibody together), preferably on the same construct. Such a trispecific molecule is preferably a triparatopic construct in which each different antibody recognizes a different epitope (here, three different epitopes) on the same antigen (here, CD163). Such antibodies are sometimes referred to herein as triparatopic anti-CD163 antibodies. Advantageously, the triparatopic construct of the present invention can highly effectively inhibit both PRRSV-1 and PRRSV-2 infection, even in the presence of high concentrations of soluble CD163, such as those found in pigs suffering from infectious diseases (or complex (multi-pathogen) diseases), as discussed elsewhere herein.

[0095] Thus, a preferred embodiment of the present invention provides a combination of a membrane-specific antibody (or binding protein) of the invention, such as the H17B11 antibody set forth in Table A, or an antibody comprising the three CDRs of SEQ ID NOs: 2, 3 and 4, or a sequence substantially homologous thereto, with one or more additional anti-CD163 antibodies or binding proteins. Preferably, one, two or more anti-CD163 antibodies or binding proteins are used in such a combination, i.e., a total of two, three or more different anti-CD163 antibodies (or binding proteins) are used in combination, one of the antibodies (or binding proteins) being a membrane-specific antibody of the invention.

[0096] When more than one anti-CD163 antibody (or binding protein) is used, each antibody (or binding protein) preferably binds to a different epitope on CD163, and all antibodies (or binding proteins) in the combination are each capable of binding to a CD163 target molecule. Preferably, these combinations are provided on the same construct, for example, linked by a suitable linker. This format is particularly suitable for the single domain antibodies (or binding proteins) of the invention, such as the VHH antibodies described herein.

[0097] Preferably, such combinations are provided in a single construct, such as a biparatopic construct in which two (e.g., only two) such antibodies or binding proteins are provided together in a single construct, or a triparatopic construct in which three (e.g., only three) such antibodies or binding proteins are provided together in a single construct. Preferred anti-CD163 antibodies (or binding proteins) for use in combination with the membrane-specific antibodies of the invention, particularly membrane-specific antibodies based on the CDR sequences disclosed in Table A, or sequences substantially homologous thereto, comprise at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, said antigen-binding domain comprising a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (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 containing one or two amino acid substitutions compared to the corresponding CDR sequence; (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 containing one, two, three, or four amino acid substitutions compared to the corresponding CDR sequence; and (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 containing 1, 2, 3, or 4 amino acid substitutions compared to the corresponding CDR sequence.

[0098] In a preferred embodiment, the additional anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, and the antigen-binding domain comprises a 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 of RYVMG (SEQ ID NO: 10); (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AISWSGRAPYADSVKG (SEQ ID NO: 11), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GEGAIKWTTLDAYDY (SEQ ID NO: 12).

[0099] Viewed another way, preferred anti-CD163 antibodies (or binding proteins) for use in combination with the membrane-specific antibodies of the invention, in particular membrane-specific antibodies based on the CDR sequences disclosed in Table A, or sequences substantially homologous thereto, comprise at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, said antigen-binding domain comprising a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (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 one, two, or three amino acid substitutions compared to the corresponding CDR sequence; (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 comprises one, two, or three amino acid substitutions compared to the CDR sequence; and (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 CDR sequence in question.

[0100] In a preferred embodiment, the additional anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, and the antigen-binding domain comprises a 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 of GRTPSRYV (SEQ ID NO: 26); (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of ISWSGRA (SEQ ID NO: 27), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of AGGEGAIKWTTLDAYDY (SEQ ID NO: 28).

[0101] In certain preferred embodiments of the invention, the additional 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 some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0102] In preferred embodiments, the additional 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 some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0103] In preferred embodiments, the additional anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO: 9. In some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0104] An exemplary and preferred such antibody is the H03E11 antibody shown in Tables B or D. Another preferred anti-CD163 antibody (or binding protein) for use in combination with the membrane-specific antibodies of the 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, said antigen-binding domain comprising a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of DYTIG (SEQ ID NO: 18) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to the corresponding CDR sequence; (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of CINSITSNTYYADSVKG (SEQ ID NO: 19), or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, three, or four amino acid substitutions compared to the CDR sequence; and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of 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 CDR sequence in question.

[0105] In a preferred embodiment, the additional anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, and the antigen-binding domain comprises a 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 of DYTIG (SEQ ID NO: 18); (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of CINSITSNTYYADSVKG (SEQ ID NO: 19), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO: 20).

[0106] Viewed another way, another preferred anti-CD163 antibody (or binding protein) for use in combination with the membrane-specific antibodies of the 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, e.g., porcine CD163, said antigen-binding domain comprising a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (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 comprises one, two, or three amino acid substitutions compared to the corresponding CDR sequence; (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 comprises one, two, or three amino acid substitutions compared to the CDR sequence; and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence AADSGLFSGSSCLKYRAMRFGS (SEQ ID NO: 36), or a sequence substantially homologous thereto, wherein the substantially homologous sequence contains one, two, three, or four amino acid substitutions compared to the CDR sequence in question.

[0107] In a preferred embodiment, the additional anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, and the antigen-binding domain comprises a 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 of GFTLDDYT (SEQ ID NO: 34); (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of INSITSNT (SEQ ID NO: 35), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence AADSGLFSGSSCLKYRAMRFGS (SEQ ID NO: 36).

[0108] In certain preferred embodiments of the invention, the additional 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 some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0109] In preferred embodiments, the additional 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 (e.g., at least 85%, 90%, 95%, or 98% identity). In some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0110] In preferred embodiments, the additional anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO: 17. In some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0111] An exemplary and preferred such antibody is the H03D03 antibody shown in Tables C or E.

[0112] In combinations in which three anti-CD163 antibodies (or binding proteins) are used, the three specific antibodies (or binding proteins) defined above based on the CDR sequences disclosed in Table A, Table B (or D), and Table C (or E), respectively, or sequences substantially homologous thereto, are preferred combinations, e.g., in the triparatopic constructs of the invention.

[0113] In such embodiments, a first preferred anti-CD163 antibody (or binding protein) for use in combination with the membrane-specific antibodies of the 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, said antigen-binding domain comprising a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (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 containing one or two amino acid substitutions compared to the corresponding CDR sequence; (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 containing one, two, three, or four amino acid substitutions compared to the corresponding CDR sequence; and (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 containing 1, 2, 3, or 4 amino acid substitutions compared to the corresponding CDR sequence.

[0114] In a preferred embodiment, said first additional anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, and said antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of RYVMG (SEQ ID NO: 10); (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AISWSGRAPYADSVKG (SEQ ID NO: 11), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GEGAIKWTTLDAYDY (SEQ ID NO: 12).

[0115] An alternative first additional anti-CD163 antibody (or binding protein) for use in such embodiments 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 comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (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 one, two, or three amino acid substitutions compared to the corresponding CDR sequence; (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 comprises one, two, or three amino acid substitutions compared to the CDR sequence; and (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 CDR sequence in question.

[0116] In certain preferred embodiments of the invention, the first additional 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 some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0117] In preferred embodiments, the first additional 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 some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0118] In preferred embodiments, the first additional anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO: 9. In some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0119] An exemplary and preferred such first additional antibody is the H03E11 antibody shown in Table B or D.

[0120] In such embodiments, a second preferred anti-CD163 antibody (or binding protein) for use in combination with the two antibodies (or binding proteins) as outlined above comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, said antigen-binding domain comprising a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of DYTIG (SEQ ID NO: 18) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to the corresponding CDR sequence; (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of CINSITSNTYYADSVKG (SEQ ID NO: 19), or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, three, or four amino acid substitutions compared to the CDR sequence; and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of 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 CDR sequence in question.

[0121] In a preferred embodiment, said second additional anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, and said antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of DYTIG (SEQ ID NO: 18); (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of CINSITSNTYYADSVKG (SEQ ID NO: 19), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO: 20).

[0122] An alternative second, additional anti-CD163 antibody (or binding protein) for use in such embodiments comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, said antigen-binding domain comprising a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (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 comprises one, two, or three amino acid substitutions compared to the corresponding CDR sequence; (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 comprises one, two, or three amino acid substitutions compared to the CDR sequence; and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence AADSGLFSGSSCLKYRAMRFGS (SEQ ID NO: 36), or a sequence substantially homologous thereto, wherein the substantially homologous sequence contains one, two, three, or four amino acid substitutions compared to the CDR sequence in question.

[0123] In certain preferred embodiments of the invention, the second additional 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 some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0124] In preferred embodiments, the additional 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 (e.g., at least 85%, 90%, 95%, or 98% identity). In some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0125] In preferred embodiments, the second additional anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO: 17. In some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0126] An exemplary and preferred such second additional antibody is the H03D03 antibody shown in Tables C or E.

[0127] Binding proteins or constructs or combinations are preferred, for example binding proteins or constructs or combinations comprising multiple antibodies based on the antibody sequences set out in Table A, optionally in combination with antibody sequences set out in Table B (or D) and / or Table C (or E). The present invention is exemplified by monoclonal antibodies that are VHH antibodies (single domain antibodies), the sequences of which are set out in Tables A, B, C, D and E herein. The VH CDR domains and VH domains of each of these VHH antibodies are set out in Tables A to E herein. Antibodies (or binding proteins) comprising these sets of VH CDR domains, or VH domains, and in particular multi-antibody constructs or binding proteins comprising such domains (or sequences substantially homologous thereto), are preferred embodiments of the present invention.

[0128] In embodiments in which multiple antibodies, such as those described above, are provided in combination on a single construct, the antibodies (or binding proteins) can be provided in any order. Thus, references herein to a "first" antibody, a "second" antibody, etc., should not be construed to specify where these antibodies are located on any given construct. A preferred exemplary triparatopic construct (Tri-2) comprises (from N- to C-terminus) an antibody based on a sequence in Table B (or D), followed by an antibody based on a sequence in Table C (or E), followed by an antibody based on a sequence in Table A. Another preferred exemplary triparatopic construct (Tri-10) comprises (from N- to C-terminus) an antibody based on a sequence in Table A, followed by an antibody based on a sequence in Table B (or D), followed by an antibody based on a sequence in Table C (or E). In some embodiments of the present invention, membrane-specific antibodies of the present invention, e.g., antibodies based on a sequence in Table A, can be located at the N- or C-terminus of a plurality of antibodies present. Preferred antibodies are VHH antibodies, e.g., those shown in Tables A to E.

[0129] When provided as a single construct, eg, a single protein chain, the different antibodies in the construct are conveniently joined by peptide linkers.

[0130] In an alternative embodiment, 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.

[0131] Thus, said combination of antibodies (or binding proteins) or biparatopic constructs according to this aspect of the invention preferably comprises: The first antibody (or binding protein) comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, and the antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region is (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of DYTIG (SEQ ID NO: 18) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to the corresponding CDR sequence; (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of CINSITSNTYYADSVKG (SEQ ID NO: 19), or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, three, or four amino acid substitutions compared to the CDR sequence; and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of DSGLFSGSSCLKYRAMRFGS (SEQ ID NO: 20), or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, three, or four amino acid substitutions compared to the CDR sequence of interest; and The second antibody (or binding protein) comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, wherein said antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (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 containing one or two amino acid substitutions compared to the corresponding CDR sequence; (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 containing one, two, three, or four amino acid substitutions compared to the corresponding CDR sequence; and (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 comprises a variable heavy chain (VH) CDR3 that is a sequence containing 1, 2, 3, or 4 amino acid substitutions compared to the corresponding CDR sequence.

[0132] An alternative first anti-CD163 antibody or binding protein for use in such embodiments comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, wherein said antigen-binding domain comprises a heavy chain variable region comprising three complementarity-determining regions (CDRs), said heavy chain variable region comprising: (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 comprises one, two, or three amino acid substitutions compared to the corresponding CDR sequence; (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 comprises one, two, or three amino acid substitutions compared to the CDR sequence; and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence AADSGLFSGSSCLKYRAMRFGS (SEQ ID NO: 36), or a sequence substantially homologous thereto, wherein the substantially homologous sequence contains one, two, three, or four amino acid substitutions compared to the CDR sequence in question.

[0133] 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 some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs; or 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 (e.g., at least 85%, 90%, 95%, or 98% identity). In some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs; or The first anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO: 17. In some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0134] An exemplary and preferred such antibody is the H03D03 antibody shown in Tables C or E. In alternative such embodiments, the second anti-CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to CD163, e.g., porcine CD163, and the antigen-binding domain comprises a 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 of GRTPSRYV (SEQ ID NO: 26) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, or three amino acid substitutions compared to the corresponding CDR sequence; (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 comprises one, two, or three amino acid substitutions compared to the CDR sequence; and (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 CDR sequence in question.

[0135] In certain preferred embodiments, the second additional 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 some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs; or The second additional 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 some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs; or The second additional anti-CD163 antibody or binding protein comprises a VH domain having the amino acid sequence of SEQ ID NO: 9. In some embodiments, such an antibody (or binding protein) also comprises a VL domain comprising up to three light chain CDRs, preferably three light chain CDRs.

[0136] An exemplary and preferred such second additional antibody is the H03E11 antibody shown in Table B or D.

[0137] The various antibodies (or binding proteins) or combinations thereof described above, or constructs containing said antibodies (or binding proteins) or combinations thereof, have the ability to inhibit PRRSV-1 and / or PRRSV-2 infection and can therefore be used to treat or prevent PRRSV-1 and / or PRRSV-2 infection.

[0138] A preferred use of the antibody (or binding protein)-containing constructs or combinations of the present invention is the treatment or prevention of pathogenic infections involving CD163, particularly PRRSV infections. Typically, the antibody (or binding protein)-containing constructs and combinations of the present invention inhibit (or block or reduce) pathogen (e.g., PRRSV) infection, e.g., inhibit (or block or reduce) the ability of a pathogen, e.g., PRRSV, to cause infection (e.g., infect suitable host cells). Preferably, the inhibition or reduction is a measurable inhibition or reduction, and more preferably a significant inhibition or reduction, e.g., a statistically significant inhibition or reduction, such as a probability value of ≦0.05 or <0.05. In certain embodiments, the antibody (or binding protein)-containing construct or combination of the present invention can inhibit (or block or reduce) the ability of a pathogen, such as PRRSV, to infect a host cell by 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%, for example, completely or 100%. Typically, such inhibition rates (and other inhibition rates as described herein) are compared to (or relative to) an appropriate control assay or control level, such as a control assay or control level (e.g., a negative control or background level or assay) in the absence of a binding protein or antibody (anti-CD163 antibody). Thus, a 0% inhibition (control) level (or conversely, a 100% or maximum infection level) is typically the level in the absence of a binding protein or antibody (anti-CD163 antibody).

