CD163 antibody or binding protein

Abstract

The present disclosure provides a monoclonal antibody that binds to porcine CD163 for use in treating or preventing porcine reproductive and respiratory syndrome (PRRS) virus infection in pigs. A preferred antibody comprises an antigen-binding domain that binds to porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the heavy chain variable region comprises a variable heavy chain (VH) CDR2 comprising the amino acid sequence XYAD, XYAE, or XYAN, where X can be any amino acid. Nucleic acid molecules, expression vectors, and compositions are also provided.

Description

Technical Field

[0001] The present invention generally relates to binding proteins that bind to CD163 (cluster of differentiation 163), specifically antibodies, and particularly to binding proteins and antibodies that bind to porcine CD163. Such anti-CD163 binding proteins and antibodies are used for treatment and protection in the treatment or prevention of infectious diseases such as porcine reproductive and respiratory syndrome (PRRS) virus infection, for example, to reduce the incidence and severity. Binding protein- and antibody-based compositions, methods, and kits are also provided.

[0002] Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating viral swine diseases in the world, causing significant economic losses to the swine industry. The causative agent is the PRRS virus (PRRSV), an enveloped RNA virus classified in the family Arteriviridae within the order Nidovirales. PRRSV has restricted host and cell tropism, with porcine alveolar macrophages (PAM) being an important target cell. Clinical symptoms are diverse and include dyspnea and respiratory diseases in young and neonatal pigs, late-term abortions and stillbirths in non-pregnant and pregnant sows, early pregnancy fetal resorption, and poor growth in finishing pigs. Decreased or lost pregnancies, neonatal pig deaths, and reduced growth rates in all PRRSV-infected pigs are estimated to cost pork producers in the United States alone over $650 million annually.

[0003] All currently known PRRSV isolates are classified into one of two genotypes (or species), type 1 (PRRSV-1) or type 2 (PRRSV-2), both of which cause chronic infections and similar clinical symptoms but have only approximately 60% identity at the nucleotide level. Genotype 1 is of European origin and tends to be found in European PRRSV isolates or strains, while genotype 2 is of North American origin and tends to be found in isolates or strains from Asia or the Americas (see review by Stoian and Rowland, 2019, Vet. Sci., 6, 9). Each genotype has great diversity, and numerous strains have been identified since 2006, including new highly pathogenic strains that have emerged especially in China and Vietnam. Similar highly pathogenic strains have also emerged in other locations spreading from the Malay Peninsula to southern Russia, and these are increasing the threat to pig populations (An et al., 2011, Emerging Infect Dis 17(9):1782). In China alone, more than 20 million pigs were culled annually in 2006 and 2007 due to PRRS virus infection (An et al., 2010, Emerging Infect Dis 16(2):365). More recently, case reports of virulent strains causing major outbreaks in Europe are threatening the increased emergence of the PRRS virus (Sinn et al., 2016, Porcine Health Management(2):28).

[0004] The scavenger receptor CD163 is an important mediator of PRRSV infection and thus has an important role in PRRSV infection. CD163 is a 130 kDa type I transmembrane protein with a signal peptide followed by nine scavenger receptor cysteine-rich (SRCR) domains, each approximately 100 amino acids in length, and a 35-amino acid proline-serine-threonine (PST)-rich region separating SRCR domain 6 (SRCR6) and SRCR7. A second PST-rich region connects SRCR9 to the transmembrane domain and a short cytoplasmic tail containing a functional internalization motif. Surface expression of CD163 is limited to cells of the monocyte-macrophage lineage. The SRCR5 domain of CD163 has been shown to play an important role in the occurrence of PRRSV infection of porcine alveolar macrophages (Gorp et al., 2010, J. of Virology, March, 3101-3105).

[0005] The exact mechanism of PRRSV infection is unknown. However, as part of this mechanism, PRRSV is thought to enter the endosomal compartment of cells, and the interaction between CD163 and the GP2-GP3-GP4 heterotrimer of PRRSV mediates viral uncoating and the release of the viral genome into the cytoplasm.

[0006] One proposed treatment option for PRRSV involves certain gene knockouts 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). This has been shown to function very effectively, but this treatment is complex and time-consuming in that it can treat a significant proportion of the pig population. Furthermore, and importantly, there is considerable resistance in many markets regarding the desirability of technologies involving genetic modification of animals, such as animal products produced from such animals.

[0007] The most common medical intervention used to limit the economic impact of PRRS is vaccination. Vaccines are used routinely in all regions where the disease is prevalent. Two types of vaccines are routinely used, either killed virus vaccines (inactivated) or (most commonly) modified live vaccines (MLV). However, current vaccines are only partially effective and are most valuable when deployed within an integrated approach to disease management where biosecurity and livestock production decisions are closely coordinated. The reasons behind the lack of vaccine efficacy are complex, but, combined with the biology of the virus (tropism for alveolar macrophages and high mutability), the high genetic diversity of the PRRSV population is such that the best results are seen when the vaccine strain and circulating strains are well matched immunologically (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 can be pathogenic.

[0008] Currently, there are no antiviral treatment options for PRRSV infections.

[0009] Accordingly, there is a clear need for alternative, preferably improved, treatment and prevention options for PRRSV infection (or other CD163-mediated infections) that can be readily used to treat or prevent infection in a significant number of animals.

[0010] The present invention provides one such alternative therapeutic or prophylactic option in the form of binding proteins and antibodies directed against porcine CD163 that are capable of acting to reduce or prevent PRRSV infection.

[0011] Surprisingly, in contrast to, for example, polyclonal antibody preparations that target multiple different epitopes on porcine CD163, a single type of antibody (monoclonal antibody) that targets the same epitope on porcine CD163 has been shown to be effective in significantly reducing or preventing PRRSV infection. Furthermore, a subset of these anti-CD163 antibodies has been confirmed to exhibit different inhibition of infection by type 1 and / or type 2 PRRSV.

[0012] Accordingly, the inventors have provided anti-CD163 antibodies that bind to CD163, particularly porcine CD163, and are capable of inhibiting its activity or function. Such antibodies (or other binding proteins, for example, containing the CD163 antigen-binding domain described herein) can inhibit the ability of CD163 to interact with other proteins, such as viral proteins, thereby inhibiting the infection of cells such as porcine alveolar macrophages. Such antibodies (or other binding proteins, for example, containing the CD163 antigen-binding domain described herein) can be conveniently and advantageously used for treating or preventing infectious diseases in pigs, particularly PRRSV infections.

[0013] In one embodiment, the present invention provides a binding protein, such as an antibody, such as a monoclonal antibody, that binds to CD163, such as porcine CD163, for use in treating or preventing infections in pigs, such as PRRS virus infection or CD163-mediated infection. Accordingly, in particular, the present invention provides a monoclonal antibody that binds to porcine CD163 for use in treating or preventing PRRS virus infection in pigs. However, the antibodies of the present invention can be used for treating or preventing any pathological condition in pigs in which CD163 has been shown to play a role, in which case the binding and inhibition of this protein can be a useful therapeutic tool.

[0014] As discussed elsewhere in this specification, preferred antibodies (or binding proteins) of the invention and antibodies suitable for use in the therapeutic methods described herein have the ability to bind to the SRCR5 domain of CD163 and, for example, have an epitope in the SRCR5 domain of CD163. Further, preferred antibodies (or binding proteins) have the ability to inhibit type 1 and / or type 2 PRRSV infection, more preferably the ability to inhibit type 1 and type 2 PRRSV infection. Further, some preferred antibodies have the ability to inhibit type 2 PRRSV infection, preferably the ability to specifically inhibit type 2 PRRSV infection.

[0015] The ability to inhibit type 1 and / or type 2 PRRSV infection Family 40 In a further embodiment, the invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region comprises: (i) A variable heavy-chain (VH) CDR1 comprising the amino acid sequence of RYVMG (SEQ ID NO: 2) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one or two amino acid substitutions as compared to a given CDR sequence, the variable heavy-chain (VH) CDR1, (ii) A variable heavy-chain (VH) CDR2 comprising the amino acid sequence of GIAWSGRAPYADSVKG (SEQ ID NO: 3) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions as compared to a given CDR sequence, the variable heavy-chain (VH) CDR2, and (iii) A variable heavy-chain (VH) CDR3 comprising the amino acid sequence of GEGAIRWTTLDAYDY (SEQ ID NO: 4) or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions as compared to a given CDR sequence, the variable heavy-chain (VH) CDR3.

[0016] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region comprises the following: (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 comprising one or two amino acid substitutions as compared to a given CDR sequence, the variable heavy-chain (VH) CDR1, (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 comprising one, two, three or four amino acid substitutions as compared to a given CDR sequence, the variable heavy-chain (VH) CDR2, 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 comprising one, two, three or four amino acid substitutions as compared to a given CDR sequence, the variable heavy-chain (VH) CDR3.

[0017] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region comprises the following: (i) A variable heavy-chain (VH) CDR1 comprising the amino acid sequence of RYVMG (SEQ ID NO: 18), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one or two amino acid substitutions as compared to a given CDR sequence, the variable heavy-chain (VH) CDR1, (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of GIAWSGRAPYADSVKG (SEQ ID NO: 19), or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions as compared to a given CDR sequence, a variable heavy chain (VH) CDR2, and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GEGAILWTTPGAYNY (SEQ ID NO: 20), or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence is a sequence comprising one, two, three, or four amino acid substitutions as compared to a given CDR sequence, a variable heavy chain (VH) CDR3.

[0018] In a preferred embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the aforementioned antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the aforementioned heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of RYVMG (SEQ ID NO: 2), (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of GIAWSGRAPYADSVKG (SEQ ID NO: 3), and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GEGAIRWTTLDAYDY (SEQ ID NO: 4).

[0019] In a preferred embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the aforementioned antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the aforementioned heavy chain variable region comprises the following: (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).

[0020] In a preferred embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the aforementioned antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the aforementioned heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of RYVMG (SEQ ID NO: 18), (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of GIAWSGRAPYADSVKG (SEQ ID NO: 19), and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GEGAILWTTPGAYNY (SEQ ID NO: 20).

[0021] In a further embodiment of the present invention, the VH CDR2 has or comprises the amino acid sequence of X1I X3W S G R A P Y A D S V K G (SEQ ID NO: 73). In these embodiments, X1 or X3 can be any amino acid. Preferably, one or more, most preferably all, of these X residues are selected from the following groups: X1 is G or A, and X3 is A or S. Thus, a preferred VH CDR2 has or comprises the amino acid sequence of G / AIA / SWSGRAPYADSVKG (SEQ ID NO: 74). For example, preferred VH CDR2 sequences of this embodiment have or comprise SEQ ID NO: 3, 11, or 19.

[0022] In a further embodiment of the present invention, the VH CDR3 has or comprises the amino acid sequence of G E G A I X6W T T X 10 X 11 A Y X 14 Y (SEQ ID NO: 75). In these embodiments, X6, X 10 X 11 and X 14can be any amino acid. Preferably, one or more, most preferably all, of these X residues are selected from the following groups: X6 is R or K or L, and X 10 is L or P, and X 11 is D or G, and X 14 is D or N. Thus, a preferred VH CDR3 has or contains the amino acid sequence (SEQ ID NO: 76) of G E G A I R / K / L W T T L / P D / G A Y D / N Y. For example, preferred VH CDR3 sequences of this embodiment have or contain SEQ ID NO: 4, 12, or 20.

[0023] In a further embodiment, the present invention provides an antibody (or binding protein) comprising: a VH region comprising the VH CDR1 of SEQ ID NO: 2 or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence is a sequence comprising one or two (preferably one) amino acid changes as compared to a given CDR sequence, a VH region comprising the VH CDR1 of SEQ ID NO: 2, the VH CDR2 of SEQ ID NO: 73, and the VH CDR3 of SEQ ID NO: 75. In some such embodiments, VH CDR1 is preferably SEQ ID NO: 2. In some such embodiments, VH CDR2 is preferably SEQ ID NO: 3, 11, or 19. In some such embodiments, VH CDR3 is preferably SEQ ID NO: 4, 12, or 20.