[0139] Such an ability to inhibit infection can be determined or tested by any suitable assay, examples of which will be readily apparent to those skilled in the art. Suitable assays include, for example, in vitro or ex vivo assays, including the use of CD163-expressing host cells, such as PAM or recombinant CD163-expressing host cells, as discussed elsewhere herein. Such cells can be contacted with PRRSV or other suitable pathogens to induce infection. Suitable assays are typically performed in the presence of serum, such as porcine serum (either from healthy or infected pigs, see below) or fetal bovine serum (FBS), or soluble CD163. The appropriate percentage of serum to use can be readily determined by one skilled in the art. For example, the assays described in the Examples section used levels of 10% FBS and 80% porcine serum.

[0140] FBS generally does not contain soluble CD163. However, porcine serum contains at least soluble CD163. Therefore, to test the ability of the antibody (or binding protein)-containing constructs or combinations of the present invention to inhibit PRRSV infection in the presence of soluble CD163, porcine serum, typically 80% porcine serum, was used. Soluble CD163 levels are generally significantly elevated in pigs suffering from infectious diseases, such as pigs undergoing an inflammatory response. Therefore, to test the ability of the antibody (or binding protein)-containing constructs or combinations of the present invention to inhibit PRRSV infection in the presence of high (but physiological) levels of soluble CD163, porcine serum (typically 80% porcine serum) from infected pigs (e.g., pigs infected with Lawsonia intracellularis) was used. Similarly, sera from pigs infected with other bacteria or viruses that produce elevated levels of soluble CD163 can also be used. Methods for determining the level of soluble CD163 in relevant serum samples can be routinely performed to establish the level of soluble CD163 present. In the pig serum assay described herein, the level of soluble CD163 was determined to be 0.40 mg / L in assays using serum from healthy pigs and 4.50 mg / L in infected pigs. Thus, such levels of soluble CD163 are exemplary and preferred, and in some embodiments of the invention, the levels of inhibition described herein are those observed in the presence of such concentrations of soluble CD163.

[0141] The ability of constructs or combinations comprising the antibodies (or binding proteins) of the present invention to inhibit or reduce such infection can be easily analyzed, for example, compared (or relative) to a 100% infection level established in a control assay. Suitable exemplary infection assays are described in the Examples section.

[0142] Any suitable concentration of constructs or combinations comprising the antibodies (or binding proteins) of the invention can be used to inhibit or reduce infection. Exemplary constructs or combinations of the invention have the ability to cause inhibition, e.g., levels of inhibition as outlined herein, with antibodies, particularly VHHs, 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 200, 300 or 400 μg / ml, for example between 50 or 100 and 200, 300 or 400 μg / ml. Preferred constructs or combinations of the invention have the ability to cause inhibition with antibodies, in particular VHHs, such as the levels of inhibition as outlined herein, when used at a concentration of 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 at a concentration of up to 100, 200, 300, 400, 450, 500, 600, 700, 800 or 900 ng / ml, such as at a concentration between 50 or 100 and 200, 300, 400 or 500 ng / ml. When a combination of antibodies (e.g., VHH antibodies) is used, these levels in some embodiments may refer to the total amount of antibodies (e.g., VHH) present, i.e., the sum of the individual concentrations of the antibodies present.

[0143] Exemplary constructs or combinations of the invention have the ability to cause inhibition with antibodies, particularly VHHs, when used at concentrations of at least 5, 10, 15, 20, 25, or 30 μM or more, for example at the levels outlined herein. Preferred constructs or combinations of the invention have the ability to cause inhibition, such as the levels of inhibition outlined herein, with antibodies, in particular VHHs, when used at a concentration of 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, or at a concentration of at least 50, 100, 200, 300, 400, 500, 600, 700, 800 or 900 nM, such as between 50 or 100 and 200, 300, 400 or 500 nM. More preferred constructs of the invention have the ability to cause inhibition with antibodies, particularly VHHs, when used at a concentration of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nM, such as concentrations up to 10, 20, 30, 40 or 50 nM, for example at a concentration between 5 or 10 and 30, 40 or 50 nM. When a combination of antibodies (e.g. VHH antibodies) is used, these levels in some embodiments may refer to the total amount of antibodies (e.g. VHHs) present, i.e. the sum of the individual concentrations of the antibodies present.

[0144] In some embodiments, antibody (or binding protein)-containing constructs or combinations of the present invention can inhibit (block or reduce) the ability of PRRSV-1 or PRRSV-2 to cause infection (e.g., infect CD163-expressing host cells). In some embodiments, binding proteins or antibodies of the present invention can inhibit (block or reduce) the ability of both PRRSV-1 and PRRSV-2 to cause infection (e.g., infect CD163-expressing host cells). Note that antibody (or binding protein)-containing constructs or combinations of the present invention target CD163 on host cells, as opposed to PRRSV (or other pathogens) themselves. This provides the important advantage of being able to inhibit infection by any virus, such as PRRSV, that utilizes the same binding region on CD163 for infection or pathogenesis. In this way, antibodies and the like of the present invention can provide a means to block many strains or isolates of PRRSV, including highly pathogenic strains or isolates, provided that they use CD163 to infect cells. The utilization of CD163 is believed to be common to infections by multiple PRRSV strains. Thus, the constructs and combinations of the present invention have broad utility. This contrasts with known approaches to PRRSV, such as vaccination, which are strain-specific and whose effectiveness (or lack thereof) may vary depending on the strain. Thus, the constructs and combinations of the present invention offer significant advantages and flexibility over such prior methods.

[0145] Preferred constructs or combinations of the present invention have the ability to almost completely inhibit PRRSV-1 infection, or to completely inhibit it, for example, at least 90%, 95%, or 98% inhibition may be observed, or 100% inhibition may be observed. Alternatively, at least 50%, 60%, 70%, 75%, or 80% inhibition may be observed. In some embodiments, antibodies capable of showing at least 80% inhibition of PRRSV-1 infection, more preferably at least 85%, 90%, 95%, or 98% inhibition, are preferred.

[0146] Preferred constructs or combinations of the present invention have the ability to almost completely inhibit PRRSV-2 infection, or to completely inhibit it, for example, at least 90%, 95%, or 98% inhibition may be observed, or 100% inhibition may be observed. Alternatively, at least 50%, 60%, 70%, 75%, or 80% inhibition may be observed. In some embodiments, antibodies capable of showing at least 80% inhibition of PRRSV-2 infection, more preferably at least 85%, 90%, 95%, or 98% inhibition, are preferred.

[0147] Other preferred constructs or combinations of the present invention have the ability to inhibit 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, for example, in the presence of relatively low levels of soluble CD163, for example, at levels up to or around 0.40 mg / L.

[0148] Other preferred constructs or combinations of the present invention have the ability to inhibit 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, e.g., at levels up to or around 4.50 mg / L.

[0149] In some 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 some embodiments, the binding proteins or antibodies of the present invention have the ability to specifically inhibit (or block or reduce) the ability of PRRSV-2 to cause infection (e.g., infect CD163-expressing host cells or specifically inhibit PRRSV-2 infection). Thus, exemplary antibodies can inhibit PRRSV-2 infection by at least 25%, 30%, 35%, 40%, 45%, or 50% (e.g., inhibit the ability of PRRSV-2 to infect host cells by at least 25%, 30%, 35%, 40%, 45%, or 50%).

[0150] An exemplary construct or combination comprises three VHH antibodies as shown in Tables A, B (or D), and C (or E).

[0151] In certain embodiments, the constructs or combinations of the present invention are 50 In some embodiments, the IC (e.g., for inhibiting PRRSV1 and / or PRRSV2 infection of a host cell, e.g., a PAM) is 10.0 nM or less, 9.5 nM or less, 9.0 nM or less, 8.5 nM or less, 8.0 nM or less, 7.5 nM or less, 7.0 nM or less, 6.5 nM or less, 6.0 nM or less, 5.5 nM or less, 5 nM or less, 4.5 nM or less, 4.0 nM or less, 3.5 nM or less, or 3.0 nM or less. 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. Particularly exemplary IC 50 The values ​​are also shown in the examples.

[0152] In certain embodiments, the constructs or combinations of the present invention are 90In some embodiments, the IC (e.g., for inhibiting PRRSV1 and / or PRRSV2 infection of a host cell, e.g., a PAM) is 30.0 nM or less, 25.0 nM or less, 20.0 nM or less, 15.0 nM or less, 12 nM or less, 10.0 nM or less, 9.5 nM or less, 9.0 nM or less, 8.5 nM or less, 8.0 nM or less, 7.5 nM or less, 7.0 nM or less, 6.5 nM or less, 6.0 nM or less, 5.5 nM or less, 5 nM or less, 4.5 nM or less, 4.0 nM or less, 3.5 nM or less, or 3.0 nM or less. 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. Particularly exemplary IC 90 The values ​​are also shown in the examples.

[0153] Preferred IC as above 50 Value or IC 90 The values ​​are preferably those determined in a suitable viral infectivity assay, eg as described above or in the Examples section.

[0154] In an alternative embodiment of the invention, the constructs or combinations of the invention can be used to reduce or prevent the risk of PRRSV infection.

[0155] Preferably, the above capabilities and properties are observed at a measurable or significant level, more preferably at a statistically significant level, when compared to an appropriate control level. Suitable significance levels are discussed elsewhere herein. More preferably, one or more of the above capabilities and properties are observed at a level that is measurably better, or more preferably significantly better (preferably statistically significantly better), when compared to capabilities observed for antibodies of the prior art.

[0156] In any statistical analysis referred to herein, preferably, a statistically significant difference relative to a relevant control or other comparison entity or measurement has a probability value of ≦0.1 or <0.1, preferably ≦0.05 or <0.05. Suitable methods for determining statistical significance are well known and described in the art, and any of these may be used.

[0157] In some embodiments, the constructs or combinations of the present invention have one or more, preferably two or more, or three or more, and most preferably all, of the functional properties described herein, particularly preferred functional properties.

[0158] As used throughout the application, the terms "a" and "an" are used to mean "at least one," "at least a first," "one or more," or "multiple" components or steps to which they refer, unless an upper limit is specifically stated thereafter. Thus, by way of example, "an antibody" as used herein means "at least a first antibody."

[0159] Furthermore, when the terms "comprising," "containing," "having," or "having," or other equivalent terms are used herein, in some more specific embodiments, for example, in the definitions of CDR or FR sequences herein, these terms include the terms "consisting of" or "consisting essentially of," or other equivalent terms.

[0160] Nucleic acid molecules comprising a nucleotide sequence encoding a binding protein or antibody of the invention (or for use in the invention), or encoding a construct or combination as defined herein, or a portion or fragment thereof, or a nucleic acid molecule substantially homologous thereto, form a further aspect of the invention.

[0161] Preferred nucleic acid molecules are those encoding a VHH antibody or VH region or domain of the invention (e.g., those encoding SEQ ID NO: 1, 9, or 17). Other preferred nucleic acid molecules are those encoding the set of three CDR sequences defined in any one of Tables A, B, C, D, or E, or sequences substantially homologous thereto. Preferred such nucleic acid molecules also encode the appropriate framework regions, e.g., FR1, FR2, FR3, and FR4 regions, preferably the set of FR sequences defined in any one of Tables A, B, C, D, or E, or sequences substantially homologous thereto.

[0162] The nucleic acid molecules of the invention are, for example, DNA or RNA molecules.

[0163] The term "substantially homologous," as used herein in reference to an amino acid or nucleic acid sequence, includes sequences having at least 55%, 60%, 65%, 70%, or 75%, preferably at least 80%, even more preferably at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the disclosed amino acid or nucleic acid sequence. In certain embodiments, an antibody (or binding protein) of the invention comprises one or at least one heavy chain variable region (or VH domain) comprising 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%, and most preferably at least about 97%, 98%, or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NOs: 1, 9, 17.

[0164] Therefore, the substantially homologous sequences of the present invention contain single or multiple base or amino acid changes (addition, substitution, insertion or deletion) to the sequences of the present invention. At the amino acid level, preferred substantially homologous sequences contain up to five, for example, only one, two, three, four or five, preferably one, two, three or four, preferably one, two or three, more preferably one or two amino acid changes in one or more of the framework regions and / or one or more of the CDRs constituting the sequences of the present invention. The changes can be conservative or non-conservative amino acids. Preferably, the changes are substitutions, preferably conservative amino acid substitutions.

[0165] In certain embodiments, when a given starting sequence is relatively short (e.g., 5 amino acids in length), there may be fewer amino acid substitutions in a sequence substantially homologous thereto compared to the number of amino acid substitutions that may optionally be made in the 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 some embodiments, preferably has one or two (more preferably one) amino acid changes compared to the starting sequence. Thus, in some embodiments, the number of amino acid changes in a substantially homologous sequence (e.g., a substantially homologous CDR sequence) can be adjusted to suit the length of a given starting CDR sequence. For example, different numbers of altered amino acids can be present depending on the length of a given starting CDR sequence to achieve a particular percentage of sequence identity in each CDR, for example, at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. Thus, by way of example, in a CDR sequence herein that is 20 or 22 amino acids in length, there may be up to 8, for example only 1, 2, 3, 4, 5, 6, 7 or 8, or only 1, 2, 3, 4, 5, 6 or 7, or only 1, 2, 3, 4, 5 or 6, or only 1, 2, 3, 4 or 5, preferably only 1, 2, 3 or 4, preferably only 1, 2 or 3, more preferably only 1 or 2, altered amino acids.

[0166] 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 of the CDR regions or one or more of the FR regions disclosed in Tables A or B or C or D or E. Thus, in some 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.

[0167] In some embodiments, in antibodies with a substantially homologous sequence compared to a given sequence, or with a degree of sequence identity compared to a given sequence, the altered amino acid residues are not in the CDR regions. For example, in some embodiments, in antibodies with a VH domain that has a degree of sequence identity with a given VH domain sequence of a particular antibody of the invention (or for use) (e.g., as disclosed in Table A, Table B, or Table C, Table D, or Table E), the altered (or variant) residue(s) are not in the CDR regions. Thus, in some embodiments, in antibodies with a substantially homologous sequence compared to a given sequence, or with a degree of sequence identity compared to a given sequence, the altered amino acid residue(s) are present in one or more framework regions.

[0168] As will be apparent from elsewhere herein, in other embodiments, in antibodies having substantially homologous sequences compared to a given sequence, or antibodies having a degree of sequence identity compared to a given sequence, the altered amino acid residue(s) may be in the CDR regions.

[0169] In some embodiments, in antibodies (or binding proteins) that have substantially homologous sequences relative to a given sequence or that have a degree of sequence identity relative to a given sequence, the three VH CDR amino acid sequences (i.e., all three VH CDR sequences combined) are considered to be the full CDR complement of the antibody, and the amino acid sequence of the full CDR complement of said antibody is at least 60%, 65%, or 70%, preferably at least 75%, or 80%, or at least 85%, or 90%, or at least 95% identical to the corresponding full CDR complement (or full CDR complement) of a given starting (or reference) antibody. The starting (or reference) antibody can have the CDR sequences of an antibody disclosed in Table A, B, or C, D, or E.