[0024] In one embodiment, the present invention provides an antibody (or binding protein) comprising: A VH region comprising a VH CDR1 of SEQ ID NO: 2 or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one or two (preferably one) amino acid changes as compared to a given CDR sequence, the VH CDR1 of SEQ ID NO: 2, the CDR2 of SEQ ID NO: 74, and the VH CDR3 of SEQ ID NO: 76. In some such embodiments, the VH CDR1 is preferably SEQ ID NO: 2. In some such embodiments, the VH CDR2 is preferably SEQ ID NO: 3, 11 or 19. In some such embodiments, the VH CDR3 is preferably SEQ ID NO: 4, 12 or 20.

[0025] In a further embodiment of the invention, the antibody (or binding protein) comprises: A VH region comprising a VH CDR1 of SEQ ID NO: 2 or a sequence comprising one or two (preferably one) amino acid changes as compared to a given CDR sequence, a VH CDR2 of SEQ ID NO: 73 or a sequence substantially homologous thereto, and a VH CDR3 of SEQ ID NO: 75 or a sequence substantially homologous thereto. In such embodiments, the aforementioned substantially homologous sequences comprise one, two, three or four, preferably one, two or three, preferably one or two (more preferably one) amino acid changes as compared to a given CDR sequence.

[0026] In a further embodiment of the invention, the antibody (or binding protein) comprises: A VH region comprising a VH CDR1 of SEQ ID NO: 2 or a sequence comprising one or two (preferably one) amino acid changes as compared to a given CDR sequence, a VH CDR2 of SEQ ID NO: 74 or a sequence substantially homologous thereto, and a VH CDR3 of SEQ ID NO: 76 or a sequence substantially homologous thereto. In such embodiments, the aforementioned substantially homologous sequences comprise one, two, three or four, preferably one, two or three, preferably one or two (more preferably one) amino acid changes as compared to a given CDR sequence.

[0027] Family 70 In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region comprises the following: (i) A variable heavy-chain (VH) CDR1 comprising the amino acid sequence of TYSMG (SEQ ID NO: 26), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one or two amino acid substitutions compared to a given CDR sequence, the variable heavy-chain (VH) CDR1, (ii) A variable heavy-chain (VH) CDR2 comprising the amino acid sequence of AHRWSGSAYYAEHADSVEG (SEQ ID NO: 27), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions compared to a given CDR sequence, the variable heavy-chain (VH) CDR2, and (iii) A variable heavy-chain (VH) CDR3 comprising the amino acid sequence of GVGSAAQYRY (SEQ ID NO: 28), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions compared to a given CDR sequence, the variable heavy-chain (VH) CDR3.

[0028] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region comprises the following: (i) A variable heavy-chain (VH) CDR1 comprising the amino acid sequence of PGSMG (SEQ ID NO: 34), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one or two amino acid substitutions compared to a given CDR sequence, the variable heavy-chain (VH) CDR1, (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of AHRWSGSAYYADYADSVEG (SEQ ID NO: 35) or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions compared to a given CDR sequence, a variable heavy chain (VH) CDR2, and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GVGSAAQYTY (SEQ ID NO: 36) or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions compared to a given CDR sequence, a variable heavy chain (VH) CDR3.

[0029] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the aforementioned antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the aforementioned heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of TYSMG (SEQ ID NO: 42) or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence is a sequence comprising one or two amino acid substitutions compared to a given CDR sequence, a variable heavy chain (VH) CDR1, (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of AHRWSGSAYYAEHADSVEG (SEQ ID NO: 43) or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions compared to a given CDR sequence, a variable heavy chain (VH) CDR2, and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GVGSEAQYRY (SEQ ID NO: 44) or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions compared to a given CDR sequence, a variable heavy chain (VH) CDR3.

[0030] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein said antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and said heavy-chain variable region comprises the following: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of TYSMG (SEQ ID NO: 26), (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AHRWSGSAYYAEHADSVEG (SEQ ID NO: 27), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GVGSAAQYRY (SEQ ID NO: 28).

[0031] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein said antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and said heavy-chain variable region comprises the following: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of PGSMG (SEQ ID NO: 34), (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AHRWSGSAYYADYADSVEG (SEQ ID NO: 35), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GVGSAAQYTY (SEQ ID NO: 36).

[0032] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein said antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and said heavy-chain variable region comprises the following: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of TYSMG (SEQ ID NO: 42), (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AHRWSGSAYYAEHADSVEG (SEQ ID NO: 43), and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GVGSEAQYRY (SEQ ID NO: 44).

[0033] In a further embodiment of the invention, the VH CDR1 has or comprises the amino acid sequence of X1X2S M G (SEQ ID NO: 77). In these embodiments, X1 or X2 can be any amino acid. Preferably, one or more, most preferably all, of these X residues are selected from the following groups: X1 is T or P, and X2 is Y or G. Thus, a preferred VH CDR1 has or comprises the amino acid sequence of T / P Y / G S M G (SEQ ID NO: 78). For example, preferred VH CDR1 sequences of this embodiment have or comprise SEQ ID NO: 26, 34 or 42.

[0034] In a further embodiment of the invention, the VH CDR2 has or comprises the amino acid sequence of A H R W S G S A Y Y A X 12 X 13 A D S V E G (SEQ ID NO: 79). In these embodiments, X 12 or X 13 can be any amino acid. Preferably, one or more, most preferably all, of these X residues are selected from the following groups: X 12 is E or D, and X 13 is H or Y. Thus, a preferred VH CDR2 has or comprises the amino acid sequence of A H R W S G S A Y Y A E / D H / Y A D S V E G (SEQ ID NO: 80). For example, preferred VH CDR2 sequences of this embodiment have or comprise SEQ ID NO: 27, 35, or 43.

[0035] In a further embodiment of the invention, the VH CDR3 has or comprises the amino acid sequence of G V G S X5A Q Y X9Y (SEQ ID NO: 81). In these embodiments, X5 and X9 can be any amino acid. Preferably, one or more, most preferably all, of these X residues are selected from the following groups: X5 is A or E, and X9 is R or T. Accordingly, a preferred VH CDR3 has or comprises the amino acid sequence of G V G S A / E A Q Y R / T Y (SEQ ID NO: 82). For example, preferred VH CDR3 sequences of this embodiment have or comprise SEQ ID NO: 28, 36 or 44.

[0036] In one embodiment, the invention provides an antibody (or binding protein) comprising: A VH region comprising VH CDR1 of SEQ ID NO: 77, VH CDR2 of SEQ ID NO: 79, and VH CDR3 of SEQ ID NO: 81. In some such embodiments, VH CDR1 is preferably SEQ ID NO: 26, 34 or 42. In some such embodiments, VH CDR2 is preferably SEQ ID NO: 27, 35 or 43. In some such embodiments, VH CDR3 is preferably SEQ ID NO: 28, 36 or 44.

[0037] In one embodiment, the invention provides an antibody (or binding protein) comprising: A VH region comprising VH CDR1 of SEQ ID NO: 78, VH CDR2 of SEQ ID NO: 80, and VH CDR3 of SEQ ID NO: 82. In some such embodiments, VH CDR1 is preferably SEQ ID NO: 26, 34 or 42. In some such embodiments, VH CDR2 is preferably SEQ ID NO: 27, 35 or 43. In some such embodiments, VH CDR3 is preferably SEQ ID NO: 28, 36 or 44.

[0038] In other embodiments of the invention, the antibody (or binding protein) comprises: A VH region comprising a sequence having one or two (preferably one) amino acid changes compared to the VH CDR1 of SEQ ID NO: 77 or a given CDR sequence, a VH CDR2 of SEQ ID NO: 79 or a sequence substantially homologous thereto, and a VH CDR3 of SEQ ID NO: 81 or a sequence substantially homologous thereto. In such embodiments, the aforementioned substantially homologous sequences are sequences comprising one, two, three or four, preferably one, two or three, preferably one or two (more preferably one) amino acid changes compared to a given CDR sequence.

[0039] In other embodiments of the invention, the antibody (or binding protein) comprises: A VH region comprising a sequence having one or two (preferably one) amino acid changes compared to the VH CDR1 of SEQ ID NO: 78 or a given CDR sequence, a VH CDR2 of SEQ ID NO: 80 or a sequence substantially homologous thereto, and a VH CDR3 of SEQ ID NO: 82 or a sequence substantially homologous thereto. In such embodiments, the aforementioned substantially homologous sequences are sequences comprising one, two, three or four, preferably one, two or three, preferably one or two (more preferably one) amino acid changes compared to a given CDR sequence.

[0040] One or more of the CDR sequences are X X In embodiments of the invention where the residue (or another type of alternative residue as defined herein) is included, then, compared to a given CDR sequence, CDRs having sequences that are substantially homologous thereto and that include one, two, three or four, preferably one, two or three (more preferably one or two, or one) amino acid changes or amino acid substitutions are also encompassed by the invention. In some such embodiments, the aforementioned changes or substitutions at the amino acid residue may include one or more of the X X residues, or may be at residues other than the X X residues. In other such embodiments, the aforementioned changes are in a mixture of X X residues and non-X X residues.

[0041] Clone 150 (#15) In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein said antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and said heavy-chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of SYSMG (SEQ ID NO: 50), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence comprising one or two amino acid substitutions as compared to a given CDR sequence, the variable heavy chain (VH) CDR1, (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of AITWNGYITNYADSVKG (SEQ ID NO: 51), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions as compared to a given CDR sequence, the variable heavy chain (VH) CDR2, and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of TTFSTTSPISRTYNY (SEQ ID NO: 52), or a sequence substantially homologous thereto, wherein said substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions as compared to a given CDR sequence, the variable heavy chain (VH) CDR3.

[0042] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein said antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and said heavy-chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of SYSMG (SEQ ID NO: 50), (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of AITWNGYITNYADSVKG (SEQ ID NO: 51), and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of TTFSTTSPISRTYNY (SEQ ID NO: 52).

[0043] Clone 70 (#23) In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity determining regions (CDRs), and the heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of TYAMG (SEQ ID NO: 58), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one or two amino acid substitutions compared to a given CDR sequence, the variable heavy chain (VH) CDR1, (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of IISFGGTFYADSVKG (SEQ ID NO: 59), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions compared to a given CDR sequence, the variable heavy chain (VH) CDR2 and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GRTLSKRADSYAS (SEQ ID NO: 60), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions compared to a given CDR sequence, the variable heavy chain (VH) CDR3.

[0044] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity determining regions (CDRs), and the heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of TYAMG (SEQ ID NO: 58), (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of IISFGGTFYADSVKG (SEQ ID NO: 59), and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GRTLSKRADSYAS (SEQ ID NO: 60).

[0045] Clone 144 (#1) In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of MYAMS (SEQ ID NO: 66), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one or two amino acid substitutions compared to a given CDR sequence, the variable heavy chain (VH) CDR1, (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of AINTSGRYSRYADSVKG (SEQ ID NO: 67), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions compared to a given CDR sequence, the variable heavy chain (VH) CDR2 and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of TDKGNWALAMSYDY (SEQ ID NO: 68), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence comprising one, two, three or four amino acid substitutions compared to a given CDR sequence, the variable heavy chain (VH) CDR3.

[0046] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of MYAMS (SEQ ID NO: 66), (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of AINTSGRYSRYADSVKG (SEQ ID NO: 67), and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of TDKGNWALAMSYDY (SEQ ID NO: 68).

[0047] All antibodies (or binding proteins) described in the above section have the ability to inhibit type 1 and type 2 PRRSV infections and can therefore be used for the treatment or prevention of type 1 and / or type 2 PRRSV infections.

[0048] Ability to inhibit type 2 PRRSV infection As described above, other anti-CD163 antibodies and binding proteins of the present invention have the ability to inhibit type 2 PRRSV infection, preferably specifically inhibit (or only or preferentially inhibit type 2) type 2 PRRSV infection. For example, it inhibits type 2 PRRSV infection but does not inhibit (or does not significantly inhibit) type 1 PRRSV infection. Accordingly, a further embodiment of the present invention provides an antibody (or binding protein) that can specifically inhibit type 2 PRRSV infection. Examples of such "type 2" antibodies or binding proteins are described below. In other preferred embodiments, these "type 2" antibodies and binding proteins that can inhibit type 2 PRRSV infection can be used in combination with the above antibodies, thereby inhibiting type 1 and / or type 2 PRRSV infections, preferably inhibiting type 1, or type 1 and type 2 PRRSV infections.