[0170] The altered residues can be conservative or non-conservative amino acid substitutions, or a mixture thereof. In such embodiments, preferred modifications are conservative amino acid substitutions.

[0171] In all embodiments, binding proteins, e.g., antibodies, comprising substantially homologous sequences retain the ability to bind to CD163, e.g., porcine CD 163. Preferably, binding proteins, e.g., antibodies, comprising substantially homologous sequences retain one or more (preferably all) of the other properties described herein in connection with the H17B11 (Table A), H03E11 (Table B), or H03D03 (Table C) antibodies, as appropriate.

[0172] The CDRs of the antibodies (or binding proteins) of the present invention are preferably separated by suitable framework regions as found in naturally occurring antibodies and / or effective engineered antibodies. H (e.g., VHH), V LThe individual CDR sequences are preferably provided within or incorporated into a suitable framework or scaffold to enable antigen (here, CD163) binding. Such framework sequences or regions may correspond to naturally occurring framework regions FR1, FR2, FR3, and / or FR4, as appropriate for forming a suitable scaffold, or may correspond to consensus framework regions identified, for example, by comparing various naturally occurring framework regions. Alternatively, non-antibody scaffolds or frameworks, such as T cell receptor frameworks, may be used.

[0173] Suitable sequences that can be used for the framework region are well known and documented in the art, and any of these may be used. Preferred sequences for the framework region are one or more of the framework regions that make up (or are used in) the VHH antibodies of the present invention, preferably one or more of the framework regions of the H17B11, H03E11, or H03D03 VHH antibodies disclosed in Table A, Table B (or D), and Table C (or E), respectively, or framework regions that are substantially homologous thereto, particularly framework regions that allow antigen specificity to be maintained, for example framework regions that result in a substantially identical or identical three-dimensional structure of the antibody.

[0174] In certain preferred embodiments, all four variable heavy chain (SEQ ID NOs: 5, 6, 7 and 8) framework regions (FR), or FR regions substantially homologous thereto, are found in antibodies (or binding proteins) of the invention, particularly antibodies (or binding proteins) based on the CDRs of SEQ ID NOs: 2, 3 and 4.

[0175] In other preferred embodiments, all four variable heavy chain (SEQ ID NOs: 13, 14, 15 and 16) framework regions (FR), or FR regions substantially homologous thereto, are found in antibodies (or binding proteins) of the invention, particularly antibodies (or binding proteins) based on the CDRs of SEQ ID NOs: 10, 11 and 12.

[0176] In other preferred embodiments, all four variable heavy chain (SEQ ID NOs: 21, 22, 23 and 24) framework regions (FR), or FR regions substantially homologous thereto, are found in antibodies (or binding proteins) of the invention, particularly antibodies (or binding proteins) based on the CDRs of SEQ ID NOs: 18, 19 and 20.

[0177] In other preferred embodiments, all four variable heavy chain (SEQ ID NOs: 29, 30, 31 and 32) framework regions (FR), or FR regions substantially homologous thereto, are found in antibodies (or binding proteins) of the invention, particularly antibodies (or binding proteins) based on the CDRs of SEQ ID NOs: 26, 27 and 28.

[0178] In other preferred embodiments, all four variable heavy chain (SEQ ID NOs: 37, 38, 39 and 40) framework regions (FR), or FR regions substantially homologous thereto, are found in antibodies (or binding proteins) of the invention, particularly antibodies (or binding proteins) based on the CDRs of SEQ ID NOs: 34, 35 and 36.

[0179] The CDR sequences of particular antibodies of the invention are shown in Tables A, B, C, D and E. In some embodiments, the CDR sequences of the antibodies of the present invention may be CDR sequences in the VH (VHH) domain of the antibodies of the present invention identified using any suitable method (or tool), for example, 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 identified using the IMGT numbering scheme (e.g., Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); www.imgt.org)).

[0180] Using routine methods in the art, such as alanine scanning mutagenesis and / or analysis of the crystal structure of an antigen-antibody complex, it can be determined which amino acid residues in the CDRs do not contribute, or do not contribute significantly, to antigen binding, and are therefore good candidates for alteration or substitution in embodiments of the invention involving substantially homologous sequences.

[0181] Once an addition, deletion, substitution, or insertion of one or more amino acids in the amino acid sequence of a parent antibody to form a new antibody has been identified, said parent antibody being one of the antibodies of the invention as defined elsewhere herein, testing the resulting new antibody to identify an antibody that binds to CD163 in accordance with the invention, e.g., a membrane-specific antibody as described elsewhere herein, can be performed using techniques routine in the art. Such methods can be used to form a plurality of new antibodies that can be tested for their ability to bind to CD163, e.g., exhibit membrane-specific binding as described elsewhere herein. Preferably, said addition, deletion, substitution, or insertion of one or more amino acids occurs in one or more of the CDR domains.

[0182] For example, the above manipulations can be conveniently carried out by genetic modification at the nucleic acid level, whereby a nucleic acid molecule encoding the appropriate binding protein and its domain is modified so that the resulting expressed protein's amino acid sequence is then modified in an appropriate manner. Testing the ability of one or more modified antibodies to bind to CD163, for example, 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.

[0183] The novel antibodies produced, obtained or obtainable by these methods form a further aspect of the present invention.

[0184] 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 proteins or nucleic acid molecules of the invention in substantially the same way. For example, any substantially homologous antibody should retain the ability to bind to CD163 as described above. Preferably, a substantially homologous antibody should retain one or more (or all) of the functional capabilities of the starting antibody, e.g., the ability to exhibit membrane-specific binding as described elsewhere herein, if appropriate.

[0185] Preferably, any substantially homologous antibody should retain the ability to specifically bind to the same epitope of CD163 recognized by the starting antibody in question, e.g., the same epitope recognized by one or more CDR domains of the antibodies of the invention described herein or the VH (VHH) domain of the invention, e.g., bind to the same epitope as one or more of the various antibodies of the invention (e.g., one or more of the VHH antibodies set out in Tables A, B, C, D, or E, respectively). Thus, preferably, any substantially homologous antibody should retain the ability to compete with one or more of the various antibodies of the invention (e.g., the VHH antibodies set out in Tables A, B, C, D, or E, respectively) for binding to CD163 in a suitable assay.

[0186] Binding to the same epitope / antigen can be readily tested using methods well known and described in the art, such as binding assays, e.g., competition assays, or by analysis of the crystal structure of the antigen-antibody complex. Retention of other functional properties can also be readily tested using methods well known and described in the art or herein.

[0187] 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 as the antibodies and antibody fragments of the present invention, e.g., whether it binds to the same epitope, or whether it binds with the same or comparable affinity, such as competitive or ELISA assays as described elsewhere herein. For example, to determine whether an antibody (or binding protein) binds to a membrane-bound form of CD163, e.g., porcine CD163, an assay capable of measuring antibody binding to cells expressing CD163, such as a FACS assay, is preferred. SPR, e.g., a BIAcore assay, can also be readily used to determine whether an antibody, e.g., a "substantially homologous" antibody, can bind to CD163, e.g., whether it can bind to a soluble form of CD163 (or whether it cannot bind). Indeed, SPR (BIAcore) assays are suitable for determining whether an antibody (or binding protein) does not bind or does not significantly bind to a soluble form of CD163, e.g., porcine CD163. Those skilled in the art will be aware of other suitable methods and variations.

[0188] As outlined below, competitive binding assays can be used to test whether an antibody, e.g., a "substantially homologous" antibody, retains the ability to specifically bind to substantially the same epitope of CD163 recognized by one or more of the antibodies of the invention set forth in the various sequence listings herein, or has the ability to compete with one or more of the various antibodies of the invention set forth in the various sequence listings herein. The method described below is only one example of a suitable competitive assay. Those of skill in the art will be aware of other suitable methods and variations.

[0189] In an exemplary competitive assay, binding of various effective concentrations of an antibody of the present invention to CD163 (e.g., a membrane-bound form of CD163) is assessed in the presence of various concentrations of a test antibody (e.g., a substantially homologous antibody). The amount of binding inhibition induced by the test antibody can then be assessed. Test antibodies that increase in concentration with the antibody of the present invention (i.e., increasing the concentration of the test antibody results in a corresponding decrease in the amount of the antibody of the present invention binding to CD163 (e.g., a membrane-bound form of CD163)) are evidence of binding to substantially the same epitope. Preferably, the test antibody significantly reduces the amount of the antibody of the present invention binding to CD163 (e.g., a membrane-bound form of CD163). Preferably, the test antibody reduces the amount of the antibody of the present invention binding to CD163 (e.g., a membrane-bound form of CD163) by at least about 95%. ELISA and flow cytometry assays can be used to assess binding inhibition in such competitive assays, although other suitable techniques will be known to those of skill in the art. Flow cytometry assays are particularly preferred when membrane-specific antibodies of the present invention are involved.

[0190] Such antibodies (monoclonal antibodies) have the ability to specifically bind to an epitope on CD163 that is substantially identical (or the same) as the epitope on CD163 recognized by an antibody of the invention, e.g., a membrane-specific antibody of the invention, or an overlapping epitope on CD163, or have the ability to compete with an antibody of the invention (e.g., VHH antibody H17B11 as shown in Table A). Antibodies that have the ability to compete with an antibody of the invention, e.g., a membrane-specific antibody of the invention (such as VHH antibody H17B11 as shown in Table A), are a further embodiment of the invention.

[0191] As used herein, the term "competing antibody" refers to an antibody that binds to nearly, substantially, or essentially the same epitope as a "reference antibody," or even the same epitope. A "competing antibody" includes antibodies with overlapping epitope specificity. Thus, a competing antibody can effectively compete with a reference antibody for binding to CD163 (e.g., membrane-bound CD163). Preferably, a competing antibody can bind to the same epitope as the reference antibody. Alternatively, a competing antibody preferably has the same epitope specificity as the reference antibody.

[0192] A "reference antibody", as used herein, is an antibody capable of binding to CD163 in accordance with the present invention, preferably having a VH domain as defined herein, more preferably a VHH antibody having a VH domain or comprising SEQ ID NO: 1, 9 or 17 (or the relevant three CDR sequences of said sequences) as outlined in Table A, B, C, D or E. A preferred reference antibody is a VHH antibody having a VH domain or comprising SEQ ID NO: 1 (or the relevant three CDR sequences of said sequences) as outlined in Table A. In other words, a preferred reference antibody is a membrane-specific antibody as defined in Table A.

[0193] Identifying one or more competing antibodies or antibodies that bind to the same epitope is now technically straightforward given the availability of reference antibodies such as those outlined in the sequence listing herein. In particular, it is preferred to identify one or more competing antibodies that bind to the same epitope as the VHH antibodies of Table A and exhibit membrane-specific binding activity. It will be appreciated that, since the identification of a competing antibody or an antibody that binds to the same epitope can be determined in comparison with the reference antibody, it is not necessary to actually determine the epitope to which either or both of the antibodies bind in order to identify a competing antibody or an antibody that binds to the same epitope. However, epitope mapping, if necessary, can be performed using standard techniques.

[0194] Thus, a further aspect provides an antibody (or binding protein) comprising an antigen binding domain that binds or specifically binds to CD163, e.g. porcine CD163, wherein said antibody (antigen binding domain) binds to the same epitope as a VHH antibody of Table A (or an antibody having CDRs defined in Table A, or CDRs substantially homologous thereto), and exhibits membrane-specific binding activity as described elsewhere herein.

[0195] To the best of the inventors' knowledge, no antibody (e.g., monoclonal antibody) has been described in the art that can bind (or specifically bind) to porcine CD163, wherein the antibody binds to the membrane-bound form of porcine CD163 on cells but does not significantly bind to the soluble form of porcine CD163 as described herein. Preferably, the antibody binds within the SRCR5 domain of CD163.

[0196] Thus, the individual membrane-specific monoclonal antibodies described herein, e.g., in Table A, are both unique and advantageous. Furthermore, the epitopes to which such antibodies bind, as well as antibodies that bind to these same epitopes, are of interest. Thus, it is believed that the antibodies of the present invention can bind to novel epitopes, e.g., conformational epitopes, in the SRCR5 region of porcine CD163 on cells, conferring membrane-specific binding.

[0197] Substantially homologous sequences of the proteins of the invention include, but are not limited to, conservative amino acid substitutions, or changes that do not affect, for example, the VH, VL, or CDR domains of an antibody, such as antibodies to which tag sequences, toxins, or other moieties have been added that do not contribute to antigen binding, or changes to convert one type or format of binding protein, antibody molecule, or fragment to another type or format of binding protein, antibody molecule, or fragment (e.g., converting a VHH to a Fab or scFv or whole antibody, or vice versa), or converting an antibody molecule to an antibody molecule of a particular class or subclass (e.g., converting an antibody molecule to an IgG or subclass thereof, e.g., IgG2).

[0198] As used herein, a "conservative amino acid substitution" refers to an amino acid residue being replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In another example, families of amino acid residues can be grouped based on hydrophobic or hydrophilic side groups.

[0199] Homology can be evaluated by any convenient method. However, to determine the degree of homology between sequences, computer programs that perform multiple alignment of sequences, such as Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994), are useful. If necessary, the Clustal W algorithm can be used with the BLOSUM62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992) and a gap opening penalty of 10 and a gap extension penalty of 0.1, resulting in the highest degree of match between the two sequences, with at least 50% of the total length of one of the sequences involved in the alignment. Other methods that can be used to align sequences include 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), which results in the highest degree of correspondence between two sequences and determines the number of identical amino acids between the two sequences. Other methods for calculating the percentage identity between two amino acid sequences are generally recognized in the art, such as the method described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math., 48:1073, 1988, Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects). Generally, computer programs are used for such calculations.

[0200] Programs that compare and align pairs of sequences, 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. Additionally, the European Bioinformatics Institute's Dali server provides structure-based alignments of protein sequences (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).

[0201] By providing a reference point, sequences according to the invention having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology, sequence identity, etc. can be determined using the ALIGN program with default parameters (e.g., available on the internet at the GENESTREAM network server, IGH, Montpellier, France).

[0202] As used herein, the terms "antibody" and "immunoglobulin" broadly refer to any immunological binding agent containing an antigen-binding domain, including polyclonal and monoclonal antibodies. However, monoclonal antibodies are preferred. In other words, in some embodiments, the antibodies of the present invention are not polyclonal. Depending on the type of constant domain in their heavy chains, whole antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. The antibodies of the present invention can be in any of these classes. Some of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, and IgG4. For example, camelid antibodies are IgG antibodies and often have IgG2 or IgG3 constant domains. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0203] Generally, when whole antibodies rather than antigen-binding regions are used in the present invention, IgGs are preferred because they are the most common antibodies in the physiological situation and they are most easily made in the laboratory.

[0204] The "light chains" of mammalian antibodies are assigned to one of two clearly distinct types, kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains and a few amino acids in the framework regions of their variable domains.

[0205] As used herein, the term "heavy chain complementarity determining region" ("heavy chain CDR") refers to a region of hypervariability within the heavy chain variable region (VH domain) of an antibody molecule or within a VHH antibody molecule. The heavy chain variable region has three CDRs, designated heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, from the amino terminus to the carboxy terminus. The heavy chain variable region also has four framework regions (FR1, FR2, FR3, and FR4, from the amino terminus to the carboxy terminus). These framework regions separate the CDRs.

[0206] As used herein, the "heavy chain variable region" (V H The term antibody heavy chain variable region (Hb domain) refers to the variable region of an antibody molecule.

[0207] As used herein, the term "light chain complementarity determining region" ("light chain CDR") refers to the light chain variable region (V L The light chain variable region refers to the region of hypervariability within the light chain (domain). The light chain variable region has three CDRs, designated light chain CDR1, light chain CDR2, and light chain CDR3, from the amino terminus to the carboxy terminus. The light chain variable region also has four framework regions, designated FR1, FR2, FR3, and FR4, from the amino terminus to the carboxy terminus. These framework regions separate the CDRs.

[0208] As used herein, the "light chain variable region" (V L The term (domain) refers to the variable region of the light chain of an antibody molecule. As will be understood by those skilled in the art, immunological binding reagents encompassed by the term "antibody" include or extend to all antibodies and antigen-binding fragments thereof, including whole antibodies, dimeric, trimeric and multimeric antibodies, bispecific antibodies, chimeric antibodies, recombinant antibodies and engineered antibodies, and fragments thereof.

[0209] Thus, the term "antibody" is used to refer to any antibody-like molecule having an antigen-binding region (e.g., an antigen-binding region comprising CDRs and any FRs derived from an antibody molecule, or a region corresponding thereto), and this term includes Fab', Fab, F(ab')2, single domain antibody (DAB), TandAb dimer, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, linear antibody, minibody, diabody, bispecific antibody fragment, bibody, tribody (scFv-Fab fusion, bispecific or trispecific, respectively). ; sc-diabodies; kappa (lambda) bodies (scFv-CL fusions); BiTEs (bispecific T cell engagers, scFv-scFv tandems that attract T cells); DVD-Ig (dual variable domain antibodies, bispecific format); SIPs (small immune proteins, a type of minibody); SMIPs ('small modular immunopharmaceuticals' scFv-Fc dimers); DARTs (ds stabilized diabodies 'dual affinity retargeting'); antibody fragments containing an antigen-binding domain such as small antibody mimetics containing one or more CDRs etc.

[0210] Techniques for preparing and using various antibody-based constructs and fragments are well known in the art.

[0211] Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be produced by treating antibodies with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges and produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAb, TandAb, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques or chemically synthesized. Techniques for producing antibody fragments are well known and described in the art.

[0212] In all embodiments of the invention, single-domain antibodies (also referred to 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, capable of binding to an antigen (although single VL domains capable of binding to an antigen have been described and can be used). Thus, in some such preferred embodiments, an antibody (or antigen-binding domain) of the invention comprises (or consists of) one (or a single, or only, or only one) heavy chain variable region (VH or VHH), although in some embodiments, several of these individual heavy chain variable regions, identical or different in sequence, can be present together in the same construct or molecule.

[0213] 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, such as a camelid such as a llama, or a shark, with a desired antigen, then cloning the VH domain of the produced antibody into a suitable expression vector and selecting binders. Libraries of VH domains (e.g., phage display libraries of human VH domains) are also available or can be generated and then screened.

[0214] Due to their relatively small size, single domain antibodies may have a relatively short half-life, for example, a relatively short plasma half-life.Therefore, such antibodies may be modified to extend or increase their half-life, and such modified antibodies (binding proteins) constitute part of the present invention.Techniques for doing this are well known and described in the art, and any of these can be used. Examples include attaching, conjugating, or fusing an antibody (binding protein) to albumin / serum albumin (or another protein or entity that itself has a long (or longer) half-life, e.g., an alternative entity or protein that has a longer half-life than the antibody to which it is fused, or that can act to extend the half-life of the protein (such as an antibody) to which it is bound), or attaching, conjugating, or fusing an antibody (binding protein) to another protein or entity (e.g., an antibody, e.g., a VHH antibody) that can interact with a protein or entity that itself has a long (or longer) half-life (such as, for example, an antibody that binds to IgG, e.g., porcine IgG, e.g., a VHH antibody), or attaching or conjugating an antibody (binding protein) to PEG (or other polymer, e.g., a hydrophilic polymer), or attaching, conjugating, or fusing an antibody (binding protein) to an antibody or other protein or entity that binds FcRn. In this regard, fusion to the IgG Fc region is an established strategy for extending the half-life of therapeutic proteins. Thus, preferred antibodies (binding proteins) comprise an Fc region or domain, e.g., are fused to an Fc region or domain (i.e., are Fc fusions). Such Fc regions or domains are known in the art and generally comprise the CH2 and CH3 domains of antibody heavy chains, which associate to form homodimers. These regions can be derived from any suitable source or species, e.g., a source or species different from the host species used to generate the antibody, e.g., by immunization or from a source or species different from the location from which the antibody is derived, but preferably correspond to or are derived from a porcine Fc region or domain.

[0215] Thus, in some embodiments of the invention, the antibody (or binding protein) comprises, is conjugated or otherwise fused to an entity capable of extending half-life, preferably albumin or an IgG Fc region.

[0216] In some embodiments of the invention, the antibody (or binding protein) comprises, is conjugated to, or is otherwise fused to, an entity capable of extending half-life, such as a further antibody capable of extending half-life, such as a VHH antibody.

[0217] In certain embodiments, antibodies or antibody fragments of the present invention comprise all or a portion 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, e.g., an IgG2 heavy chain constant region, or a portion thereof. Furthermore, antibodies or antibody fragments can comprise all or a portion of a kappa light chain constant region or a lambda light chain constant region, or a portion thereof. All or a portion of such constant regions can be naturally occurring or wholly or partially synthetic. Suitable sequences for such constant regions are well known and described in the art. When the full complement of constant regions from the heavy and light chains is included in an antibody of the present invention, such an antibody is typically referred to herein as a "full-length" or "whole" antibody. In some embodiments, IgG2 antibodies are preferred. In other embodiments, it is preferred that no constant region, e.g., a heavy or light chain constant region, is present, e.g., the variable domain or heavy chain variable domain (VH) is the only part of the antibody that is present.

[0218] The antibody or antibody fragment can be naturally occurring or wholly or partially synthetically produced.

[0219] Many antibodies or antibody fragments contain an antibody light chain variable region (V) that contains three CDR domains. L ) and an antibody heavy chain variable region (V) containing three CDR domains H The VL and VH generally form an antigen-binding site.

[0220] However, it is well documented in the art that the presence of three CDRs from the light chain variable domain and three CDRs from the heavy chain variable domain of an antibody is not necessarily required for antigen binding, and therefore constructs smaller than the above-mentioned classical antibody fragments are known to be effective.

[0221] For example, camelid antibodies have a broad antigen-binding repertoire but lack light chains. Furthermore, results using single-domain antibodies containing only a VH domain or only a VL domain have shown that these domains can bind to antigens with acceptably high affinity and have other advantages, such as small size and ease of production. Therefore, single-domain antibodies (e.g., VHH antibodies) as described and exemplified herein, in which three CDRs can effectively bind to antigens, are preferred.

[0222] The antibodies, binding proteins, and nucleic acid molecules of the present invention are generally "isolated" or "purified" molecules insofar as they are distinguished from any such components that may be present in situ within the human or animal body (e.g., camelids) or within tissue samples derived from the human or animal body (e.g., camelids). However, these sequences may correspond to or be substantially homologous to sequences found in the human or animal body (e.g., camelids). Thus, the terms "isolated" or "purified," as used herein with respect to nucleic acid molecules or sequences and proteins or polypeptides, such as antibodies, refer to such molecules when they are isolated, purified, or substantially free from their natural environment, e.g., when isolated or purified from the human or animal body (where they are, in fact, naturally occurring), or when produced by a technological process, i.e., including recombinantly and synthetically produced molecules.

[0223] It should be noted that the antibodies of the present invention are artificial constructs in that they do not exist in nature and do not correspond to molecules occurring in nature. For example, preferred antibodies are single domain antibodies that can be produced by engineering or recombinantly, and even in species that naturally produce such antibodies, such as camelids, such species do not produce antibodies against CD163, particularly porcine CD163, unless induced to do so experimentally, for example, by immunization. In other words, the antibodies of the present invention are non-native.

[0224] As used herein, the term "fragment" refers to biologically relevant fragments, e.g., fragments that contribute to antigen binding, e.g., fragments that form part of the antigen-binding site and / or contribute to the functional properties of a CD163 antibody. Particular preferred fragments are those that comprise the heavy chain variable region (V H The VH domain comprises or consists of a VH CDR.

[0225] Those skilled in the art will appreciate 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 in any of several ways well known and described in the art, but are most preferably prepared using recombinant methods.

[0226] Nucleic acid fragments encoding the heavy and light chain variable regions of the antibodies of the present invention can be obtained or produced by any suitable method, for example, by cloning or synthesis, as appropriate. Once nucleic acid fragments encoding the heavy and / or light chain variable regions of the antibodies of the invention have been obtained, these fragments can be further manipulated by standard recombinant DNA techniques, e.g., to convert the variable region fragments into full-length antibody molecules with appropriate constant region domains, or into a particular format of antibody fragment discussed elsewhere herein, e.g., single domain antibodies such as VHHs, Fab fragments, scFv fragments, or formats in which multiple antibodies are present (e.g., single domain antibodies / VHH antibodies), such as the biparatopic and triparatopic constructs described herein. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding the antibody molecules of the invention are generally incorporated into one or more appropriate expression vectors to facilitate production of the antibodies of the invention, or for example, to facilitate selection or screening, e.g., by incorporation into a phage display vector.

[0227] Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), as long as the vector is compatible with the host cell used. An expression vector is "suitable for transforming a host cell," which means that the expression vector contains a nucleic acid molecule of the present invention and a regulatory sequence selected based on the host cell used for expression, operably linked to the nucleic acid molecule. Operable linked is intended to mean that the nucleic acid is linked to a regulatory sequence in a manner that allows expression of the nucleic acid.

[0228] Thus, the present invention contemplates expression vectors, e.g., recombinant expression vectors that contain or include a nucleic acid molecule of the invention or a fragment thereof, as well as the necessary regulatory sequences for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the invention.

[0229] An expression vector can be introduced into a host cell to produce a transformed host cell. The terms "transformed," "transfected," "transformation," and "transfection" 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. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al., 1989 (Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989) and other laboratory texts.

[0230] Suitable host cells include a wide 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. Additionally, the proteins of the present invention can be expressed in prokaryotic cells such as E. coli.

[0231] Other expression vectors include RNA or mRNA expression vectors, such as self-amplifying RNA expression vectors, which can be used, for example, to express an antibody or construct or combination of the invention in a subject to be treated.

[0232] The proteins of the invention can also be prepared by chemical synthesis using well-known techniques in protein chemistry, such as solid phase synthesis. Yet another aspect provides an expression construct or expression vector or expression system (e.g., viral or bacterial or other expression construct, vector, or system) comprising one or more of the nucleic acid fragments or segments or molecules of the invention. Preferably, the expression construct or vector or system is recombinant. Preferably, the aforementioned construct or vector or system further comprises the necessary regulatory sequences for transcription and / or translation of the protein sequence encoded by the nucleic acid molecule of the invention. Preferred constructs, etc., are those that allow for long-term or persistent expression of the antibody (or binding protein) of the invention in the host target species, e.g., in pigs. Such expression can be transient, e.g., by episomal integration, or more permanent, e.g., via genomic integration, provided that a sufficient level and length of expression is achieved for a therapeutic or biological effect to be observed. Again, self-amplifying RNA expression vectors would be a suitable example.

[0233] Yet another aspect provides host cells (e.g., mammalian host cells or bacterial host cells or yeast host cells) or viruses, or other delivery vehicles (e.g., lipid-based delivery vehicles such as liposomes or lipid nanoparticles) comprising one or more expression constructs or expression vectors of the invention. Host cells or viruses or delivery vehicles comprising one or more of the nucleic acid molecules of the invention are also provided. Host cells (e.g., mammalian host cells or bacterial host cells, yeast host cells) or viruses expressing an antibody (or binding protein) or construct or combination of the invention, or delivery vehicles comprising an expression construct or nucleic acid molecule of the invention form yet another aspect.

[0234] Such expression constructs or vectors or systems, or host cells or viruses or delivery vehicles, or other nucleic acid products or fragments encoding the antibodies (or binding proteins) or constructs or combinations of the invention can be administered to a subject as a therapeutic agent to enable the production of the antibodies (or binding proteins) of the invention in situ within the subject, thereby exerting their therapeutic effect.

[0235] Yet another aspect of the present invention provides a method for producing (or manufacturing) an antibody, binding protein, protein construct, or combination of the present invention, comprising culturing a host cell of the present invention. Preferred methods include (i) culturing, e.g., in a culture medium, host cells comprising one or more recombinant expression vectors or one or more nucleic acid sequences of the present invention under conditions suitable for expression of the encoded antibody or binding protein; and optionally (ii) isolating or obtaining the antibody or binding protein from the host cells or the growth or culture medium / supernatant. Such production (or manufacturing) methods may also include a step of purifying the antibody or binding protein product and / or formulating the antibody or product into a composition comprising at least one additional component, such as a pharmaceutically acceptable carrier or excipient.

[0236] In embodiments, when an antibody or binding protein of the invention is composed of multiple polypeptide chains (e.g., certain fragments such as Fab fragments or whole antibodies), all polypeptides are preferably expressed in a host cell from either the same or different expression vectors so that the complete protein of the invention, e.g., antibody protein, assembles within the host cell and can be isolated or purified therefrom.

[0237] In another aspect, the invention provides a method of binding CD163 comprising contacting a composition comprising CD163 with an antibody of the invention.

[0238] In yet another aspect, the present invention provides a method for detecting CD163, comprising contacting a composition suspected of containing CD163 with an antibody of the present invention under conditions effective to allow the formation of a CD163 / antibody complex, and detecting the complex so formed.