[0049] Clone 57 (#11) Accordingly, in a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the aforementioned antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the aforementioned heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of VYGTG (SEQ ID NO: 84) or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence is a sequence comprising one or two amino acid substitutions compared to a given CDR sequence, the variable heavy chain (VH) CDR1, (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of GISGTTGSTLYADSVKG (SEQ ID NO: 85), or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence contains one, two, three, or four amino acid substitutions compared to a given CDR sequence, a variable heavy chain (VH) CDR2, and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GGRVYITTSSWAY (SEQ ID NO: 86), or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence contains one, two, three, or four amino acid substitutions compared to a given CDR sequence, a variable heavy chain (VH) CDR3.

[0050] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the aforementioned antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the aforementioned heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of VYGTG (SEQ ID NO: 84), (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of GISGTTGSTLYADSVKG (SEQ ID NO: 85), and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of GGRVYITTSSWAY (SEQ ID NO: 86).

[0051] Clone 41 (#12) In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the aforementioned antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the aforementioned heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of RYAMG (SEQ ID NO: 92), or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence contains one or two amino acid substitutions compared to a given CDR sequence, a variable heavy chain (VH) CDR1, (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of AIAWSTGSTYYANSVKG (SEQ ID NO: 93), or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence comprises one, two, three, or four amino acid substitutions compared to a given CDR sequence, the variable heavy chain (VH) CDR2, and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of ETRYCSGFGCLDPRTYGS (SEQ ID NO: 94), or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence comprises one, two, three, or four amino acid substitutions compared to a given CDR sequence, the variable heavy chain (VH) CDR3.

[0052] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the aforementioned antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the aforementioned heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of RYAMG (SEQ ID NO: 92), (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of AIAWSTGSTYYANSVKG (SEQ ID NO: 93), and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of ETRYCSGFGCLDPRTYGS (SEQ ID NO: 94).

[0053] Clone 171 (#14) In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the aforementioned antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the aforementioned heavy chain variable region comprises the following: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of TDTMA (SEQ ID NO: 100), or a sequence substantially homologous thereto, wherein the aforementioned substantially homologous sequence comprises one or two amino acid substitutions compared to a given CDR sequence, the variable heavy chain (VH) CDR1, (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of GIGRSGGSIYYADAVKG (SEQ ID NO: 101), or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, three, or four amino acid substitutions compared to a given CDR sequence, and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of RQRIGLVVGALGYDY (SEQ ID NO: 102), or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, three, or four amino acid substitutions compared to a given CDR sequence.

[0054] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the heavy chain variable region comprises: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of TDTMA (SEQ ID NO: 100), (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of GIGRSGGSIYYADAVKG (SEQ ID NO: 101), and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of RQRIGLVVGALGYDY (SEQ ID NO: 102).

[0055] Clone 29 (#17) In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the heavy chain variable region comprises: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of DYTIG (SEQ ID NO: 108), or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one or two amino acid substitutions compared to a given CDR sequence. (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of CINSITSNTYYADSVKG (SEQ ID NO: 109), or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, three or four amino acid substitutions compared to a given CDR sequence, and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of DSGLFSGSSCLKYRAMRFGS (SEQ ID NO: 110), or a sequence substantially homologous thereto, wherein the substantially homologous sequence comprises one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0056] In a further embodiment, the present invention provides a binding protein, such as an antibody, comprising an antigen-binding domain that binds to CD163, such as porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the heavy chain variable region comprises: (i) A variable heavy chain (VH) CDR1 comprising the amino acid sequence of DYTIG (SEQ ID NO: 108), (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence of CINSITSNTYYADSVKG (SEQ ID NO: 109), and (iii) A variable heavy chain (VH) CDR3 comprising the amino acid sequence of DSGLFSGSSCLKYRAMRFGS (SEQ ID NO: 110).

[0057] Other embodiments Certain preferred embodiments of the present invention provide an antibody (or binding protein) that binds to CD163, such as porcine CD163, comprising a VH domain having the amino acid sequence of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57 or 65, 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.

[0058] In preferred embodiments, the present invention provides an antibody (or binding protein) that binds to CD163, such as porcine CD163, comprising a VH domain having the amino acid sequence of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57 or 65, 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.

[0059] In preferred embodiments, the present invention provides an antibody (or binding protein) that binds to CD163, such as porcine CD163, comprising a VH domain having the amino acid sequence of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57 or 65. 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.

[0060] Certain preferred embodiments of the present invention provide an antibody (or binding protein) that binds to CD163, such as porcine CD163, comprising a VH domain having the amino acid sequence of SEQ ID NO: 83, 91, 99 or 107, 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.

[0061] In a preferred embodiment, the present invention provides an antibody (or binding protein) that binds to CD163, such as porcine CD163, comprising a VH domain having the amino acid sequence of SEQ ID NO: 83, 91, 99 or 107, or a sequence having at least 80% sequence identity therewith (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.

[0062] In a preferred embodiment, the present invention provides an antibody (or binding protein) that binds to CD163, such as porcine CD163, comprising a VH domain having the amino acid sequence of SEQ ID NO: 83, 91, 99 or 107. 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.

[0063] Other preferred embodiments are in the form of an immunoglobulin (Ig), such as an IgG form, or in a form comprising all or part of the immunoglobulin constant region of various antibodies (or binding proteins) as defined herein, such as an IgG constant region, for example in the form of a full-length Ig or IgG form. Of course, it is understood that a complete IgG antibody typically comprises two substantially identical heavy chains and two substantially identical light chains. Preferred forms comprising part of the immunoglobulin constant region are forms comprising an Fc region or domain, such as an Fc fusion. Such Fc regions or domains are known in the art and generally comprise the CH2 and CH3 domains of the antibody heavy chain, which associate to form a homodimer. These regions can be from any suitable source or species, for example a source or species different from the host species used to produce the antibody by immunization, or different from the location from which the antibody is derived, but preferably corresponding to or derived from a porcine Fc region or domain. Since such Fc regions are homodimers (or form homodimers), they can be conveniently used to dimerize two polypeptide chains. Thus, by linking or fusing one or more single-domain antibodies (e.g., VHH antibodies) of the invention to each chain of the Fc region, when the two chains of the Fc region dimerize, they can be used to provide multiple copies of the single-domain antibodies (e.g., VHH antibodies) of the invention in a single construct or molecule. When multiple single-domain antibodies (e.g., VHH antibodies) of the invention are sequentially linked or fused to each chain of the Fc region, these antibodies can be the same antibody (e.g., two or more copies of the same VHH can be provided on each chain) or different antibodies. Thus, for example, Fc fusions can be used to provide constructs comprising multiple copies of the same single-domain antibody of the invention, or multiple copies of different single-domain antibodies of the invention. Such constructs generally comprise multiple copies of the same antibody of the invention (e.g., multiple copies of a single single-domain antibody or VHH antibody, or multiple copies of multiple different single-domain antibodies or VHH antibodies), and thus such constructs can, for example, exhibit improved binding of CD163 due to the avidity effect.

[0064] Binding proteins, for example, antibodies based on the antibody sequences of 49 (#18), 47 (#19), 48 (#20), 76 (#2), 77 (#16), 78 (#8), 150 (#15), 70 (#23), or 144 (#1) described in Tables A, B, C, D, E, F, G, H, or I are preferred. The present invention is exemplified by monoclonal antibodies that are VHH antibodies (single domain antibodies), the sequences of which are shown in Tables A, B, C, D, E, F, G, H, and I herein. The VH CDR domains and VH domains of each of these VHH antibodies are shown in Tables A-I herein. Antibodies (or binding proteins) comprising the VH CDR domain, or these sets of VH domains, or IgG sequences (or sequences substantially homologous thereto) comprising such domains are preferred embodiments of the present invention.

[0065] Furthermore, binding proteins, for example, antibodies based on the antibody sequences of 57 (#11), 41 (#12), 171 (#14), 29 (#17) described in Tables 1, 2, 3, or 4 are preferred. The present invention is exemplified by monoclonal antibodies that are VHH antibodies (single domain antibodies), the sequences of which are shown in Tables 1, 2, 3, and 4 herein. The VH CDR domains and VH domains of each of these VHH antibodies are shown in Tables 1, 2, 3, and 4 herein. Antibodies (or binding proteins) comprising these sets of VH CDR domains, or VH domains, or IgG sequences comprising such domains (or sequences substantially homologous thereto) are preferred embodiments of the present invention.

[0066] Specific examples of substantially identical sequences are sequences having at least 60% or 65% identity to the disclosed amino acid sequences. In certain embodiments, the antibodies (or binding proteins) of the invention comprise at least one heavy chain variable region comprising an amino acid sequence region having at least about 60%, 65%, 70% or 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90% or 95%, most preferably at least about 97%, 98% or 99% amino acid sequence identity to the amino acid sequences of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57 or 65.

[0067] Other specific examples of substantially identical sequences are sequences having at least 60% or 65% identity to the disclosed amino acid sequences. In certain embodiments, the antibodies (or binding proteins) of the invention comprise at least one heavy chain variable region comprising an amino acid sequence region having at least about 60%, 65%, 70% or 75%, more preferably at least 80%, more preferably at least about 85%, more preferably at least about 90% or 95%, most preferably at least about 97%, 98% or 99% amino acid sequence identity to the amino acid sequences of SEQ ID NO: 83, 91, 99 or 107.

[0068] Other preferred examples of substantially identical sequences are sequences that include conservative amino acid substitutions of the disclosed amino acid sequences.

[0069] Other preferred examples of substantially identical sequences are sequences that include one, two, three or four, preferably one, two or three, preferably one or two (more preferably one) amino acid changes in one or more of the disclosed CDR regions or one or more of the FR regions. Such changes can be conservative amino acid substitutions or non-conservative amino acid substitutions, or mixtures thereof.

[0070] In such embodiments, preferred changes are conservative amino acid substitutions.

[0071] In all embodiments, a binding protein, such as an antibody, comprising substantially identical sequences retains the ability to bind to CD163, such as porcine CD163. Preferably, a binding protein, such as an antibody, comprising substantially identical sequences retains one or more (preferably all) of the other properties described herein with respect to the 49 (#18), 47 (#19), 48 (#20), 76 (#2), 77 (#16), 78 (#8), 150 (#15), 70 (#23) or 144 (#1) antibodies.

[0072] In all embodiments, a binding protein, such as an antibody, comprising substantially identical sequences retains the ability to bind to CD163, such as porcine CD163. Preferably, a binding protein, such as an antibody, comprising substantially identical sequences retains one or more (preferably all) of the other properties described herein with respect to the 57 (#11), 41 (#12), 171 (#14) or 29 (#17) antibodies.

[0073] Further examples of substantially identical amino acid sequences according to the invention are described elsewhere in this specification.

[0074] The CDRs of the antibodies (or binding proteins) of the invention are preferably separated by appropriate framework regions such as those found in naturally occurring antibodies and / or effective engineered antibodies. Thus, the V H (e.g., VHH), V L and the individual CDR sequences are preferably provided within or incorporated into a framework or scaffold appropriate to enable antigen (CD163 herein) binding. Such framework sequences or regions may correspond to naturally occurring framework regions, FR1, FR2, FR3 and / or FR4, as necessary to form an appropriate scaffold, or may correspond to a consensus framework region identified, for example, by comparing various naturally occurring framework regions. Alternatively, a non-antibody scaffold or framework, such as a T cell receptor framework, can be used.

[0075] Suitable arrays that can be used in the framework region are well known and described in the art, and any of these can be used. Preferred arrays for the framework region are one or more of the framework regions of the VHH antibodies of the present invention, preferably the 49 (#18), 47 (#19), 48 (#20), 76 (#2), 77 (#16), 78 (#8), 150 (#15), 70 (#23), or 144 (#1) VHH antibodies disclosed in Tables A, B, C, D, E, F, G, H, and I, or framework regions substantially homologous thereto, particularly framework regions that allow maintenance of antigen specificity, for example, one or more of the framework regions that result in substantially the same or the same 3D structure of the antibody.

[0076] Other preferred arrays for the framework region, particularly for the "type 2" antibodies of the present invention, are one or more of the framework regions of the VHH antibodies of the present invention, preferably the 57 (#11), 41 (#12), 171 (#14), or 29 (#17) VHH antibodies disclosed in Tables 1, 2, 3, and 4, or framework regions substantially homologous thereto, particularly framework regions that allow maintenance of antigen specificity, for example, one or more of the framework regions that result in substantially the same or the same 3D structure of the antibody.