[0239] Compositions comprising at least a first antibody (or binding protein), construct, or combination of the present invention, or a nucleic acid molecule, expression vector, or host cell of the present invention, constitute further aspects of the present invention. Formulations (compositions) comprising one or more antibodies, constructs, or combinations of the present invention, or a nucleic acid molecule, expression vector, or host cell of the present invention, admixed with a suitable diluent, carrier, or additive, constitute preferred embodiments of the present invention. Such formulations may be for use in pharmaceutical applications, such as animal health applications, or veterinary applications, such as livestock breeding applications; therefore, the compositions of the present invention are preferably pharmaceutically acceptable or acceptable for administration to non-human animals, such as mammals, preferably pigs. Suitable diluents, additives, and carriers are known to those skilled in the art.

[0240] Compositions according to the invention may be presented in a form suitable for, for example, oral, nasal, parenteral (e.g., intramuscular, subcutaneous or intradermal), intravenous, topical or rectal administration, with intramuscular administration being particularly convenient. The active compounds defined herein (e.g., antibodies of the present invention) can be presented in conventional pharmaceutical dosage forms such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules, or sustained-release forms. Conventional pharmaceutical additives and conventional production methods can be used to prepare these forms.

[0241] Injectable solutions can be produced in conventional manner, for example, by the addition of preservatives, such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solution can then be filled into injection vials or ampoules.

[0242] Suitable genetic modifications can be determined by one of skill in the art. The pharmaceutical composition may further comprise additional active ingredients (eg, as described elsewhere herein) in connection with a co-administration or combination regimen.

[0243] A further aspect of the present invention provides anti-CD163 antibodies (or binding proteins), constructs, or combinations as defined herein, or nucleic acid molecules, expression vectors, or host cells of the present invention, for use in therapy, particularly in the treatment or prevention of any disease or condition associated with CD163, or where CD163 plays a role, e.g., a causative role (e.g., a causative role, in whole or in part), or an essential role. For example, the anti-CD163 antibodies of the present invention can be used to treat or prevent any infectious disease caused by a virus or other pathogen, which is associated with CD163 or in which CD163 plays a role, e.g., a causative role (e.g., a causative role, in whole or in part), or an essential role. Examples include PRRSV infection and Simian hemorrhagic fever virus (SHFV). In other words, according to the present invention, the anti-CD163 antibodies (or binding proteins), constructs, or combinations can target, inhibit, or reduce the function of CD163, particularly CD163 expressed on or within PAM or other CD163-positive cells. Thus, the anti-CD163 antibodies (or binding proteins) or constructs or combinations defined herein can be used to treat or prevent any disease or condition in which inhibiting CD163 or blocking or reducing CD163 function is useful.

[0244] A preferred embodiment provides an anti-CD163 antibody (or binding protein), construct, or combination of the present invention for use in the treatment or prevention of infectious diseases in pigs, preferably viral infections in pigs. Particularly preferred is the treatment or prevention of PRRSV infection. In embodiments in which pigs are treated, the anti-CD163 antibody (or binding protein), construct, or combination of the present invention typically is, comprises, or encodes an anti-pig CD163 antibody (or binding protein).

[0245] CD163 is thought to be the receptor for all PRRS virus strains. However, there are two species of PRRSV: PRRSV-1 and PRRSV-2. While PRRSV-1 and PRRSV-2 viruses share some phenotypic similarities, there are differences between the virus species. The antibodies (or binding proteins), constructs, combinations, etc. of the present invention can be used to treat or prevent PRRSV-1 and / or PRRSV-2 infection, e.g., PRRSV-1 and PRRSV-2 infection, or to treat or prevent (e.g., specifically treat or prevent) PRRSV-2 infection.

[0246] The binding protein or antibody or construct or combination in the therapeutic methods and uses of the present invention is administered to a subject (animal, or mammal, e.g., pig) in need of treatment in a pharmaceutically, therapeutically, or physiologically effective amount. Thus, the above-mentioned methods and uses may include the additional step of identifying a subject in need of treatment.

[0247] Treatment of a disease or condition (e.g., treating an existing disease) according to the present invention includes curing the disease or condition, or reducing or alleviating the disease (e.g., reducing the severity of the disease) or symptoms of the disease.

[0248] The therapeutic methods and uses of the present invention are suitable for disease prevention as well as active disease treatment (e.g., treatment of existing diseases). Therefore, prophylactic and meta-protective (treatment in the face of a disease outbreak, e.g., treatment of a group of subjects after diagnosis of an infectious disease and / or treatment of clinical disease in a part of the group, with the aim of preventing the spread of an infectious disease to animals in close contact and / or at serious risk) treatment are also encompassed by the present invention. For this reason, in the methods and uses of the present invention, treatment also includes prophylaxis, meta-protection, or, where appropriate, prevention.

[0249] Such preventative (or protective) aspects can be conveniently performed on healthy or normal, or at-risk subjects, and can include both complete and significant prevention. Similarly, significant prevention includes scenarios in which the severity of the disease or symptoms of the disease are reduced (e.g., measurably or significantly reduced) compared to the severity or symptoms expected in the absence of treatment.

[0250] Preferred subjects for treatment are patients who are infected with or at risk of being infected with PRRSV. As described elsewhere herein, however, the therapeutic methods of the present invention can also be performed on subjects with other (non-PRRSV) infections, such as other bacterial or viral infections, or complex (multi-pathogen) infections. In particular, such treatments are suitable for treating subjects who have measurable levels of soluble CD163 in their serum or who have elevated, e.g., significantly elevated, levels of soluble CD163 above those found in healthy subjects.

[0251] For example, clinical signs of PRRSV infection include fetal resorption in pregnant sows or gilts, stillbirths and late abortions, and respiratory disease and syndromes, such as respiratory distress, in all pigs, particularly young pigs and young piglets. Other symptoms include anorexia (which often leads to reduced growth rate and weight loss or reduced weight gain (e.g., reduced average daily weight gain)), fever, lethargy, respiratory distress (e.g., pneumonia or pneumonia / pulmonary lesions), reproductive failure and diarrhea (especially in young piglets), central nervous system (CNS) signs, and death. It has been reported that subjects infected with PRRSV are susceptible to endemic diseases such as meningitis, Glasser's disease, exudative dermatitis, scabies, and bacterial bronchopneumonia, as well as PCV2 infection (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: March 29, 2019). Many of these diseases are typically managed with antimicrobial products such as antibiotics. As a result, the present invention has a role in reducing the use of antimicrobial products on farms.

[0252] Thus, the antibodies or binding proteins or constructs or combinations of the invention may be used to treat or prevent clinical diseases or symptoms, such as those associated with PRRSV infection or downstream endemic diseases as outlined above, or to prevent a virus, such as PRRSV, circulating (e.g., viral load in serum) or infection (e.g., a first infection) or a new infection (e.g., a second or subsequent infection), such as a PRRSV infection (e.g., a first PRRSV infection) or a new PRRSV infection (e.g., a second or subsequent PRRSV infection).

[0253] Therefore, preferred subjects for treatment according to the present invention include all types of pigs (sometimes referred to as swine), for example, any pig, swine, or swine species, including pigs of all ages and species, provided that they are susceptible to or susceptible to infection with the pathogens defined herein, particularly PRRSV. Piglets, particularly young piglets or piglets born from infected sows (up to 80% of which die), are particularly preferred subjects, as are nursery pigs (e.g., weaned pigs up to 12 weeks of age) and growing or fattening pigs (e.g., pigs up to slaughter age), particularly growing pigs. Preweaned piglets and piglets up to 4 weeks of age (especially piglets from infected sows, which may transmit the infection via the mammary secretions of infected sows) are also preferred subjects for treatment, as are gilts, sows, and pregnant sows. Preferred subjects include all types of pigs, and therefore it should be understood that subjects for treatment in all embodiments and aspects include groups of subjects, such as herds and litters of the same species kept together, but do not exclude treatment of individual animals.

[0254] In some embodiments, for example, when prevention is concerned, the subject is at risk of being affected by the disease or condition in question, for example, at risk of being infected with a pathogen or virus (e.g., PRRSV) as described above and developing the disease. Such a subject may be a healthy subject, or a subject that does not show symptoms of the disease, or any other suitable "at risk" subject. In another embodiment, the subject is a subject that has, or is suspected of having (or developing), or potentially has (or is developing) the disease or condition in question as described above.

[0255] Alternatively, the present invention provides a method of treating or preventing a disease or condition associated with CD163, or where CD163 has a role, e.g., a causative role (e.g., a wholly or partially causative role) or an essential 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 invention as defined herein. Suitable diseases or conditions or subjects are described elsewhere herein.

[0256] Treatment or prevention of infectious diseases in pigs is preferred, preferably treatment or prevention of viral infections in pigs. Particularly preferred is treatment or prevention of PRRSV infection, such as PRRSV-1 and / or PRRSV-2 infection, for example treatment or prevention of PRRSV-1 and PRRSV-2 infection, or treatment or prevention (e.g., particularly treatment or prevention) of PRRSV-2 infection.

[0257] Thus, according to a further aspect, there is provided a method for treating or preventing PRRSV infection in pigs, for example, a method for treating or preventing PRRSV-1 and / or PRRSV-2 infection in pigs, the method comprising administering to a subject in need thereof a therapeutically effective amount of a monoclonal antibody that binds to porcine CD163. Suitable CD163 antibodies (or binding proteins) or constructs or combinations for use in such methods are described herein.

[0258] Thus, a further aspect provides a method for treating or preventing a PRRSV infection in a subject, preferably a pig, e.g., a method for treating or preventing a PRRSV-1 and / or PRRSV-2 infection in said subject, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody, binding protein, or combination of the invention. Such treatment can alternatively comprise administration of one or more nucleic acid molecules, expression vectors, or host cells of the invention.

[0259] Therapeutically effective amounts can be determined based on clinical evaluations and can be readily monitored. The therapeutic use embodiments of the invention described herein apply mutatis mutandis to this aspect of the invention.

[0260] Furthermore, alternatively, the present invention provides the use of an anti-CD163 antibody (or binding protein) or construct or combination of the present invention, e.g., a monoclonal antibody of the present invention, as defined herein, in the manufacture of a medicament for use in therapy. Preferred therapeutic applications are described elsewhere herein, particularly for use in the treatment or prevention of any disease or condition associated with CD163 or in which CD163 plays a role, e.g., a causative role (e.g., a causative role in whole or in part) or an essential role. For example, an anti-CD163 antibody (or binding protein) of the present invention can be used to treat or prevent any infectious disease caused by a virus or other pathogen, which is associated with CD163 or in which CD163 plays a role, e.g., a causative role (e.g., a causative role in whole or in part) or an essential role. In other words, according to the present invention, an anti-CD163 antibody (or binding protein) can target, inhibit, or reduce the function of CD163, particularly CD163 expressed on or within PAM or other CD163-positive cells. Thus, the anti-CD163 antibodies (or binding proteins) or constructs or combinations defined herein can be used to treat or prevent any disease or condition in which inhibiting CD163 or blocking or reducing CD163 function is useful.

[0261] A preferred embodiment provides the use of an anti-CD163 antibody (or binding protein) of the invention in the manufacture of a medicament for use in the treatment or prevention of an infection in a pig, preferably a viral infection in a pig. Particularly preferred is the treatment or prevention of a PRRSV infection, e.g., a PRRSV-1 and / or PRRSV-2 infection, e.g., the treatment or prevention of a PRRSV-1 and PRRSV-2 infection, or the treatment or prevention (e.g., particularly the treatment or prevention) of a PRRSV-2 infection.

[0262] Thus, a further aspect provides the use of a monoclonal antibody that binds to porcine CD163 in the manufacture of a medicament for use in treating or preventing PRRS virus infection, preferably PRRSV-1 and / or PRRSV-2 infection, in pigs. Suitable CD163 antibodies (or binding proteins) or constructs or combinations for such use are described herein.

[0263] Thus, a further aspect provides the use of an antibody, binding protein or combination of the invention in the manufacture of a medicament for use in therapy in a subject, preferably for use in the treatment or prevention of PRRSV infection in a subject, for example for use in the treatment or prevention of PRRSV-1 and / or PRRSV-2 infection in a subject, preferably a pig. Such use may alternatively comprise the use of one or more nucleic acid molecules, expression vectors or host cells of the invention.

[0264] The therapeutic use embodiments of the invention described herein apply mutatis mutandis to this aspect of the invention.

[0265] In some embodiments, antibodies (or binding proteins) of the invention can be used in combination. For example, a membrane-specific antibody of the invention (e.g., an antibody defined in Table A) can be used in combination with one, two, or more alternative anti-CD163 antibodies (e.g., an antibody defined in Table B (or D) and / or an antibody defined in Table C (or E)). Preferred combinations of anti-CD163 antibodies are those in which the individual anti-CD163 antibodies of the combination bind to different epitopes on the CD163 molecule, e.g., biparatopic (two different epitopes) or triparatopic (three different epitopes) constructs as described herein.

[0266] Any combination of VHH antibodies shown in Tables A, B (or D), and C (or E) can be used. Preferred combinations include: A VHH antibody of Table A and a VHH antibody of Table B (or D). A VHH antibody from Table A and a VHH antibody from Table C (or E). A VHH antibody of Table B (or D) and a VHH antibody of Table C (or E). A VHH antibody of Table A, a VHH antibody of Table B (or D), and a VHH antibody of Table C (or E).

[0267] In all of the above combinations, antibodies having the three CDRs shown in Tables A to E, or antibodies having sequences substantially homologous thereto, can also be used, where appropriate.

[0268] Preferred combinations are those that result in an improved or increased, preferably a significant improvement or increase, therapeutic effect compared to either a single active agent (monotherapy), or a single antibody, or an antibody (e.g., VHH) of the invention administered as a single anti-CD163 agent. Other preferred combinations are those in which the individual anti-CD163 antibodies of the combination bind to different epitopes on the CD163 molecule. As described elsewhere herein, in some embodiments of the invention, the antibodies (or binding proteins) of the invention can also be used in combination with an entity capable of extending half-life, such as an additional antibody capable of extending half-life, e.g., a VHH antibody, or any other suitable half-life extending entity.

[0269] In such combination therapies using two or more antibodies (or binding proteins) of the present invention, the second (or subsequent, e.g., third) anti-CD163 antibody may be administered to a subject substantially simultaneously with the first anti-CD163 antibody of the present invention, such as from a single pharmaceutical composition, or from two pharmaceutical compositions administered closely together (at the same time or similar times). Alternatively, the second (or subsequent, e.g., third) anti-CD163 antibody of the present invention may be administered to a subject prior to or following administration of the first anti-CD163 antibody of the present invention. As used herein, "prior to or following" refers to "alternating," such that the second antibody is administered to a subject at a different time from the administration of the first anti-CD163 antibody component. Generally, the two (or more) components may be administered effectively spaced in time or together to allow each component to exert its respective therapeutic effect, i.e., the components are administered in "biologically effective amounts" at "biologically effective time intervals" and as part of the same therapeutic regimen.