[0077] In certain preferred embodiments, all four of the variable heavy chain (SEQ ID NOs: 5, 6, 7, and 8) framework regions (FRs), or FR regions substantially homologous thereto, are found in the antibodies of the present invention, as needed.

[0078] In other preferred embodiments, all four of the variable heavy chain (SEQ ID NOs: 13, 14, 15, and 16) framework regions (FRs), or FR regions substantially homologous thereto, are found in the antibodies of the present invention, as needed.

[0079] In other preferred embodiments, optionally, all four of the variable heavy chain (SEQ ID NOs: 21, 22, 23, and 24) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0080] In other preferred embodiments, optionally, all four of the variable heavy chain (SEQ ID NOs: 29, 30, 31, and 32) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0081] In other preferred embodiments, optionally, all four of the variable heavy chain (SEQ ID NOs: 37, 38, 39, and 40) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0082] In other preferred embodiments, optionally, all four of the variable heavy chain (SEQ ID NOs: 45, 46, 47, and 48) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0083] In other preferred embodiments, optionally, all four of the variable heavy chain (SEQ ID NOs: 53, 54, 55, and 56) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0084] In other preferred embodiments, optionally, all four of the variable heavy chain (SEQ ID NOs: 61, 62, 63, and 64) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0085] In other preferred embodiments, optionally, all four of the variable heavy chain (SEQ ID NOs: 69, 70, 71, and 72) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0086] In certain preferred embodiments, particularly with respect to the "type 2" antibodies of the present invention, all four of the variable heavy chain (SEQ ID NOs: 87, 88, 89, and 90) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0087] In other preferred embodiments, optionally, all four of the variable heavy chain (SEQ ID NOs: 95, 96, 97, and 98) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0088] In other preferred embodiments, optionally, all four of the variable heavy chain (SEQ ID NOs: 103, 104, 105, and 106) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0089] In other preferred embodiments, optionally, all four of the variable heavy chain (SEQ ID NOs: 111, 112, 113, and 114) framework regions (FRs), or FR regions that are substantially homologous thereto, are found in the antibodies of the present invention.

[0090] As described above, the present invention provides a binding protein, such as an antibody, that binds to (or specifically recognizes or specifically binds to) CD163. CD163 is also known as M130, MM130, SCAR1, macrophage-related antigen, hemoglobin scavenger receptor, or scavenger receptor cysteine-rich type 1 protein M130. Preferred binding proteins of the present invention are antibodies, particularly VHH antibodies. However, the embodiments described herein in relation to antibodies, such as VHH antibodies, apply equally, with the necessary modifications, to other types of binding proteins or vice versa.

[0091] A preferred binding protein is any single polypeptide chain that can bind (e.g., specifically bind) to porcine CD163. 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 generally comprising CDR regions (and optionally FR regions), such that the CDR regions (and optionally FR regions) of the antibodies of the present invention can be grafted onto a suitable scaffold or framework, such as an immunoglobulin scaffold, are used.

[0092] However, in other embodiments, a non-immunoglobulin-based single-chain binding protein / scaffold protein can be used that can be selected for its ability to specifically bind to a particular target antigen (CD163 or porcine CD163) itself. Such molecules are also referred to as antibody mimics (or antibody mimetics). Examples of suitable non-immunoglobulin-based single-chain binding proteins are known in the art and have been described, for example, adnectin (e.g., a compound available from Therapeutics, Inc., Waltham, MA); affimer (e.g., available from Avacta); ankyrin repeat protein or DARPin (e.g., available from Molecular Partners AG, Zurich, Switzerland); lipocalin, such as anticalin (e.g., available from Pieris Proteolab AG, Freising, Germany); human A-domain (e.g., avimer); staphylococcal protein A (e.g., available from Affibody AG, Sweden); thioredoxin; and gamma-B-crystallin or ubiquitin-based molecules, such as affilin (e.g., available from Scil Proteins GmbH, Halle, Germany), including, for example, fibronectin (or fibronectin-based molecules) based on the 10th module of the fibronectin type III domain. Such molecules can also be used as scaffolds onto which suitable CDRs mediating target antigen binding can be grafted. For example, the CDR regions (and optionally the FR regions) of the antibodies of the present invention can be grafted onto a suitable non-immunoglobulin scaffold.

[0093] In other embodiments of the invention, nucleic acid-based molecules such as aptamers can be used provided that such molecules can be selected for their ability to specifically bind to a particular target antigen (CD163 or porcine CD163) itself. Thus, when binding proteins are referred to herein, these embodiments can be extended to other types of binding entities or moieties such as nucleic acid-based molecules.

[0094] Preferred non-antibody binding proteins (or binding moieties) of the present invention have the ability to bind to the same epitope as the anti-CD163 antibody of the present invention, and such binding proteins (or binding moieties) can be selected, for example, using the antibody of the present invention as a reference antibody, by, for example, a competitive assay as described elsewhere herein.

[0095] CD163 is a 130 kDa type I transmembrane protein and has a signal peptide followed by nine scavenger receptor cysteine-rich (SRCR) domains, each of which is approximately 100 amino acids in length, and a proline-serine-threonine (PST)-rich region of 35 amino acids separates SRCR domain 6 (SRCR6) and SRCR7. A second PST-rich region connects SRCR9 to the transmembrane domain and a short cytoplasmic tail containing a functional internal trafficking motif. Surface expression of CD163 is limited to cells of the monocyte-macrophage system.

[0096] Particularly relevant to the present invention is that CD163 is expressed on the surface of porcine alveolar macrophages (PAM) and is thought to play an important role in the ability of various pathogens, particularly porcine reproductive and respiratory syndrome virus (PRRSV), to cause disease in pigs.

[0097] Accordingly, the binding proteins or antibodies of the present invention bind or are capable of binding to CD163. According to the present invention, CD163 can be derived from any species, for example, any mammalian species such as pigs (porcine), humans, cows (bovine), dogs (canine), cats (feline), sheep (ovine), horses (equine), mice and monkeys. In a preferred embodiment, CD163 is porcine CD163 and the antibody binds or is capable of binding to (or specifically recognizes or specifically binds to) porcine CD163.

[0098] In certain embodiments, the antibody can cross-react (or bind) with CD163 of other species. Thus, in some embodiments, the antibody can bind to porcine CD163, along with one or more other species, e.g., one or more other mammalian species of CD163, such as those described above. In some embodiments, the antibody can also bind to porcine CD163 and human CD163. In other words, the antibody can cross-react with both porcine CD163 and human CD163. In other embodiments, the antibody can bind to porcine CD163 but does not bind to (or binds significantly less or does not cross-react with) human CD163.

[0099] The binding proteins and antibodies of the present invention can bind to any suitable form of CD163, particularly forms of CD163 that include the SRCR5 domain. Thus, such forms can include full-length CD163, or non-full-length forms of CD163, e.g., truncated forms of CD163, or other variant forms of CD163 that include a subset of SRCR domains but generally include the SRCR5 domain. Preferred and convenient forms of CD163 to which the binding proteins and antibodies of the present invention can bind include recombinant CD163, e.g., recombinant porcine CD163, or CD163 when expressed on the cell surface (cell surface-expressed CD163). Thus, such cell surface forms often represent the native or natural form of CD163, e.g., the form found in cells that naturally express or overexpress CD163.

[0100] Suitable cell types that naturally express CD163 are well known to those skilled in the art and include monocytes and macrophages. A preferred cell type is PAM. Alternatively, CD163 can be expressed or overexpressed by recombinant means (or other engineering means) in cell types that do not normally express CD163, i.e., cells that express a recombinant form of CD163 can be used.

[0101] Exemplary forms of CD163 that can be used herein to evaluate the binding ability of binding proteins and antibodies, for example, recombinant CD163, are constructs that include full-length CD163 or subsets of different CD163 SRCR domains such as CD163-SRCR1-9, CD163-SRCR4-7, or CD163-SRCR5-6. Other combinations of CD163 SRCR domains and fragments containing subsets of different CD163 SRCR domains can likewise be used, provided that all or a portion (preferably all) of the SRCR5 domain is present. In some embodiments, the antibody does not (or does not significantly) bind to a CD163 molecule that includes a deletion of the SRCR5 domain or a deletion within the domain, or a mutation within the domain. The porcine form is preferably used to evaluate the antibodies of the present invention, but equivalent forms from other species, such as other mammalian species, can also be used, for example, to evaluate cross-reactivity.

[0102] The sequences of CD163 from 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, the Uniprot number for porcine CD163 is Q2VL90 and the Uniprot number for human CD163 is Q86VB7.

[0103] Accordingly, preferred binding proteins or antibodies of the present invention have the ability to bind to the SRCR5 domain of CD163 or an epitope within the SRCR5 domain, preferably the porcine SRCR5 domain.

[0104] The sequence of the porcine SRCR5 domain is shown below. This corresponds to residues 477-577 of Uniprot Q2VL90: PRLVGGDIPCSGRVEVQHGDTWGTVCDSDFSLEAASVLCRELQCGTVVSLLGGAHFGEGSGQIWAEEFQCEGHESHLSLCPVAPRPDGTCSHSRDVGVVCS (SEQ ID NO: 115).

[0105] The sequence of porcine CD163 is shown below. This corresponds to the entire sequence of Uniprot Q2VL90.

[0106] Methods for assessing binding (or the ability to bind) to CD163 in a suitable form are well known to those skilled in the art, and any suitable method can be used.

[0107] Convenient and suitable methods for assessing binding include in vitro binding assays such as ELISA assays for assessing the binding of antibodies to immobilized antigens such as immobilized forms of CD163 as described above. Those skilled in the art are proficient in ELISA assays and will be able to readily establish suitable conditions for assessing the ability of a binding protein or antibody to bind to CD163 in such assays. Particularly preferred ELISA assays are described in the Examples section. Alternatively, or in addition, the binding of antibodies to CD163 expressed on the cell surface can be evaluated by any suitable means including, for example, flow cytometry assays (e.g., FACS analysis) using cells expressing a recombinant form of PAM or CD163, such as the forms described elsewhere herein. Particularly preferred flow cytometry assays are described in the Examples section. Another method for testing the ability of an antibody to bind to CD163 on the cell surface is immunohistochemistry.

[0108] In certain embodiments, the binding protein or antibody of the invention binds to CD163 (e.g., porcine CD163 or human CD163) (as determined) in a surface plasmon resonance (SPR) assay (e.g., a BIACore assay). Suitable SPR assays are known in the art. In certain preferred SPR assays, a suitable form of CD163 is captured (or immobilized) on a solid support (e.g., a sensor chip) via, for example, amine coupling (e.g., 2000 response units (RU) of CD163 are 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.

[0109] Such SPR assay methods can be conveniently used to measure the binding rate of antibody-antigen interactions, for example, to determine the association rate (ka), dissociation rate (kd), and affinity (KD). In certain embodiments, the measurements are performed at 25 °C in a suitable buffer at pH 7.4, such as a standard HEPES-EDTA buffer like HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.0005% surfactant P20, sold by GE Healthcare Life Sciences). The rate parameters can be determined or calculated by any suitable model or software, for example, by fitting the sensogram experimental data assuming a 1:1 interaction using BIAevaluation software. Particularly preferred SPR assays are described in the Examples section of this specification.

[0110] Thus, in particularly preferred embodiments, the binding protein or antibody of the present invention binds to CD163 (e.g., porcine or human CD163, preferably porcine CD163) as determined (when evaluated) in a surface plasmon resonance (SPR) assay (e.g., a BIACore assay).

[0111] In certain preferred embodiments, when the antibody of the present invention is in the VHH format, it has a high binding affinity for CD163 (e.g., porcine CD163), for example, having a K D (equilibrium dissociation constant) in the range of 50 nM or less (better).

[0112] Thus, preferably, in the case of the VHH format, the antibody of the present invention has a binding affinity for CD163 (e.g., porcine CD163) corresponding to a K of less than 100 nM, less than 80 nM, less than 60 nM, less than 50 nM, less than 45 nM, less than 40 nM, less than 35 nM, less than 30 nM, less than 25 nM, less than 20 nM, less than 15 nM, or less than 10 nM, more preferably less than 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5 or 1.0 nM. D Exemplary specific binding affinities are disclosed in the Examples. Exemplary forms of CD163 that can be used to evaluate such binding affinity include recombinant porcine CD163 containing SRCR4-7 or recombinant porcine CD163 containing SRCR1-9. Suitable exemplary forms are described in the Examples section, for example, constructs pCD163-SRCR4-7huFc or pCD163-SRCR1-9huFc. Thus, the above binding affinity can be observed when the antibody of the present invention is assayed using these constructs or when assayed, for example, in an SPR assay.

[0113] As described above, in some embodiments of the present invention, the antibody can bind to porcine CD163 but not (or substantially not) to human CD163. In other words, they preferentially bind to porcine CD163 as opposed to human CD163.

[0114] A preferred use of the binding protein or antibody of the present invention is in the treatment or prevention of pathogen infections involving CD163, and in particular, PRRSV infection. Typically, the binding protein or antibody of the present invention inhibits (or blocks or reduces) pathogen (e.g., PRRSV) infection, e.g., inhibits (or blocks or reduces) the ability of a pathogen, e.g., PRRSV, to cause infection (e.g., infect a suitable host cell). Preferably, the inhibition or reduction is a measurable inhibition or reduction, more preferably a statistically significant inhibition or reduction, e.g., a statistically significant inhibition or reduction with a p-value of ≦0.05 or <0.05. In certain embodiments, the binding protein or antibody of the present invention inhibits (or blocks or reduces) the ability of a pathogen, e.g., PRRSV, to infect a host cell by 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%. Typically, such % inhibition (and other percentage inhibition levels described herein) is compared to (or relative to) an appropriate control assay or control level, e.g., a control assay or control level in the absence of the binding protein or antibody (anti-CD163 antibody) (e.g., a negative control or background level or assay). Thus, a 0% inhibition (control) level (or conversely a 100% or maximum infection level) is typically the level in the absence of the binding protein or antibody (anti-CD163 antibody).

[0115] Such an ability to inhibit infection can be determined or tested in any suitable assay, examples of which will be readily derivable by those skilled in the art. Suitable assays can be, for example, in vitro or ex vivo assays and can include, for example, the use of CD163-expressing host cells such as PAM or recombinant CD163-expressing host cells as discussed elsewhere in this specification. Such cells can be contacted with PRRSV or other suitable pathogens at levels that cause infection of the cells. Suitable assays can typically be performed in the presence of serum, such as porcine serum or fetal bovine serum (FBS). The appropriate percentage of serum to use can be readily determined by those skilled in the art, for example, levels of 10% FBS and 80% porcine serum were used in the assays described in the Examples section. Next, the ability of the binding proteins or antibodies of the present invention to inhibit or reduce such infection can be readily analyzed, for example, compared to (or against) a 100% infection level set by a control assay. Suitable and exemplary infection assays are described in the Examples section.

[0116] To inhibit or reduce infection, any suitable concentration of the binding protein or antibody can be used. Exemplary antibodies of the present invention, when used at concentrations of at least 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 300 or 400 μg / ml, for example, up to concentrations of 200, 300 or 400 μg / ml, for example, concentrations between 50 or 100 and 200, 300 or 400 μg / ml, have the ability to cause inhibition, such as the inhibition levels outlined herein, particularly with VHHs. When combinations of antibodies (e.g., VHH antibodies) are used, in some embodiments these levels can refer to the total amount of antibody (e.g., VHH) present, i.e., the sum of the individual concentrations of the antibodies present.

[0117] In some embodiments, the binding protein or antibody of the present invention can inhibit (or block or reduce) the ability of type 1 or type 2 PRRSV to cause infection (e.g., infect CD163-expressing host cells). In some embodiments, the binding protein or antibody of the present invention can inhibit (or block or reduce) the ability of both type 1 PRRSV and type 2 PRRSV to cause infection (e.g., infect CD163-expressing host cells). It should be noted that the binding protein or antibody of the present invention targets host cell CD163, in contrast to PRRSV (or other pathogenic entities) itself. This provides an important advantage that it can inhibit infection by any virus that uses the same binding region on CD163 for infection or lesion formation, such as PRRSV. Thus, antibodies such as those of the present invention can provide a means to block many strains or isolates of PRRSV, including highly pathogenic strains or isolates, provided that CD163 is used to infect cells. The use of CD163 is thought to be common in infections by multiple PRRSV strains. Therefore, the antibodies of the present invention have broad utility. This is in contrast to some known approaches to PRRSV, which can be strain-specific, for example vaccination, and their effectiveness (or whether they are truly effective) can vary depending on the strain. Thus, the antibodies of the present invention provide important advantages and flexibility over such conventional methods.

[0118] Preferred antibodies of the present invention have the ability to almost completely inhibit type 1 PRRSV infection, for example, at least 90% inhibition can be observed. Alternatively, at least 50%, 60%, 70%, 75%, or 80% inhibition can be observed. In some embodiments, antibodies having the ability to show at least 80% inhibition of type 1 PRRSV infection, more preferably at least 85%, 90% or 95% inhibition are preferred.

[0119] Preferred antibodies of the present invention have the ability to inhibit type 2 PRRSV infection by at least 50%, at least 55% or at least 60%, more preferably at least 65%, at least 70%, at least 75%, or at least 80% inhibition.

[0120] Some preferred antibodies of the present invention have the ability to inhibit both type 1 and type 2 PRRSV infections, for example, at the levels described above and elsewhere in this specification. Such antibodies may also be referred to herein as "dual" antibodies. Thus, exemplary antibodies may be capable of at least 50% inhibition of type 2 PRRSV in combination with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition of type 1 PRRSV. Alternative exemplary antibodies may be capable of at least 55% or 60% inhibition of type 2 PRRSV in combination with at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% inhibition of type 1 PRRSV. Alternative exemplary antibodies may be capable of at least 65%, 70% or 75% inhibition of type 2 PRRSV in combination with at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% inhibition of type 1 PRRSV. In some embodiments, preferred antibodies of the present invention may be capable of at least 65%, 70% or 75% inhibition of type 2 PRRSV in combination with at least 90% or 95% inhibition of type 1 PRRSV.

[0121] Exemplary "dual" antibodies in the form of VHH antibodies are 49 (#18), 47 (#19), 48 (#20), 76 (#2), 77 (#16), 78 (#8), 150 (#15), 70 (#23), and 144 (#1), respectively, as shown in Tables A, B, C, D, E, F, G, H, and I.

[0122] In some embodiments, the binding protein or antibody of the present invention can inhibit (or block or reduce) the ability of type 2 PRRSV to infect host cells. In some embodiments, the binding protein or antibody of the present invention has the ability to specifically inhibit (or block or reduce) the ability of type 2 PRRSV to cause infection (e.g., infect CD163-expressing host cells or specifically inhibit type 2 PRRSV infection). Such binding proteins or antibodies preferentially inhibit or reduce type 2 PRRSV infection as opposed to type 1 PRRSV infection. Thus, exemplary antibodies may be able to inhibit type 2 PRRSV infection by at least 40%, 45%, or 50% (e.g., inhibit the ability of type 2 PRRSV to infect host cells by at least 40%, 45%, or 50%).

[0123] In other embodiments, such binding proteins or antibodies do not inhibit or reduce type 1 PRRSV infection (e.g., do not significantly inhibit or reduce it) (e.g., do not inhibit or reduce the ability of type 1 PRRSV to infect host cells or do not significantly inhibit or reduce it). By way of pure example, such antibodies that do not significantly inhibit or reduce type 1 PRRSV infection may not be able to reduce such infection by less than 10%, or less than 5%, or less than 2%, preferably not at all (0%). Such antibodies may be referred to herein as "type 2 specific" or "type 2 only" antibodies. Exemplary such antibodies in the form of VHH antibodies are 57 (#11), 41 (#12), 171 (#14), and 29 (#17), as shown in Tables 1, 2, 3, and 4, respectively.

[0124] The comparison of the inhibition of type 1 and type 2 PRRSV can be easily performed using appropriate assays. For example, using the same concentration of test antibody or binding protein, keeping the assay conditions the same, one assay is performed using type 1 PRRSV and another assay is performed using type 2 PRRSV. Appropriate controls for evaluating such inhibition are also described elsewhere herein.

[0125] In certain embodiments, the antibody of the present invention has an IC of 350 μg / ml or less, 300 μg / ml or less, 280 μg / ml or less, 260 μg / ml or less, 240 μg / ml or less, 220 μg / ml or less, 200 μg / ml or less, 190 μg / ml or less, 180 μg / ml or less, 170 μg / ml or less, 160 μg / ml or less, 150 μg / ml or less, 140 μg / ml or less, 130 μg / ml or less, 120 μg / ml or less, 110 μg / ml or less, 100 μg / ml or less, 90 μg / ml or less, or 80 μg / ml or less 50 (e.g., for inhibiting PRRSV1 infection of host cells, e.g., PAM). In some embodiments, the IC 50 is 80 - 350, 300, 250 or 200 μg / ml, or 80 - 160 μg / ml, or 80 - 120 μg / ml, or 100 - 200 μg / ml, or 100 - 160 μg / ml, or 100 - 120 μg / ml. Specific exemplary IC 50 values are also shown in the examples.

[0126] In certain embodiments, the antibody of the present invention has an IC of 300 μg / ml or less, 280 μg / ml or less, 260 μg / ml or less, 240 μg / ml or less, 220 μg / ml or less, 210 μg / ml or less, 200 μg / ml or less, 180 μg / ml or less, 170 μg / ml or less, 160 μg / ml or less, 150 μg / ml or less, 140 μg / ml or less, 130 μg / ml or less, 120 μg / ml or less, 110 μg / ml or less or 100 μg / ml or less 50 (e.g., for inhibiting PRRSV2 infection of host cells, e.g., PAM). In some embodiments, the IC 50 is 100 or 150 or 200 - 300 μg / ml, or 200 - 260 μg / ml, or 200 - 220 μg / ml, or 220 - 300 μg / ml, or 220 - 260 μg / ml, or 220 - 240 μg / ml. Specific exemplary IC 50 values are also shown in the examples.

[0127] The above - mentioned preferred IC 50The value is preferably determined by a suitable viral infectivity assay, such as those determined in the above or the Examples section.

[0128] The bispecific antibodies described herein exhibit good ability to inhibit type 2 PRRSV infection, but it is generally observed that the inhibition of type 2 PRRSV infection is not as complete or as high as the level observed for the inhibition of type 1 PRRSV infection. Without wishing to be bound by theory, there may be multiple epitopes on CD163 involved in type 2 infection. Thus, in a preferred embodiment of the invention, for example, the bispecific antibodies and type 2 specific antibodies described herein can be used in combination. Such combinations can be particularly useful when the treatment or prevention of type 2 PRRSV infection is needed or desired.

[0129] In an alternative embodiment of the invention, the binding proteins or antibodies of the invention can be used to reduce or prevent the risk of PRRSV infection.

[0130] Preferably, the above capabilities and characteristics are observed at measurable or significant levels, more preferably at statistically significant levels, when compared to appropriate control levels. Appropriate significance levels are discussed elsewhere in this document. More preferably, one or more of the above capabilities and characteristics are observed at levels that are measurably or more preferably significantly (preferably statistically significantly) superior to the capabilities observed for antibodies of the prior art.

[0131] In any statistical analysis referred to herein, preferably, the statistically significant difference relative to the relevant control or other comparative entity or measurement has a p-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 can be used.

[0132] In some embodiments, the binding protein or antibody of the invention has one or more, preferably two or more, or three or more, most preferably all of the functional characteristics described herein, particularly the preferred functional characteristics.

[0133] When used throughout the application, the terms "a" and "an" are used in the sense of referring to "at least one", "at least a first", "one or more", or "a plurality" of components or steps, unless the upper limit is specifically stated thereafter. Thus, as used herein, the term "antibody" means "at least a primary antibody".

[0134] Furthermore, when the terms "comprising", "comprises", "having" or "has", 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.

[0135] A nucleic acid molecule comprising a nucleotide sequence encoding the binding protein or antibody of the invention or a portion or fragment thereof as defined herein, or a nucleic acid molecule substantially homologous thereto, forms a further aspect of the invention.

[0136] Preferred nucleic acid molecules are those encoding the VHH antibody or VH region or domain of the invention (e.g., those encoding SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57 or 65). Other preferred nucleic acid molecules are those encoding a set of three CDR sequences defined by any one of Tables A, B, C, D, E, F, G, H or I. Preferred such nucleic acid molecules also encode appropriate framework regions, e.g., FR1, FR2, FR3 and FR4 regions, preferably a set of FR sequences defined by any one of Tables A, B, C, D, E, F, G, H or I.