[0270] As described elsewhere herein, preferably, combinations of anti-CD163 antibodies (or binding proteins) of the invention (and optionally half-life extending entities) can be conveniently administered as part of the same molecule or construct, where appropriate, and can be joined or linked, for example, by artificial linkers. This mode of administration can be particularly suitable for VHH antibodies (or other types of antibody molecules composed of a single polypeptide chain), the individual antibodies of which are conveniently joined by suitable peptide (or other) linkers, for example non-native peptides or artificial linkers in a single polypeptide chain comprising multiple VHH (or other) antibodies, either VHH antibodies of the invention or in combination with other VHHs or other antibodies. Suitable linkers are well known and described in the art. Exemplary linkers include GS linkers, such as one or more repeats of the G4S linker (GGGGS, SEQ ID NO: 43). The linker used in the construct used in the accompanying examples is SEQ ID NO: 44, GGGGSGGGGS, i.e., two repeats of GGGGS. Five-repeat linkers are also used. However, it will be understood that linkers (spacers) of other sequences and lengths, such as other GS linkers or other suitable linkers, can also be used.

[0271] In such embodiments, the antibodies are generally linked together using appropriate techniques, e.g., spacing, so that each component can exert its respective effect, e.g., binding to CD 163. For example, in embodiments in which the anti-CD163 antibodies of the invention bind to different epitopes on CD163, combinations of such antibodies are preferred, with the constructs appropriately designed so that each individual antibody is capable of binding to CD163, e.g., its CD163 epitope.

[0272] Thus, in some embodiments, an anti-CD163 antibody (or binding protein) of the present invention can be used as the sole active agent in a therapeutic regimen (monotherapy), or multiple anti-CD163 antibodies of the present invention can be used, for example, in the above-described combinations. In some embodiments, an anti-CD163 antibody (or binding protein) of the present invention (or a suitable combination) can be used as the sole active anti-CD163 agent or the sole active anti-CD163 antibody in a therapeutic regimen, or they can be the sole effective anti-PRRSV agent in a therapeutic regimen. However, in some embodiments, additional anti-CD163 agents or anti-PRRSV agents can be used.

[0273] Thus, an anti-CD163 binding protein or antibody of the present invention (or a suitable combination) can be combined with one or more further (additional CD163-targeting or non-CD163-targeting) active agents, for example, at least a second therapeutic or biological agent, the first being an anti-CD163 binding protein or antibody of the present invention (or a combination of such binding proteins or antibodies).

[0274] The anti-CD163 antibodies (or binding proteins) of the invention (or suitable combinations) can be combined, for example, with other therapeutic agents or vaccines useful for treating or preventing the disease in question, e.g., PRRSV, or other diseases, as described elsewhere herein.

[0275] Suitable exemplary dosing regimens for such combination treatments can be as described elsewhere herein for combinations of anti-CD163 antibodies.

[0276] The present invention further includes kits comprising one or more of the antibodies, or constructs, or compositions of the invention, or one or more nucleic acid molecules encoding the antibodies or constructs of the invention, or one or more recombinant expression vectors comprising the nucleic acid sequences of the invention, or one or more host cells or viruses comprising the recombinant expression vectors or nucleic acid sequences of the invention. Preferably, the kits are for use in the methods and uses described herein, e.g., the therapeutic methods described herein. Preferably, the kits include instructions for use of the kit components. Preferably, the kits are for treating a disease or condition described elsewhere herein, and optionally include instructions for use of the kit components to treat such disease or condition. Equivalent embodiments of the binding proteins of the invention are also provided.

[0277] The antibodies (or binding proteins) of the invention defined herein can also be used as molecular tools for in vitro or in vivo applications and assays. Because antibodies (and binding proteins) have an antigen-binding site, they can function as members of specific binding pairs, and these molecules can be used in any assay where a specific binding pair member is required.

[0278] Thus, a further aspect of the present invention provides reagents comprising the antibodies (or binding proteins) of the invention as defined herein and the use of such antibodies (or binding proteins) as molecular tools, for example in in vitro or in vivo assays.

[0279] The term "reduction" or "reduction" (or equivalent terms) as used herein includes a measurable decrease or reduction when compared to an appropriate control. Suitable controls would be readily identified by one skilled in the art and would include untreated or placebo-treated subjects or healthy subjects, or samples or assays in which the antibody (or binding protein) of the invention is not present. Preferably, the decrease or reduction is significant, e.g., clinically or statistically significant. The term "increase" (or equivalent term) as used herein includes a measurable increase or elevation compared to an appropriate control. Suitable controls would be easily identified by one skilled in the art and would include untreated or placebo-treated subjects or healthy subjects, or samples or assays in which the antibody (or binding protein) of the present invention is not present. Preferably, the increase is clinically or statistically significant.

[0280] Preferably, such an increase (and indeed other increases, improvements or positive effects as referred to elsewhere herein) or such a decrease (and indeed other decreases, reductions or negative effects as referred to elsewhere herein) is (as appropriate) a measurable increase, decrease etc., more preferably it is a significant increase, decrease etc., preferably a clinically significant or statistically significant increase, decrease etc., relative to an appropriate control level or value (e.g. when compared to an untreated or placebo-treated subject, or compared to a healthy or normal subject, or compared to the same subject prior to treatment, or compared to a sample or assay in which the antibody (or binding protein) of the invention is not present), e.g. with a probability value of ≦0.05 or <0.05.

[0281] Methods for determining the statistical significance of differences between test groups or levels of specific parameters are well known and documented in the art. For example, herein, statistical comparisons are performed using significance tests such as the Stuparameter Mann-Whitney U-rank-sum test, the chi-square test, Fisher's exact test, one-way ANOVA, or two-way ANOVA. A probability value of ≤0.05 or <0.05 indicates a decrease or increase in the level of a specific parameter or a difference between test groups of subjects is generally considered statistically significant.

[0282] Table of Amino Acid Sequences and Their Sequence Identifiers (SEQ ID NOs) Disclosed herein All amino acid sequences are written herein from N-terminus to C-terminus, as is customary in the art. [Table A] [Table B] [Table C] [Table D] [Table E]

[0283] The invention is further described in the following non-limiting examples with reference to the following figures. [Brief explanation of the drawings]

[0284] [Figure 1] Dose-response FACS binding assay of pPAM WT cells with biparatopic antibody candidates. [Figure 2] FACS competition assay of pPAM WT cells and example biparatopic antibody candidates in the presence of increasing concentrations of soluble CD163 protein. [Figure 3] Infection assay: Infection with monomeric VHH, PRRSV-1 BOR57. [Figure 4] Infection assay: Infection with monomeric VHH, PRRSV-2 MN184. [Figure 5] Infection assay: Infection with biparatopic VHH, PRRSV-1 BOR57. [Figure 6] Infection assay: dose response of biparatopic 03E11 + 03D03 2(G4S), PRRSV-1 LT3, Sigma RPMI, 10% FBS. [Figure 7] Infection assay: Biparatopic 03E11 + 03D03 2(G4S) dose response, PRRSV-1 LT3, Sigma RPMI, 80% low CD163 porcine serum. [Figure 8]Infection assay: Biparatopic 03E11 + 03D03 2(G4S) dose response, PRRSV-1 LT3, Sigma RPMI, 80% high CD163 porcine serum. [Figure 9] Infection assay: Biparatopic 03E11 + 03D03 2(G4S) dose response, PRRSV-2 MN184, Sigma RPMI, 10% FBS. [Figure 10] Infection assay: Avian paratopic-10 17B11-03E11-03D03 2(G4S) dose response, PRRSV-1 LT3, Sigma RPMI 80% low CD163 porcine serum. [Figure 11] Infection assay: Avian Paratopic-10 17B11-03E11-03D03 2(G4S) dose response, PRRSV-1 LT3, Sigma RPMI 80% high CD163 porcine serum. [Figure 12] Infection assay: Avian paratopic-10 17B11-03E11-03D03 2(G4S) dose response, PRRSV-2 NA174, Sigma RPMI 80% low CD163 porcine serum. [Figure 13] Infection assay: Avian paratopic-10 17B11-03E11-03D03 2(G4S) dose response, PRRSV-2 NA174, Sigma RPMI 80% high CD163 porcine serum. [Example]

[0285] Example 1 Immunization, Library Generation, Screening, and Clone Selection Materials and Methods immunization Single-domain antibodies were obtained from llamas immunized with HEK293T cells expressing different porcine CD163 constructs and boosted three times with porcine alveolar macrophage (pPAM) WT cells. The llamas were injected with HEK293T cells expressing pCD163-SRCR-FL-PST2 (i.e., a full-length construct encompassing the beginning of SRCR1 and the end of PST2) and boosted three times. Animals received five injections at 2-week intervals. Six days after the final boost, serum was collected for antibody titers against the pCD163-SRCR-FL-PST2-His protein by ELISA. For this ELISA, recombinant proteins were coated onto 96-well plates (Maxisorp; Nunc). After blocking, diluted serum samples were added and the presence of anti-pCD163 antibodies was demonstrated using mouse anti-llama IgG (FJB, Cat. nr. FJ1203MAB01B09) followed by donkey anti-mouse IgG-HRP antibody (JIR, Cat. nr. 715-035-150). Library Construction RNA was extracted from the PBMCs of two immunized llamas (400 ml each). 40 μg of RNA was used for cDNA synthesis using random primers. This cDNA was used for primary PCR amplification using non-tag primers annealing to the leader sequence and hinge CH1 region, followed by secondary PCR amplification to introduce restriction endonuclease sites for cloning VHH genes into the pDCL1 phagemid vector. The library was electroporated into TG1 E. coli cells, and bacterial glycerol stocks of the immunized library were stored at -80°C.

[0286] choice Phages produced from the llama VHH library pool were used in two consecutive rounds of phage display selection using HEK293T cells expressing pCD163 recombinant protein, different porcine CD163 constructs, or pPAM WT cells. The first round of selection was performed with pPAM WT cells, with a prior negative counterselection against empty HEK293T WT cells. The second round of selection was performed with HEK293T pCD163-SRCR-FL-PST2-expressing cells, with a prior negative counterselection using pPAMΔ5 domain cells (cells lacking SRCR domain 5; Burkhard et al., 2017). For the selection of HEK293T cells expressing the pCD163-SRCR-FL-PST2 construct, nonspecific phages were washed away in PBS buffer, pH 7.4, followed by elution of specific phages with trypsin (total elution). For the selection round of pPAM, 5E106 cells were used. Nonspecific phages were washed away in PBS buffer, pH 7.4, followed by elution of specific phages with trypsin (total elution). Serial dilutions of the eluted phages were made and used to infect exponentially growing TG1 cells. The infected TG1 cells were plated on LBCarb100Glu2% plates, and enrichment values ​​relative to background (no selection antigen) were calculated.

[0287] ELISA screening Individual clones from the second round of selection condition output were picked into 96-well master plates and tested as periplasmic extracts (PE) for binding to pCD163-SRCR-FL-PST2-His or pCD163-SRCR1-9-huFc (i.e., constructs containing the beginning of SRCR-1 to the end of SRCR-9 but without PST2), or huCD6-pPST2-His protein by binding ELISA at pH 7.0. For PE-binding ELISA, MaxiSorp TMHigh-protein-binding capacity 96-well ELISA plates were coated overnight at 4°C with 1 μg / ml pCD163-SRCR-FL-PST2-His or huCD6-pST2-His, or pCD163-SRCR1-9-huFc protein diluted in PBS. The next day, plates were washed three 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, plates were washed three times with PBS Tween 0.05% (pH 7.4), and 20 μl of PE + 80 μl of 1% Marvel / PBS (pH 7.4) was added per well. Plates were incubated at room temperature (RT) for 1 hour with shaking. The plate was washed three times with PBS Tween 0.05% (pH 7.4) and incubated with 100 μl of anti-c-Myc antibody (Roche; Cat. nr. 11667203001) followed by secondary antibody DAM-HRP (JIR; Cat. nr. 715-035-150) in 1% Marvel / PBS (pH 7.4) for 1 h at RT with shaking. The plate was washed three times with PBS Tween 0.05% (pH 7.4) and substrate solution (TMB solution) was added. The reaction was stopped with H2SO4 and read at 450 nm on a plate reader.

[0288] Cell-based screening (FACS): Periplasmic extracts (PE) of selected clones were incubated with anti-c-myc antibody (Roche; Cat. nr. 11667203001) specific for the c-myc tag present in soluble VHH for 30 min at room temperature (RT) with agitation. The mixture (PE + anti-c-myc antibody) was added to pPAM WT or pPAMΔ5 domain (SRCR domain 5 deleted cells) and incubated with gentle shaking at 4°C for 60 min. The cells were washed three times with 150 μl / well of FACS Buffer and incubated with 50 μl / well of secondary antibody GAM-APC for 30 minutes at 4° C. with shaking in the dark. The cells were washed three times with 150 μl / well of FACS Buffer, resuspended in 75 μl / well of FACS Buffer, and then transferred to a FACS instrument (Attune TM Measurements were performed in the RL-1 channel (APC channel) at 10000 kJ / s (NxT) and a total of 10,000 cells were acquired per sample.

[0289] Sequence Method Positive binders were sent for sequencing, and clones were grouped into families based on HCDR3 sequence differences. Expression and purification of VHH candidate antibodies Synthetic genes encoding VHH variable domains with FLAG and His tags were obtained. Each DNA construct was digested with restriction enzymes, the inserts were gel-purified, and each variable domain insert was ligated into the mammalian expression vector pcDNA3.1. ExpiCHO-S cells were transfected with the VHH sequences using 40 μg of the entire DNA plasmid construct. A total volume of 25 mL of cells was used for 8 days of protein production (32°C, 5% CO2). Produced VHH antibodies were captured from the clarified supernatant using a HisTrap HP 5 mL IMAC column (GE Healthcare, Cat. nr. 17-5248-02) on an AKTA Pure 25 FPLC system. The eluted antibody peak fractions were buffer-exchanged into 1x PBS pH 7.4 and concentrated using a 3 kDa MCO spin concentrator (Amicon, Cat. nr. UFC900324). The purified protein was analyzed for the presence of the correct chain by analytical size exclusion chromatography (aSEC) and SDS-PAGE.

[0290] Dose-response ELISA using purified VHHs MaxiSorp TMHigh-protein-binding capacity 96-well ELISA plates were coated overnight at 4°C with 1 μg / ml pCD163-SRCR1-9-huFc diluted in PBS. The next day, the plates were washed three times with PBS Tween 0.05% (pH 7.4) and blocked with 250 μl / well of 4% Marvel for 1 hour at room temperature. After blocking, the plates were washed three times with PBS Tween 0.05% (pH 7.4). VHHs were diluted in PBS (pH 7.4) in 3-fold steps from 200 to 0.0034 nM and added to pCD163-SRCR1-9-huFc-coated and blocked ELISA wells for 1 h at RT. The plate was washed three times with PBS Tween 0.05% (pH 7.4) and incubated with anti-histidine-HRP (Miltenyi Biotec, Cat. no. 130-092-783) in PBS pH 7.4 for 1 hour at room temperature. The plate was washed three times with PBS Tween 0.05% (pH 7.4) and substrate solution (TMB solution) was added. The reaction was stopped with H2SO4 and read at 450 nm on a plate reader.

[0291] Affinity measurement of purified VHHs by Biacore To evaluate the affinity of the selected purified clones for the pCD163 protein, pCD163-SRCR1-9-huFc and pCD163-1-PST2-His (also referred to herein as pCD163-SRCR-FL-PST2-His) proteins were coated onto CM5 sensorships (GE Healthcare) by amine coupling. Surface plasmon resonance (SPR) (Biacore 3000, GE Healthcare) was used to measure the binding kinetics of selected single-domain antibodies at pH 7.4. 2075–2423 RU of pCD163-SRCR1-9-huFc or 2859–3286 RU of pCD163-1-PST2-His were dissolved at 20 μg / ml or 30 μg / ml in acetate buffer, pH 5.0 or pH 5.5, and immobilized on a CM5 chip using standard amine coupling techniques.

[0292] The QC for immobilization was a commercially available anti-porkine CD163 antibody (BioRad, Cat. MCA2311GA) diluted to 30 nM in HBS-EP pH 7.4 buffer. 1x HBS-EP pH 7.4 was used as the running buffer during binding kinetic measurements. Purified VHHs were injected in 2-fold dilutions from 200 nM to 12.5 nM in HBS-EP pH 7.4 at 30 μl / min for 2 min with a 1-min off-rate wash between injections. The off-rate wash was performed 300 s after the last injection of each cycle. Two 10 μl injections of 1 M NaCl, 1 mM Glycine pH 1.5 were performed between samples to return RU levels to baseline after regeneration. Using the simultaneous fitting option in the BIAevaluation software, a 1:1 binding fit with mass transfer was applied to a series of sample curves to calculate kinetic constants of the antibody-antigen interaction, including association rate (ka), dissociation rate (kd), and affinity (KD). Curves were excluded from the fit after visual inspection of residuals and consideration of the chi2 (Chi2) value. A minimum of four curves were considered for simultaneous fitting.

[0293] Results and Discussion Membrane-specific binding was confirmed, as exemplified by clone 17B11. As can be seen from Tables 1, 3, and 4, clone 17B11 did not exhibit binding to non-cell surface-bound (soluble) CD163 in ELISA, ELISA EC50, or Biacore experiments. As shown in Table 2, clone 17B11 binds to pPAM WT cells expressing CD163. Furthermore, clone 17B11 does not bind to pPAMΔ5 WT cells, indicating that its binding specifically requires the SRCR5 domain of CD163 expressed on the cell surface. Taken together, these results indicate that clone 17B11 is specific for cell surface-associated CD163. [Table 1] [Table 2] [Table 3]

[0294] [Table 4] N / D = Bond too weak to measure. The sequence of 17B11 is shown in Table A (also referred to herein as clone 39).

[0295] Example 2: Functional assays using biparatopic and triparatopic constructs containing 17B11 VHH antibodies Anti-murine CD163 clones 03E11 (H03E11) and 03D03 (H03D03) The previously selected anti-porcin CD163 VHH clones 03D03 and 03E11 were selected from a library constructed from two llamas immunized with pCD163-SRCR4-7-huFc and boosted with pCD163-SRCR1-9-huFc. Two consecutive rounds of phage display selection were performed using pCD163 recombinant protein or pPAM WT cells. The selection round for recombinant protein was performed using 10 μg / ml of pCD163-SRCR1-9-huFc or pCD163-SRCR4-7-huFc pH 7.4 (PBS buffer). After washing away nonspecific phage, specific phage were eluted with trypsin (total elution). The selection round for pPAM cells was performed as described above.

[0296] These clones were identified by PEELISA screening of recombinant protein (soluble CD163) and PEFACS screening of cells (membrane CD163). PEELISA screening was performed using pCD163-SRCR4-7-huFc or pCD163-SRCR5-6-huFc proteins at pH 7.0 (PBS). PEFACS screening was performed using pPAM WT and pPAMΔ5 cells. All of these clones showed binding to both soluble porcine CD163 (recombinant protein) and membrane-type porcine CD163 (pPAM WT), but did not show significant binding to pPAMΔ5 cells. Therefore, these clones are believed to bind to the SRCR5 domain of porcine CD163. The 03E11 antibody has been shown to inhibit PRRSV-1 or PRRSV-2 infection (see Figures 3 and 4). The sequence of 03E11 is shown in Table B (sometimes referred to herein as clone 19). The 03D03 antibody has been shown to inhibit PRRSV-2 infection (see Figure 4), and the sequence of 03D03 is shown in Table C (sometimes referred to herein as clone 17). These antibodies were used in combination with the 17B11 antibody to construct biparatopic (2 antibody) and triparatopic (3 antibody) constructs as described below.

[0297] material and method Construction of biparatopic and triparatopic antibody candidates Individual VHHs were assembled into biparatopic and triparatopic combinations using 2x(G4S) or 5x(G4S) flexible linkers. These linkers were placed between the individual antibodies in the constructs. The combinations were designed in a semi-rational manner, aiming for non-competing epitopes, high affinity and potency for both PRRSV1 and PRRSV2 subtypes, and the ability to bind cell surface CD163 and block infection in the presence of competing soluble CD163 proteins. This non-interference is important given the potential presence of high levels of soluble CD163 in the serum of animals in the field, particularly those suffering from bacterial and / or viral infections such as Lawsonia intracellularis, which are quite common and may act as a sink for therapeutic agents that do not favor cell surface CD163.

[0298] Biparatopic constructs (containing two different anti-porcine CD163 antibodies capable of binding to different epitopes on porcine CD163) were first generated and evaluated for binding to pPAM WT cells. Furthermore, binding to pPAM WT cells was measured in a competitive setting with increasing concentrations of soluble CD163. Examples of biparatopic constructs are summarized in Table 5. Examples of biparatopic constructs 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) means that each individual VHH is separated by two G4S linker repeats, and 5(G4S) means that each individual VHH is separated by five G4S linker repeats.

[0299] Triparatopic constructs (containing three anti-porcine CD163 antibodies capable of binding to different epitopes on porcine CD163) were also generated. The triparatopic constructs are summarized in Table 6 and include: 03E11 + 03D03 + 17B11 2(G4S), sometimes referred to as Tri-2; 03E11 + 17B11 + 03D03 2(G4S); 03D03 + 03E11 + 17B11 2(G4S); 03D03 + 17B11 + 03E11 2(G4S); 17B11 + 03E11 + 03D03 2(G4S), sometimes referred to as Tri-10; and 17B11 + 03D03 + 03E11 2(G4S). 2(G4S) means that individual VHHs are separated by a G4S linker with two repeats each.

[0300] Expression and purification of biparatopic and triparatopic candidate antibodies Synthetic genes encoding VHH variable domains and the appropriate combination of FLAG and His tags were ligated into the mammalian expression vector pcDNA3.1 or pcDNA3.4 with the appropriate linkers. ExpiCHO-S or HEK293T cells were transfected with the DNA plasmid constructs and cultured for 7-10 days at 32°C and 5% CO2 for protein production. The produced biparatopic and triparatopic VHH antibody constructs were captured from the clarified supernatant using a HisTrap IMAC column (GE Healthcare, Cat. nr. 17-5248-02) on an FPLC system. The eluted antibody peak fractions were buffer exchanged into 1x PBS pH 7.4 and concentrated using a 3 kDa MCO spin concentrator (Amicon, Cat. nr. UFC900324). The purified proteins were analyzed for the presence of the correct chains by analytical size-exclusion chromatography (aSEC) and SDS-PAGE.

[0301] Dose-response FACS of pPAM WT cells Biparatopic and triparatopic antibody constructs in PBS (pH 7.4) were serially diluted 3-fold from 150 nM to 0.023 nM in FACS buffer (1x PBS pH 7.4, 0.5% FBS, 0.5 mM EDTA). Biparatopic and triparatopic candidates were incubated with anti-FLAG-biotin (Sigma, Cat. nr. F9291) on ice for 30 minutes with shaking. This mixture was added to pPAM WT cells and incubated on ice for 60 minutes with gentle shaking. Cells were washed three times with 150 μl / well of FACS buffer and incubated with 50 μl / well of secondary detection reagent Streptavidin RP.E.-conjugated antibody (Invitrogen, Cat. nr. SA10044) on ice for 30 minutes with shaking, protected from light. The cells were then washed three times with 150 μl / well of FACS Buffer, resuspended in 50 μl / well of FACS Buffer, and run on a FACS instrument (Attune TM N×T) and a total of 10,000 cells were acquired per sample.

[0302] FACS competition assay using pPAM WT cells: Binding of biparatopic antibody candidates in the presence of soluble CD163 protein Biparatopic candidates were diluted to 1 nM in FACS buffer and mixed with pCD163-SRCR1-9-PST2 in FACS buffer at final concentrations of 0, 1, 10, or 100 nM. This mixture was incubated with pPAM WT cells on ice for 60 minutes with shaking. For VHH detection, anti-FLAG-Biotin antibody (Sigma, Cat. nr. F9291) was added to the cell mixture and incubated on ice for 30 minutes with shaking. Cells were washed three times with 150 μl / well of FACS buffer and incubated with the secondary detection reagent anti-mouse IgG-APC (Invitrogen, Cat. nr. A865) on ice for 30 minutes with shaking, protected from light. Cells were washed three times with 150 μl / well of FACS buffer, resuspended in 50 μl / well of FACS buffer, and analyzed on a FACS instrument (Attune TM N×T) and a total of 10,000 cells were acquired per sample.

[0303] Results and Discussion As can be seen in Figure 1 and Table 5, biparatopic VHH candidates bound very well to pPAM WT cells. As shown in Figure 2, biparatopic combinations can exhibit reduced binding to pPAM WT cells in the presence of competing soluble CD163. This is exemplified by 03E11 + 03D03 5(G4S), which significantly inhibited competing soluble CD163 at 10 nM and 100 nM. However, other biparatopic combinations, including the membrane-specific anti-CD163 17B11 VHH antibody (e.g., 03E11 + 17B11 5(G4S) and 17B11 + 03D03 5(G4S)), maintained higher levels of binding to pPAM WT cells, even in the presence of soluble CD163 protein at concentrations up to 100 nM. Thus, it was clearly demonstrated that the biparatopic combination with 17B11 maintained good levels of binding to pPAM WT cells, even in the presence of 10 nM and even 100 nM of competing soluble CD163 protein, compared with the biparatopic combination without 17B11. [Table 5]

[0304] As can be seen in Table 6, the triparatopic VHH candidates also bound very well to pPAM WT cells. [Table 6]

[0305] Example 3: Inhibition of Porcine Reproductive and Respiratory Syndrome (PRRS) virus infection of primary porcine alveolar macrophage cells by biparatopic and triparatopic constructs containing the 17B11 VHH antibody Materials and Methods PRRS virus infection protocol reagent Control antibody: Primary antibody: anti-PRRS 1AC7, Ingenasa Secondary antibody: Goat anti-mouse IgG(H+L) Alexa Fluor Plus 488, ThermoFisher, A32723 Culture medium: Complete RPMI, 10% FBS, 80% low-sCD163 porcine serum or 80% high-sCD163 porcine serum, ultraglutamine, Pen / Strep (sCD163 is soluble CD163) PAM isolation: Isolation of porcine alveolar macrophages was performed as described by Burkard et al., 2017. Virus isolation: Type 1 virus: BOR57 isolate (Roslin Institute, Edinburgh, UK) Type 1 virus: LT3 (PRRSV1 subtype 2 strain, Roslin Institute, Edinburgh, UK) Type 2 virus: MN184 US strain (Han et al 2006) Type 2 virus: NA174 (Roslin Institute, Edinburgh, UK) Infection Protocol Day 1 - Seed cells Porcine alveolar macrophage cells were seeded in 48-well plates at 20 million cells / plate in complete RPMI and incubated overnight in a CO2 incubator.

[0306] Day 2 - VHH treatment and infection challenge 1. Pretreatment (30 min before infection) a. Remove the medium from the cells b. Add 100 L of culture medium to the untreated uninfected control and the untreated infected control. c. Add 20 L of PBS to 100 L of culture medium and add to mock-treated infected controls. d. Add appropriate amount of VHH stock to 100 L of culture medium and add to treated and infected samples. e. Place the plate back into the CO2 incubator and let it sit for 30 minutes. 2. Thaw the virus stock and sonicate for 15 seconds before use. 3. Infection challenge (2 hours) a. Remove the culture medium from the cells and save the VHH-containing culture medium for overnight incubation. b. Add 100 L of culture medium to the untreated, uninfected control. c. 10 L of virus is added to 100 L of culture medium and added to untreated infected controls. d. For mock-treated infected controls, add 10 L of virus and 20 L of PBS to 100 L of culture medium. e. For treated and infected samples, add the appropriate amount of VHH stock and 10 μl of virus to 100 L of culture medium. f. Gently agitate the plate and return it to the CO2 incubator. g. Gently agitate the plate every 15 minutes for 2 hours. 4. Overnight Incubation (15 hours) a. Remove the medium from the cells b. Add 100 L of culture medium to the untreated uninfected control and the untreated infected control. c. Add 20 L of PBS to 100 L of culture medium and add to mock-treated infected controls. d. Add the VHH-containing culture medium reserved from the pretreatment step to the appropriate sample. e. Return the plate to the CO2 incubator and incubate for 15 hours.

[0307] Day 3 - Assay and measurement of viral infection 5a. Direct lysis RT-qPCR protocol for viral RNA quantification in culture supernatants a. 5 μL of culture supernatant collected at 24 hpi b. Dilute 1:2 with lysis buffer (20 mM Trizma HCl buffer pH 7.5, 300 mM NaCl, 2.5% Igepal® CA-630, 1:2000 RNasin® Plus RNase Inhibitor) and mix. c. Sample incubated at RT for 20 min d. Samples are diluted (1:5) with nuclease-free HO for use in the subsequent qRT-PCR protocol (primers optimized and validated for PRRSV). e. Measure: TCID50 / ml (%) relative to mock treatment 5b. In-well fixation and staining protocol a. Aspirate the medium from the cells b. Fix the cells in 4% formaldehyde / PBS++ (containing calcium and magnesium) solution for 30 minutes. c. Wash once with PBS++ d. Permeabilize with Triton-X (1% in PBS++) for 5 min at RT. e. Wash once with PBS++ or blocking solution (PBS++ / 5% FBS). f. Block with blocking solution (PBS++ / 5% FBS) for 20 min at RT. g. Add the primary antibody anti-PRRS 1AC7 at a concentration of 1:5000 to all wells except the unstained control and the secondary antibody only control. h. Incubate at RT for 1 h i. Wash 3 times with PBS++ j. Add secondary antibody Goat Anti-Mouse IgG (H+L) Alexa Fluor Plus 488 at 1:5000 to all wells except the unstained control. k. Incubate at RT for 45 min l. Wash three times with PBS++ Add 300L PBS++ n. Scrape the cells with a wide-bore p200 pipette tip, then scrape the edge of the well with a regular p200 tip, wash the well surface three times with a p1000 pipette, collect the cells, and transfer them to a FACs tube. o. Measurements were performed using a Fortessa x20.