[0137] In other embodiments, preferred nucleic acid molecules are those encoding the VHH antibodies or VH regions or domains of the invention (e.g., those encoding SEQ ID NO: 83, 91, 99 or 107). Other preferred nucleic acid molecules are those encoding a set of three CDR sequences defined by any one of Tables 1, 2, 3 or 4. Preferred such nucleic acid molecules also encode appropriate framework regions, e.g., FR1, FR2, FR3 and FR4 regions, preferably a set of FR sequences defined by any one of Tables 1, 2, 3 or 4 (e.g., the type 2 specific antibodies of the invention).

[0138] As used herein in the context of amino acid or nucleic acid sequences, the term "substantially identical" includes sequences having at least 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. Thus, substantially identical sequences of the invention include single or multiple base or amino acid changes (additions, substitutions, insertions or deletions) to the sequences of the invention. At the amino acid level, preferred substantially identical sequences include up to 5, e.g., just 1, 2, 3, 4 or 5, preferably 1, 2, 3 or 4, preferably 1, 2 or 3, more preferably 1 or 2 amino acid changes in one or more of the framework regions and / or one or more of the CDRs that make up the sequences of the invention. The foregoing changes can be in conservative or non-conservative amino acids. Preferably, the foregoing changes are substitutions, preferably conservative amino acid substitutions.

[0139] 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 that is substantially homologous thereto compared to the number of amino acid substitutions that can optionally be made in the sequence. It is substantially homologous to a longer starting sequence. For example, in certain embodiments, a sequence that is substantially homologous to a starting VH CDR1 sequence according to the present invention, for example, in some embodiments, a starting VH CDR1 sequence that can be 5 amino acid residues in length, preferably has 1 or 2 (more preferably 1) 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 according to the length of a given starting CDR sequence. For example, different numbers of amino acid changes may be present depending on the length of a given starting CDR sequence to achieve a particular % sequence identity in the CDR, e.g., at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0140] Using routine methods in the art such as alanine scanning mutagenesis and / or analysis of the crystal structure of an antigen-antibody complex, it is possible to determine which amino acid residues of the CDR do not contribute or contribute significantly to antigen binding, and thus are good candidates for changes or substitutions in embodiments of the invention that involve substantially homologous sequences.

[0141] Once one or more amino acid additions, deletions, substitutions, or insertions in the amino acid sequence of a parent antibody for forming a new antibody are identified, the aforementioned parent antibody is one of the antibodies of the invention as defined elsewhere herein, and testing the resulting new antibody to identify an antibody that binds to CD163 according to the present invention can be done using techniques routine in the art. Using such methods, multiple new antibodies can be formed and all of them can be tested for their ability to bind to CD163. Preferably, the aforementioned addition, deletion, substitution or insertion of one or more amino acids occurs in one or more of the CDR domains.

[0142] For example, the aforementioned operation can be conveniently carried out by genetic engineering at the nucleic acid level such that a nucleic acid molecule encoding an appropriate binding protein and its domain is modified such that the amino acid sequence of the resulting expressed protein is then modified in an appropriate manner. Testing the ability of one or more of the modified antibodies that bind to CD163 is well known in the art and can be done by any suitable method described. Suitable methods are also described elsewhere in this specification and in the Examples section.

[0143] New antibodies produced, obtained, or obtainable by these methods form a further aspect of the present invention.

[0144] The term "substantially homologous" also includes alterations or chemical equivalents of the amino acid and nucleotide sequences of the proteins or nucleic acid molecules of the present invention that perform substantially the same function as the proteins or nucleic acid molecules of the present invention in substantially the same manner. For example, any substantially homologous antibody must retain the ability to bind to CD163 as described above. Preferably, any substantially homologous antibody must retain one or more (or all) of the functional abilities of the starting antibody.

[0145] Preferably, any substantially homologous antibody must 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., one or more of the various antibodies of the invention (e.g., each of VHH antibodies 49 (#18), 47 (#19), 48 (#20), 76 (#2), 77 (#16), 78 (#8), 150 (#15), 70 (#23), or 144 (#1) shown in Tables A, B, C, D, E, F, G, H, G, and I) and must retain the ability to bind to the same epitope. Thus, preferably, any substantially homologous antibody must retain the ability to compete with one or more of the various antibodies of the invention (e.g., each of VHH antibodies 49 (#18), 47 (#19), 48 (#20), 76 (#2), 77 (#16), 78 (#8), 150 (#15), 70 (#23), or 144 (#1) shown in Tables A, B, C, D, E, F, G, H, G, and I) for binding to CD163 in a suitable assay.

[0146] In other embodiments, any substantially homologous antibody must 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 one or more of the antibodies of the invention described herein or the VH (VHH) domain of the invention, e.g., one or more of the various type 2 antibodies of the invention (e.g., each of VHH antibodies 57 (#11), 41 (#12), 171 (#14), or 29 (#17) shown in Tables 1, 2, 3, and 4) and must retain the ability to bind to the same epitope. Thus, preferably, any substantially homologous antibody must retain the ability to compete with one or more of the various type 2 antibodies of the invention (e.g., each of VHH antibodies 57 (#11), 41 (#12), 171 (#14), or 29 (#17) shown in Tables 1, 2, 3, and 4, e.g., the type 2 specific antibodies of the invention) for binding to CD163.

[0147] Binding to the same epitope / antigen is well known in the art and can be readily tested by methods described, for example, using binding assays, such as competitive assays, or by analysis of the crystal structure of the antigen - antibody complex. Retention of other functional properties is also well known in the art and can be readily tested by methods described or methods described herein.

[0148] Thus, one of ordinary skill in the art will understand that binding assays, such as the competitive assays or ELISA assays described elsewhere herein, can be used to test whether any antibody, e.g., a "substantially identical" antibody, has the same binding specificity, e.g., binds to the same epitope as the antibodies and antibody fragments of the invention, or has the same or equivalent affinity. The BIAcore assay can also be readily used to establish whether an antibody, e.g., a "substantially identical" antibody, can bind to CD163. One of ordinary skill in the art will recognize other suitable methods and variations.

[0149] As outlined below, a competitive binding assay can be used to test whether an antibody, e.g., a "substantially identical" 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 as shown in the various sequence listings herein, or has the ability to compete with one or more of the various antibodies of the invention as shown in the various sequence listings herein. The method described below is only one example of a suitable competitive assay. One of ordinary skill in the art will recognize other suitable methods and variations.

[0150] Exemplary competitive assays involve evaluating the binding of various effective concentrations of the antibody of the present invention to CD163 in the presence of various concentrations of a test antibody (e.g., a substantially homologous antibody). Next, the amount of inhibition of binding induced by the test antibody can be evaluated. A test antibody that shows an increase in competition with the antibody of the present invention with increasing concentration (i.e., an increase in the concentration of the test antibody results in a corresponding decrease in the amount of the antibody of the present invention that binds to CD163) is evidence of binding to substantially the same epitope. Preferably, the test antibody substantially reduces the amount of the antibody of the present invention that binds to CD163. Preferably, the test antibody reduces the amount of the antibody of the present invention that binds to CD163 by at least about 95%. ELISA and flow cytometry assays can be used to evaluate inhibition of binding in such competitive assays, although other suitable techniques will be known to those of skill in the art.

[0151] An antibody of the present invention (e.g., a VHH antibody 49 (#18), 47 (#19), 48 (#20), 76 (#2), 77 (#16), 78 (#8), 150 (#15), 70 (#23), or 144 (#1) as shown in Tables A, B, C, D, E, F, G, H, and I, respectively) that has the ability to specifically bind to substantially the same (or the same) epitope of CD163 or an overlapping epitope of CD163, or an antibody (monoclonal antibody) that has the ability to compete with one or more of the various antibodies of the present invention (e.g., a VHH antibody 49 (#18), 47 (#19), 48 (#20), 76 (#2), 77 (#16), 78 (#8), 150 (#15), 70 (#23), or 144 (#1) as shown in Tables A, B, C, D, E, F, G, H, and I, respectively) is a further embodiment of the present invention.

[0152] In another embodiment, an antibody that has the ability to specifically bind to a substantially identical (or the same) epitope of CD163 or an overlapping epitope of CD163 recognized by an antibody of the invention (e.g., VHH antibody 57 (#11), 41 (#12), 171 (#14), or 29 (#17) as shown in Tables 1, 2, 3, and 4, respectively), or an antibody (monoclonal antibody) that has the ability to compete with one or more of the various antibodies of the invention (e.g., VHH antibody 57 (#11), 41 (#12), 171 (#14), or 29 (#17), e.g., a type 2 specific antibody of the invention as shown in Tables 1, 2, 3, and 4, respectively) is a further embodiment of the invention. In some embodiments, a preferred such antibody is VHH antibody 171 (#14) that includes SEQ ID NO: 99 (or the three CDR sequences related to the foregoing sequences) outlined in Table 3.

[0153] As used herein, the term "competing antibody" refers to an antibody that binds to an epitope that is substantially or essentially the same as, or the same as, a "reference antibody." "Competing antibodies" include antibodies having overlapping epitope specificities. Thus, a competing antibody can effectively compete with a reference antibody for binding to 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.

[0154] As used herein, a "reference antibody" is an antibody that can bind to CD163 according to the invention, preferably has a VH domain as defined herein, more preferably has a VH domain or is a VHH antibody that includes SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, or 65 (or the three CDR sequences related to the foregoing sequences) outlined in Tables A, B, C, D, E, F, G, H, or I.

[0155] As used herein, other "reference antibodies" are antibodies that can bind to CD163 according to the present invention, preferably having a VH domain as defined herein, more preferably having a VH domain or a VHH antibody comprising SEQ ID NO: 83, 91, 99 or 107 (or the three related CDR sequences of the foregoing sequences) outlined in Tables 1, 2, 3 or 4 (e.g., type 2 specific antibodies of the present invention). In some embodiments, a preferred reference antibody is a VHH antibody comprising SEQ ID NO: 99 (or the three related CDR sequences of the foregoing sequence) outlined in Table 3.

[0156] The identification of one or more competing antibodies or antibodies that bind to the same epitope is currently a straightforward technical matter, and reference antibodies as outlined in the Sequence Listing of this specification are provided. Since the identification of competing antibodies or antibodies that bind to the same epitope can be determined in comparison to the reference antibody, it will be understood that it is not necessary in any way to actually determine the epitope to which one or both antibodies bind in order to identify competing antibodies or antibodies that bind to the same epitope. However, if desired, epitope mapping can be performed using standard techniques.

[0157] Analysis of the crystal structure of the antigen-antibody complex between the SRCR5 domain of porcine CD163 described in SEQ ID NO: 115 and the VHH antibody 171 (#14), i.e., VHH 014 (2D01) having the amino acid sequence (SEQ ID NO: 17' or 99) shown in Table 3, has been performed to determine the region (epitope) within CD163 to which this antibody binds (see Figure 6). The residues on porcine CD163 that contribute to antigen binding have been identified as S507, E509, L526, and L527 of porcine CD163 (see the Uniprot Q2VL90 sequence described in SEQ ID NO: 116). The crystal structure shows that S507 and E509 interact with L104 of VHH 014 (2D01), L526 interacts with Y59 of VHH 014 (2D01), and L527 interacts with D62 of VHH 014 (2D01).

[0158] Epitopes of CD163 Accordingly, a further aspect provides an antibody (or binding protein) comprising an antigen-binding domain that binds or specifically binds to porcine CD163, wherein said antibody (antigen-binding domain) binds to an epitope in the SRCR5 domain of porcine CD163 that comprises the amino acids S507, E509, L526 and L527 of SEQ ID NO: 116, or corresponding residues in an alternative CD163 sequence, e.g., a CD163 sequence from another species (or is defined thereby).