[0308] Serum containing soluble CD163 Low-soluble CD163 serum was collected from healthy pigs. High-soluble CD163 serum was collected from pigs with ongoing Lawsonia intracellularis infection. Lawsonia intracellularis infection induces CD163 shedding and subsequently produces high levels of soluble CD163 in serum, but Lawsonia intracellularis in serum does not interfere with PRRS virus infection assays.

[0309] Soluble CD163 serum concentrations were calculated relative to standard sera previously confirmed by ELISA. Low-CD163 sera from healthy pigs and high-CD163 sera from infected pigs were diluted 1:1 and 1:9 with PBS, respectively, and then measured by dot blot analysis using the primary detection antibody anti-PRRS 1AC7 (Ingenasa) and the secondary antibody goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 (ThermoFisher, A3272). Serum from healthy pigs contained 0.4 mg / L (±0.015 STDEV) of soluble CD163, whereas serum from infected pigs contained 4.5 mg / L (±0.45 STDEV). Medium containing 10% FBS did not contain soluble CD163.

[0310] Results and Discussion Examples of individual VHH, biparatopic, and triparatopic VHH constructs that inhibit infection of pPAM host cells by PRRS virus family members are shown below. After a 17-hour infection cycle, assays as described above were used to measure the extent of viral infection, quantified by viral growth capacity measured by FACS or RT-qPCR. These data clearly demonstrate that VHHs can inhibit productive infection of porcine alveolar macrophage cells by both PRRSV-1 (see Figure 3) and PRRSV-2 (see Figure 4) isotypes. Individual VHHs that were active in the infection assays were divided into those that were effective against both PRRSV-1 and PRRSV-2 infection (represented by 03E11) and those that showed no inhibitory activity against PRRSV-1 infection but did show inhibitory activity against PRRSV-2 infection (represented by 17B11 and 03D03).

[0311] In an infection assay using BOR57 PRRSV-1, a biparatopic combination of VHHs 03D03 (clone 17), 03E11 (clone 19), and 17B11 (clone 39) was used (Figure 5). Next, the best-performing biparatopic construct (19-17, 03E11-03D03 2(G4S)) was used in an infection assay using LT3 PRRSV-1 (Figures 6-8) or MN184 PRRSV-2 (Figure 9) in the presence of medium containing 10% FBS, low-soluble CD163 porcine serum, or high-soluble CD163 serum.

[0312] Biparatopic antibodies without membrane-specific anti-CD163 VHHs, such as 03E11+03D03(19-17), were able to reduce PRRSV-1 viral infectivity by approximately 65%–75% in medium containing only 10% FBS (see Figure 6). In medium containing 80% low-CD163 porcine serum, such antibodies reduced PRRSV-1 viral infectivity by approximately 75% (see Figure 7).

[0313] Furthermore, in media containing 80% high-soluble CD163 porcine serum, PRRSV-1 virus infectivity was reduced by only approximately 50% (see Figure 8). Biparatopic 03E11+03D03 was also susceptible to competition with soluble CD163 for binding to pPAM WT cells in the FACS competition assay described above (Figure 2). This clearly demonstrates that competing soluble CD163 may reduce the efficacy of potential therapeutic agents that are not specific for membrane-associated CD163 and are susceptible to being blocked by competing soluble CD163.

[0314] A biparatopic combination, such as 03D03+03E11, can reduce PRRSV-2 viral infectivity by approximately 40% in a medium containing 10% FBS (see Figure 9). While effective, its efficacy against PRRSV-2 infection is lower than that against PRRSV-1 infection, even in the absence of soluble CD163. Improving this efficacy would be beneficial.

[0315] Although biparatopic combinations such as 03D03 and 03E11 can show reduced efficacy in the presence of highly soluble CD163, the data also show that in the same assays, triparatopic combinations containing membrane-specific VHHs such as 17B11, exemplified by the triparatopic Tri-2 and Tri-10, can reduce PRRSV-1 and PRRSV-2 infectivity by 100%, even in the presence of highly soluble CD163. Furthermore, triparatopic combinations combining membrane-specific anti-CD163 VHHs (e.g., 17B11) with other VHHs that can individually inhibit PRRSV-2 (e.g., 03D03 or 03E11) and / or PRRSV-1 (e.g., 03E11) infection can advantageously combine these properties.

[0316] Triparatopic and membrane-specific anti-CD163 VHHs assembled from VHHs that can individually block PRRSV-2, or PRRSV-1 and PRRSV-2 infection, are exemplified by the triparatopic Tri-2 (03E11-03D03-17B11 2(G4S)) and Tri-10 (17B11-03E11-03D03 2(G4S)).

[0317] Such triparatopic compounds can completely inhibit PRRSV-1 infectivity with very low IC50 values ​​in the range of 2.81-4.02 nM in both low-soluble CD163 sera (see Figure 10 and Table 7) and high-soluble CD163 sera (see Figure 11 and Table 7). Furthermore, such triparatopic compounds can completely inhibit PRRSV-2 infectivity with very low IC50 values ​​in the range of 2.13-7.60 nM in both low-soluble CD163 sera (see Figure 12, Table 8) and high-soluble CD163 sera (see Figure 13, Table 8). These data clearly demonstrate that combining VHHs capable of individually blocking either PRRSV-1 and -2 or PRRSV-2 with membrane-specific VHHs that exhibit reduced susceptibility to competing soluble CD163 effectively blocks infection by PRRSV-1 or -2 family members. Advantageously, such combinations can enhance the blockade of PRRSV infection in the presence of elevated levels of soluble CD163, as seen during ongoing infection, where soluble CD163 is present at significant levels in serum and may act as a sink. Such multipathogenic VHHs would have clear and significant advantages as therapeutic agents for the prevention and treatment of PRRSV infection.

[0318] [Table 7]

[0319] [Table 8]

[0320] References 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 RFLP 184 isolates of porcine reproductive and respiratory syndrome virus, Virus Res., 122: 175-182.

[0321] Example 4: Efficacy of repeated administration of a triparatopic construct containing the 17B11 antibody against experimental infection with PRRSV-1 (LT3 strain) in piglets. material and method animal 26 piglets ( Sus scrofa domesticus ), healthy and approximately 5–6 weeks old at the time of challenge. Variety:Large White x Pietrain, Male and female, PCR-negative and seronegative for PRRSV-1 and PRRSV-2 before challenge Challenge The challenge strain PRRSV-1 LT3 was approximately 10 6 TCID 50 mL (Roslin institute, Edinburgh, UK), Inoculation: 5 mL administered intranasally to each piglet once on Day 0 (D0) and once at Time 0 (T0)

[0322] Treatment group: VHH(Tri2) - 12 pigs challenged with LT3 on Day 0, T0 VHH: Triparatopic 03E11+03D03+17B11 2(G4S), called "Tri2". Formulation: Solution for injection, Tri-2 protein sample recovered from P. pastoris, 111 mg / mL, purified sodium phosphate 15 mM, arginine.HCl 100 mM, pH 7.00, in a 0.2 μm filter Dosage: 10 mg / kg, intramuscular injection (neck), starting on D0: approximately 5 hours before and approximately 3 hours after T0 (challenge). Injections were repeated daily in the morning and approximately 8 hours later from Day 1 (D1) to Day 10 (D10).

[0323] Control group: Buffer - 12 piglets were challenged with LT3 on Day 0, T0. VHH: None Formulation: Solution for injection, purified sodium phosphate 15 mM, arginine HCl 100 mM, pH 7.00, 0.2 μm filter Dosage: Same as Tri2; Intramuscular injection (neck) from D0: approximately 5 hours before and approximately 3 hours after T0 (challenge). Repeat injection twice a day from D1 to D10, in the morning and approximately 8 hours later.

[0324] Clinical and autopsy observations Observation was carried out from D-2 (2 days before challenge) to D11. Rectal temperature, general health condition, clinical observation of respiratory signs, local tolerance at the injection site, and general tolerance were measured daily. Measurement of weight gain and average daily weight gain on D-7, D-2, D5 and D11; euthanasia on D11 and necropsy for scoring of gross pneumonia lesions (Halbur et al, 1995) and sample collection.

[0325] Sample collection and analysis Blood samples were collected before challenge, and on days 2, 5, 7, 9, and 11. PRRSV qPCR and serology Measurement of Tri2 concentration. Biochemical analysis to assess general resistance (pre-challenge and D11 samples only). Fecal sample collection at the same time points for PRRSV qPCR. · Nasal secretions were swab collected on D11 for PRRSV qPCR only. Autopsy samples: Bronchoalveolar fluid samples were collected for PRRSV qPCR and Tri2 assays, and pulmonary alveolar macrophages (PAMs) were collected for additional testing. Lungs were collected for histopathology to score pneumonia and PRRSV qPCR. PRRSV qPCR was performed on tonsils, inguinal lymph nodes, and spleen. Histopathology is performed at both injection sites in the neck to assess local tolerance. Additional analysis can be performed on these tissues. Additional blood samples were taken on D-2 and D-1 to assess Tri2 concentrations several hours after administration, following the same sample schedule as the safety piglets. Laboratory tests, including serum analysis, were performed by personnel blinded to treatment allocation.

[0326] Summary of results Observations at the injection site showed no abnormalities were reported during the study period, and Tri2 was well tolerated locally. Furthermore, no systemic side effects were observed in any of the clinical observation evaluations during the study period, demonstrating that Tri2 was well tolerated systemically.

[0327] Abnormal clinical signs associated with challenge were more frequent in the control group, with coughing observed sporadically only in the control group and a significantly higher incidence of hyperthermia (rectal temperature ≥ 40°C): 45 cases in the control group compared with only 15 in the Tri2 group (P < 0.001) (Table 9). Body weight and weight gain: At the start of the study, each group was consistently homogeneous. On Day 11, before necropsy, a mean difference of approximately 1 kg was observed between the control group (9.7 ± 1.01 kg) and the Tri2 group (10.7 ± 1.67 kg). From Days 2 to 11, the mean daily weight gain was significantly higher in the Tri2 group (3.0 ± 0.52 kg) compared to the control group (2.1 ± 0.71 kg) (p < 0.05) (Table 9).

[0328] The primary endpoint, PRRSV viral load in serum, was significantly lower in Tri2 piglets than in control piglets at each observation time point from D2 to D9 (Table 9). Ct is inversely proportional to viral load. A Ct of 37 to 40 is considered negative, and a decrease in Ct indicates an increase in viral load. A difference of approximately 3.3 Ct represents a one log difference in viral titer. The serological data for PRRSV are shown in Table 10, and suggest that humoral immune responses may be slightly delayed under Tri2 therapy. Total lung lesions, as measured by Halvor score, were generally milder in Tri2 piglets (5.3 ± 5.11) compared with controls (11.1 ± 16.91), but the difference in scores was not statistically significant, likely due to data variability and sample size. Severely affected piglets, i.e., pigs with a lung lesion score of 15 or greater, were observed only in the control group (4 of 12 pigs, Table 1).

[0329] [Table 9]

[0330] [Table 10]

[0331] [Table 11]

[0332] conclusion Tri2 administered to piglets at 10 mg / kg twice daily for 10 days effectively suppressed the clinical and virological effects of PRRSV infection induced by PRRSV-1(LT3) challenge after the first Tri2 administration. Compared with untreated controls, Tri2 treatment significantly reduced PRRSV viremia at all observation time points from D2 to D9 postchallenge. Seroconversion was observed in both groups, although it was slightly delayed in the treated group. Hyperthermia was significantly less frequent in piglets treated with Tri2. Over 12 days, treated piglets gained an average of 1 kg more body weight than control piglets, and this difference was statistically significant. Gross pneumonia lesions, observed and scored at necropsy 11 days postchallenge, were generally milder in the treated group. Severe lesions, defined as a score >15, were observed in four untreated piglets (33%) and in none of the treated piglets. The formulation was well tolerated after multiple intramuscular injections. 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, (i) binds to the membrane-bound form of porcine CD163 on cells; and (ii) An antibody that does not significantly bind to the soluble form of porcine CD163.

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

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

10. An antibody or binding protein comprising:

4. the heavy chain variable region (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of 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 of AADQAGWRTAGVRNTYEYDY (SEQ ID NO: 4) 4. The antibody or binding protein of claim 3, comprising:

5. 5. The antibody or binding protein of any one of claims 1 to 4, comprising a VH domain comprising 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 of porcine CD163 as the antibody of claim 3 or claim 4.

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

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

9. 9. The combination of claim 7 or claim 8, wherein the additional anti-porcine CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to porcine CD163, the antigen-binding domain comprising a heavy chain variable region comprising three complementarity-determining regions (CDRs), the heavy chain variable region comprising: (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 containing one or two amino acid substitutions compared to the corresponding CDR sequence; (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 containing one, two, three, or four amino acid substitutions compared to the corresponding CDR sequence; and (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 containing one, two, three, or four amino acid substitutions compared to the corresponding CDR sequence. and / or The additional anti-porcine CD163 antibody or binding protein comprises at least one antigen-binding domain that binds to porcine CD163, wherein the antigen-binding domain comprises a 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 of DYTIG (SEQ ID NO: 18) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to the corresponding CDR sequence; (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of CINSITSNTYYADSVKG (SEQ ID NO: 19), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to the corresponding CDR sequence; and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of DSGLFSGSSCLKYRAMRFGS (SEQ ID NO: 20), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to the corresponding CDR sequence. Including, combinations.

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

11. 11. An 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. 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 said antibody, binding protein or combination, preferably albumin or an IgG Fc region.

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

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

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

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

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

18. 16. The antibody, binding protein or combination of any one of claims 1 to 12, one or more nucleic acid molecules of claim 13, one or more expression vectors of claim 14 or one or more host cells of claim 15 for use in treatment in a subject, preferably in the treatment or prevention of a PRRSV infection in a subject.

19. 19. The antibody, binding protein, combination, nucleic acid molecule, expression vector, or host cell for use according to claim 18, wherein the subject is a pig.

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

21. Use of an antibody, binding protein or combination described in any one of claims 1 to 12, one or more nucleic acid molecules described in claim 13, one or more expression vectors described in claim 14, or one or more host cells described in claim 15 in the manufacture of a medicament for use in the treatment, preferably the treatment or prevention, of PRRSV infection in a subject.

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

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