[0159] Alternatively, a further aspect provides an antibody (or binding protein) comprising an antigen-binding domain that binds or specifically binds to porcine CD163, wherein said antibody (antigen-binding domain) binds to an epitope in the SRCR5 domain of porcine CD163 that comprises the amino acids S32, E34, L51 and L52 of SEQ ID NO: 115, or corresponding residues in an alternative CD163 sequence, e.g., a CD163 sequence from another species (or is defined thereby). The relevant residues are underlined in SEQ ID NO: 115 below. [Sequence Listing 1] JPEG0007702952000001.jpg13170

[0160] In particular, the interaction between the CDR2 of the VHH and the residues L526 and L527 (SEQ ID NO: 116) of porcine CD163 appears to be important for the antigen-antibody (antigen-binding domain) interaction. Accordingly, a further aspect of the invention provides an antibody (or binding protein) comprising an antigen-binding domain that binds or specifically binds to porcine CD163, wherein said antibody (antigen-binding domain) binds to an epitope in the SRCR5 domain of porcine CD163 that comprises the amino acids L526 and L527 of SEQ ID NO: 116, or corresponding residues in an alternative CD163 sequence, e.g., a CD163 sequence from another species (defined thereby).

[0161] In other embodiments, the present invention provides an antibody (or binding protein) comprising an antigen-binding domain that binds or specifically binds to porcine CD163, and the aforementioned antibody (antigen-binding domain) binds to an epitope in the SRCR5 domain of porcine CD163 that comprises the amino acids L526, L527, and S507, or L526, L527, and E509, or the amino acids L526, L527, S507, and E509, or corresponding residues in an alternative CD163 sequence, e.g., a CD163 sequence from another species (or is defined thereby).

[0162] In other embodiments, the aforementioned antibody (or binding protein) binds to an epitope in the SRCR5 domain of porcine CD163 that comprises one, two, three, or all of the residues S507, E509, L526, and L527 of SEQ ID NO: 116, or corresponding residues in an alternative CD163 sequence, e.g., a CD163 sequence from another species (or is defined thereby). In other words, at least one amino acid of the epitope on CD163 bound by the antibody (or binding protein) of the present invention comprises S507, E509, L526, or L527 of SEQ ID NO: 116, or corresponding residues in an alternative CD163 sequence, e.g., a CD163 sequence from another species. Such antibodies can be considered examples of antibodies that bind to overlapping epitopes.

[0163] Alternatively, the aforementioned antibody (or binding protein) binds to an epitope in the SRCR5 domain of porcine CD163 that comprises one, two, three, or all of the residues S32, E34, L51, and L52 of SEQ ID NO: 115, or corresponding residues in an alternative CD163 sequence, e.g., a CD163 sequence from another species (or is defined thereby). In other words, at least one amino acid of the epitope on CD163 bound by the antibody (or binding protein) of the present invention comprises S32, E34, L51, or L52 of SEQ ID NO: 115, or corresponding residues in an alternative CD163 sequence, e.g., a CD163 sequence from another species. Such antibodies can be considered examples of antibodies that bind to overlapping epitopes.

[0164] As far as the inventors are aware, monoclonal antibodies that can bind or specifically bind to an epitope in porcine CD163, particularly the SRCR5 domain of porcine CD163, and that can inhibit or reduce type 2 PRRSV infection are not described in the art in either a form that only inhibits or reduces type 2 PRRSV infection or a form in which the aforementioned antibodies can inhibit or reduce type 1 and type 2 PRRSV infection.

[0165] Accordingly, the individual monoclonal antibodies described herein are unusual and advantageous. Further, as noted above, the inventors believe that they have identified an epitope on porcine CD163 that is important for type 2 PRRSV infection and is thus generally a target for antibodies and binding proteins to reduce or inhibit PRRSV infection. Further, it should be noted that the residues on porcine CD163 identified herein as part of the epitope are located in a region of CD163 that is different from those previously identified as potentially important for PRRSV infection. For example, a previous report, e.g., Ma et al., 2017 (Am. Soc. For Microbiology, 91(3):e01897-16), identified residue R561 in the SRCR5 domain of CD163 as important for type 1 PRRSV infection. This residue is found in loop 5-6 of porcine CD163 located between residues Phe544 and Arg570 of CD163. In other reports, the ligand-binding pocket (LBP) of CD163 located between residues S487 and G499 of CD163 has also been speculated to be an important region for PRRSV infection. None of the four residues identified as part of the epitope in the current study are present in these regions.

[0166] Therefore, the present invention is considered to have identified novel epitopes in different parts of the SRCR5 region of porcine CD163 that are important for PRRSV infection, particularly type 2 PRRSV infection, and antibodies (or binding proteins) that bind to these epitopes or overlapping epitopes are particularly preferred. As described above, antibody 171 (#14) shown in Table 3 has been shown to bind to this epitope. In the first experiment using competitive binding assays, it has been shown that at least antibodies 57 (#11), 70 (#23), 144 (#1), and 150 (#15) can bind to the same or overlapping epitopes.

[0167] Paratope on the antibody Two L residues of CD163, L526 and L527, have been shown to interact with Y59 and D62 of the YYAD motif found in CDR2 of VHH antibody 171 (#14), i.e., VHH 014 (2D01). This VHH antibody has been shown to have an inhibitory effect on type 2 PRRSV infection or reduce infection. Note that the sequence YYAD or a sequence very similar to the sequence YYAD is found in the equivalent or corresponding region of CDR2 of all VHH antibodies described herein. All VHH antibodies described herein have been shown to have an inhibitory effect on type 2 PRRSV infection or reduce infection. Therefore, this VH CDR2 region appears to be an important feature of antibodies (e.g., VHH antibodies) that have the ability to inhibit or reduce type 2 PRRSV infection.

[0168] Therefore, preferred antibodies (or binding proteins) of the present invention include a VH CDR2 containing CDR2, particularly an amino acid sequence YAD or YAE, preferably XYAD or XYAE, wherein X can be any amino acid, preferably Y, L, P, N, F, or R, more preferably Y, F, L, N, or R, or Y, P, or L, most preferably Y. In other embodiments, the sequence can include YAN or XYAN as an alternative to YAD or YAE.

[0169] In embodiments, the X residue is located at position 59 of SEQ ID NO: 17' or 99 of VHH 014 (2D01) shown in Table 3, i.e., VHH antibody 171 (#14), or at the corresponding position in VH CDR2 of an alternative antibody (or VHH). Alternatively, the X residue is located at position 10 of SEQ ID NO: 19' or 101 (CDR2) of VHH 014 (2D01) shown in Table 3, i.e., VHH antibody 171 (#14), or at the corresponding position in VH CDR2 of an alternative antibody (or VHH), which can be, for example, at position 8 or 9 of the CDR2 region of other VHH antibodies described herein. The positions of the other residues in the XYAD or XYAE or XYAN motif can be determined accordingly with reference to these positions.

[0170] Two residues S507 and E509 of CD163 have been shown to interact with L104 of the CDR3 of VHH antibody 171 (#14), i.e., VHH 014 (2D01). This VHH antibody has been shown to have an inhibitory effect on type 2 PRRSV infection or to reduce the infection. Thus, this VH CDR3 residue (or the corresponding residue in other antibodies, such as VHH antibodies) can be an important residue in an antibody (such as a VHH antibody) having the ability to inhibit or reduce type 2 PRRSV infection.

[0171] Thus, in some embodiments, the antibody (or binding protein) of the invention comprises a VH CDR3 comprising an amino acid residue L at position 104 of SEQ ID NO: 17' or 99 of VHH 014 (2D01) shown in Table 3, i.e., VHH antibody 171 (#14), or at the corresponding position in VH CDR3 of an alternative antibody (or VHH). Alternatively, the L residue is located at position 6 of SEQ ID NO: 20' or 102 of VHH 014 (2D01) shown in Table 3, i.e., VHH antibody 171 (#14), or at the corresponding position in VH CDR3 of an alternative antibody (or VHH).

[0172] In some embodiments, the above L residue in CDR3 is present in addition to the above YAD or YAE or YAN sequence in CDR2, preferably XYAD or XYAE or XYAN, where X can be any amino acid, preferably Y, L, P, N, F, or R, more preferably Y, F, L, N or R, or Y, P or L, and most preferably Y.

[0173] In embodiments of the invention where substantially identical sequences are provided, in some embodiments, the residues YAD, YAE or YAN, or XYAD, XYAE or XYAN as defined above are maintained or present, and the variations occur outside of these residues.

[0174] Substantially identical sequences of the proteins of the invention include conservative amino acid substitutions, or changes that do not affect, for example, the VH, VL or CDR domains of an antibody, such as the addition of a tag sequence, a toxin or other component, an antibody that does not contribute to antigen binding, or a change for converting a binding protein, antibody molecule or fragment of one type or format to a binding protein, antibody molecule or fragment of another type or format (e.g., conversion from VHH to Fab or scFv or full antibody or vice versa), or conversion of an antibody molecule to an antibody molecule of a particular class or subclass of antibody molecule (e.g., conversion of an antibody molecule to IgG or its subclass, e.g., IgG2), but are not limited thereto.

[0175] As used herein, "conservative amino acid substitution" refers to an amino acid residue that has been replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged 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 other instances, families of amino acid residues can be grouped based on hydrophobic or hydrophilic side groups.

[0176] Identity can be assessed in any convenient way. However, to determine the degree of identity between sequences, computer programs that perform multiple alignments of sequences, such as Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994), are useful. If desired, the ClustalW algorithm can be used together 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, such that the highest order match between the two sequences is obtained and at least 50% of the full length of one of the sequences is involved in the alignment. Other methods that can be used to align sequences are the alignment method of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443, 1970) as modified by Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482, 1981), such that the highest order match between the two sequences is obtained and the number of identical amino acids between the two sequences is determined. Other methods for calculating the percentage of identity between two amino acid sequences are generally technically recognized and include, for example, 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).

[0177] Generally, such calculations are performed using computer programs. Programs for comparing and aligning array pairs, such as ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444-2448, 1988, Pearson, Methods in Enzymology, 183:63-98, 1990) and gapped BLAST (Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithies, Nucleic Acids Res., 12:387, 1984) are also useful for this purpose. Further, the Dali server of the European Bioinformatics Institute provides structure-based alignment 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).

[0178] By providing a reference point, sequences according to the present 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 from the GENESTREAM network server on the Internet, IGH, Montpellier, France).

[0179] As used herein, the terms "antibody" and "immunoglobulin" broadly refer to any immunological binding agent that contains 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 antibodies. Depending on the type of the constant domain of the heavy chain, all 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, IgG4, etc. The heavy chain constant domains corresponding to 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.

[0180] Generally, when whole antibodies rather than antigen-binding regions are used in the present invention, IgG is preferred because they are the most common antibodies in physiological situations and are the easiest to produce in the laboratory.

[0181] The "light chains" of mammalian antibodies are assigned to one of two distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains and some of the amino acids within the framework regions of the variable domains.

[0182] As used herein, the term "heavy chain complementarity determining region" ("heavy chain CDR") refers to the hypervariable regions within the variable domain (V H domain) of an antibody molecule or within a VHH antibody molecule. The variable domain of the heavy chain has three CDRs called heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, from the amino terminus to the carboxy terminus. The variable domain of the heavy chain also has four framework regions (FR1, FR2, FR3, and FR4 from the amino terminus to the carboxy terminus). These framework regions separate the CDRs.

[0183] As used herein, the term "heavy chain variable region" (V H domain) refers to the variable region of the heavy chain of an antibody molecule.

[0184] As used herein, the term "light chain complementarity determining region" ("light chain CDR") refers to the hypervariable regions within the variable region (V L domain) of the light chain of an antibody molecule. The variable region of the light chain has three CDRs called light chain CDR1, light chain CDR2, and light chain CDR3 from the amino terminus to the carboxy terminus. The variable region of the light chain also has four framework regions (FR1, FR2, FR3, and FR4 from the amino terminus to the carboxy terminus). These framework regions separate the CDRs.

[0185] As used herein, the term "light chain variable region" (V L domain) refers to the variable region of the light chain of an antibody molecule.

[0186] As will be understood by those skilled in the art, the immunological binding reagents encompassed by the term "antibody" include all antibodies and antigen-binding fragments thereof, including intact antibodies, dimeric, trimeric, and multimeric antibodies, bispecific antibodies, chimeric antibodies, recombinant antibodies, and modified antibodies, and fragments thereof, or extend to them.

[0187] Accordingly, the term "antibody" is used to refer to any antibody-like molecule having an antigen-binding region, and this term includes Fab’, Fab, F(ab’)2, single domain antibodies (DAB), TandAb dimers, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, biobodies, tribodies (scFv-Fab fusions, bispecific or trispecific respectively); sc-diabodies; kappa (lambda) bodies (scFv-CL fusions); BiTE (bispecific T cell engager, scFv-scFv tandem that attracts T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small molecule immune protein, a type of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds stabilized diabody "dual affinity retargeting"); antibody fragments containing antigen-binding domains such as small antibody mimetics containing one or more CDRs, etc.

[0188] Techniques for preparing and using various antibody-based constructs and fragments are well known in the art.

[0189] Antibodies can be fragmented using conventional techniques. For example, F(ab’)2 fragments can be produced by treating an antibody with pepsin. The resulting F(ab’)2 fragments can be treated to reduce disulfide bridges to 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 in the art and are described.

[0190] In all embodiments of the present invention, single domain antibodies (also referred to as VHH antibodies, sdAbs, DABs, dAbs, nanobodies, camelid antibodies, vNAR (shark) antibodies, VH antibodies or VL antibodies), in particular VHH antibodies, nanobodies, camel antibodies, and vNAR (shark) antibodies are preferred. Such antibodies comprise a single monomeric variable antibody domain, usually a VH domain, capable of binding to an antigen (although single VL domains with the ability to bind to an antigen have been described and can be used). Thus, in some such preferred embodiments, the antibody (or antigen-binding domain) of the present invention comprises one (or a single) heavy chain variable region (VH or VHH), although in some embodiments, several of these individual heavy chain variable regions, whether having the same or different sequences, can be present together in the same construct or molecule.

[0191] Such antibodies are well known in the art and can be obtained or prepared using standard techniques that have been described. For example, such antibodies can be obtained by immunizing a suitable animal, such as a camelid animal like a llama, or a shark, with the desired antigen, then cloning the VH domain of the antibodies produced into a suitable expression vector and selecting for binders. Libraries of VH domains (e.g., phage display libraries of human VH domains) are also available or can be generated and then screened.

[0192] Due to their relatively small size, single-domain antibodies can have a relatively short half-life, e.g., a relatively short plasma half-life. Thus, such antibodies are sometimes modified to extend or prolong their half-life. Techniques for doing this are well-known and described in the art, and any of these can be used. Examples include conjugating or fusing the antibody to albumin (or another protein or entity that itself has a long (or longer) half-life), or conjugating or fusing the antibody to a protein or entity that can interact with another protein or entity that itself has a long (or longer) half-life, or conjugating or fusing the antibody to PEG (or another polymer), or conjugating or fusing the antibody to an antibody that binds to FcRn, or to another protein or entity.

[0193] In certain embodiments, the antibodies or antibody fragments of the 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. Further, the antibody or antibody fragment can comprise all or a portion, or a portion thereof, of a kappa light chain constant region or a lambda light chain constant region. All or part of such constant regions can be naturally occurring or can be wholly or partially synthetic. Appropriate sequences of such constant regions are well-known and described in the art. When the complete complement of constant regions from the heavy and light chains is included in the antibodies of the invention, such antibodies are typically referred to herein as "full-length" antibodies or "whole" antibodies. In some embodiments, IgG2 antibodies are preferred.

[0194] In other embodiments, it is preferred that there is no constant region, such as a heavy or light chain constant region. For example, the variable domain or the heavy chain variable domain (VH) is the only part of the antibody present.

[0195] Antibodies or antibody fragments can be produced naturally or can be produced wholly or partly synthetically.

[0196] Many antibodies or antibody fragments contain an antibody light chain variable region (V L ) containing three CDR domains and an antibody heavy chain variable region (V H ) containing three CDR domains. The aforementioned VL and VH generally form the antigen-binding site.

[0197] However, it is well described in the art that the presence of three CDRs from the light chain variable domain of an antibody and three CDRs from the heavy chain variable domain is not necessarily required for antigen binding. Thus, constructs smaller than the above classical antibody fragments are known to be effective.

[0198] For example, camelid antibodies have a broad antigen-binding repertoire but lack a light chain. Also, results using single domain antibodies containing only the VH domain or only the 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. Thus, three CDRs can effectively bind to an antigen, and such single domain antibodies (e.g., VHH antibodies, sdAbs, DABs, dAbs, nanobodies, camelid antibodies, vNAR (shark) antibodies, VH antibodies or VL antibodies, particularly VHH antibodies, nanobodies, camelid antibodies, and vNAR (shark) antibodies) are exemplified herein and this type of antibody is preferred (e.g., VHH antibodies).

[0199] The antibodies, binding proteins, and nucleic acid molecules of the present invention are generally "isolated" or "purified" molecules, provided that they are distinguishable from any such components that may be present in situ within the body of a human or animal (e.g., a camelid) or in a tissue sample derived from the body of a human or animal (e.g., a camelid). However, these sequences may correspond to or be substantially homologous to sequences found in the body of a human or animal (e.g., a camelid). Thus, as used herein with respect to nucleic acid molecules or sequences and proteins or polypeptides, such as antibodies, the terms "isolated" or "purified" refer to such molecules when isolated, purified, or substantially free from their natural environment, e.g., when isolated or purified from the body of a human or animal (if they actually occur naturally), or when produced by a technical process, i.e., including molecules produced recombinantly and synthetically.

[0200] It should be noted that antibodies and the like of the present invention do not exist in nature and, in that respect, are artificial constructs in that they do not correspond to molecules that occur in nature. For example, preferred antibodies are single domain antibodies that can be produced by engineering or recombination, and even in species that produce such antibodies naturally, such as camelids, such species do not produce antibodies against CD163, particularly porcine CD163, unless experimentally induced to do so, e.g., by immunization. In other words, antibodies and the like of the present invention are non-natural.

[0201] As used herein, the term "fragment" refers to a biologically relevant fragment, e.g., a fragment that contributes to antigen binding, e.g., a fragment that forms part of an antigen binding site and / or contributes to the functional properties of a CD163 antibody. Certain preferred fragments comprise or consist of the variable heavy domain (V H domain or three VH CDRs) of the antibodies of the present invention.

[0202] Those skilled in the art will understand that the proteins and polypeptides of the present invention, such as heavy and light chain CDRs, heavy and light chain variable regions, antibodies and antibody fragments, are well-known in the art and can be prepared by any of several described methods, but most preferably are prepared using recombinant methods.

[0203] The nucleic acid fragments encoding the heavy and light chain variable regions of the antibodies of the present invention can be obtained or produced, if necessary, by any suitable method, for example, by cloning or synthesis.

[0204] Once the nucleic acid fragments encoding the heavy and / or light chain variable regions of the antibodies of the present invention are obtained, these fragments can be further manipulated by standard recombinant DNA techniques to, for example, convert the variable region fragments into full-length antibody molecules having appropriate constant region domains, or into specific formats of antibody fragments discussed elsewhere herein, such as single domain antibodies like VHH, Fab fragments, scFv fragments, etc. Typically, or as part of this further manipulation procedure, the nucleic acid fragments encoding the antibody molecules of the present invention are generally incorporated into one or more suitable expression vectors to facilitate the production of the antibodies of the present invention or, for example, to facilitate selection or screening, such as by incorporation into a phage display vector.

[0205] Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), provided that the vector is compatible with the host cell in which it is to be used. The expression vector is "suitable for transformation of the host cell", which means that the expression vector contains the nucleic acid molecule of the present invention and regulatory sequences operably linked to the nucleic acid molecule, which are selected based on the host cell to be used for expression. Being operably linked is intended to mean that the nucleic acid is linked to the regulatory sequences in a manner that enables expression of the nucleic acid.

[0206] Accordingly, the present invention contemplates an expression vector, for example, a recombinant expression vector containing or comprising the nucleic acid molecule of the present invention or a fragment thereof, as well as regulatory sequences necessary for transcription and translation of the protein sequence encoded by the nucleic acid molecule of the present invention.

[0207] 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 Harbour Press, Cold Spring Harbour, NY, 1989) and other laboratory texts.

[0208] Suitable host cells include a wide variety of eukaryotic and prokaryotic host cells. For example, the protein of the present invention can be expressed in yeast cells or mammalian cells. Furthermore, the protein of the present invention can be expressed in prokaryotic cells such as Escherichia coli.

[0209] The protein of the present invention can also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis.

[0210] Yet another aspect provides an expression construct or vector or system (e.g., a virus or bacterium or other expression construct, vector or system) that includes one or more of the nucleic acid fragments or segments or molecules of the present invention. Preferably, the expression construct or vector or system is recombinant. Preferably, the aforementioned construct or vector or system further includes regulatory sequences necessary for the transcription and translation of the protein sequence encoded by the nucleic acid molecule of the present invention. Preferred constructs and the like enable long-term or sustained expression of the antibody (or binding protein) of the present invention within a host target species, e.g., within a pig. Such expression can be transient, e.g., by episomal integration, or more permanent, e.g., via genomic integration, provided that sufficient levels and lengths of expression are achieved such that a therapeutic or biological effect is observed.

[0211] Yet another aspect provides a host cell (e.g., a mammalian or bacterial or yeast host cell) or virus that includes one or more of the expression constructs or vectors of the present invention. Also provided are host cells or viruses that include one or more of the nucleic acid molecules of the present invention. Host cells (e.g., mammalian host cells or bacterial host cells, or yeast host cells) or viruses that express the antibody (or binding protein) of the present invention form yet another aspect.

[0212] Such an expression construct or vector or system, or host cell or virus, or other nucleic acid product or fragment encoding the antibody (or binding protein) of the present invention can be administered to a subject as a therapeutic agent to enable the production of the antibody (or binding protein) of the present invention in situ within the subject, thereby exerting their therapeutic effects.

[0213] Yet another aspect of the invention provides a method for producing (or manufacturing) an antibody of the invention, which includes the step of culturing a host cell of the invention. A preferred method includes (i) culturing a host cell comprising one or more of the recombinant expression vectors of the invention or one or more of the nucleic acid sequences under conditions suitable for the expression of the encoded antibody or protein, and optionally (ii) isolating or obtaining the antibody or protein from the host cell or the growth medium / supernatant. Such a production (or manufacturing) method may also include a step of purifying the antibody or 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.

[0214] In embodiments, when the antibody or protein of the invention is composed of multiple polypeptide chains (e.g., specific fragments such as Fab fragments or whole antibodies), all polypeptides are preferably expressed in host cells from either the same or different expression vectors, so that the complete protein of the invention, such as an antibody protein, can assemble within the host cell and be isolated or purified therefrom.

[0215] In another aspect, the invention provides a method for binding CD163, which includes contacting a composition comprising CD163 with an antibody of the invention.

[0216] In yet another aspect, the invention provides a method for detecting CD163, which includes contacting a composition suspected of comprising CD163 with an antibody of the invention under conditions effective to permit the formation of a CD163 / antibody complex and detecting such a formed complex.

[0217] Compositions comprising at least a primary antibody (or binding protein) of the present invention constitute a further aspect of the present invention. Preparations (compositions) comprising one or more antibodies of the present invention mixed with suitable diluents, carriers or excipients constitute a preferred embodiment of the present invention. Such preparations can be for pharmaceutical use, for example, veterinary use, and thus 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, excipients and carriers are known to those skilled in the art.

[0218] The compositions according to the present invention can be presented, for example, in a form suitable for oral, nasal, parenteral, intravenous, topical or rectal administration.

[0219] The active compounds (for example, the antibodies of the present invention) defined herein can be presented in conventional pharmacological dosage forms such as tablets, coated tablets, nasal drops, solutions, emulsions, liposome formulations, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as well as ordinary production methods can be used for the preparation of these forms.

[0220] Injection solutions can be produced in a conventional manner, for example, by the addition of preservatives such as p-hydroxybenzoates or stabilizers such as EDTA. Next, the solution can be filled into injection vials or ampoules.

[0221] Suitable dosage units can be determined by those skilled in the art.

[0222] The pharmaceutical composition may further comprise additional active ingredients (for example, as described elsewhere herein) in relation to a co-administration regimen or a combination regimen.

[0223] A further aspect of the invention provides an anti-CD163 antibody (or binding protein) as defined herein for use in therapy, in particular for the treatment or prevention of any disease or condition associated with CD163, or for which CD163 plays a role, e.g. a causative role (e.g. a wholly or partially causative role), or an essential role. For example, the anti-CD163 antibodies of the invention can be used for the treatment or prevention of any infectious disease caused by a virus or other pathogen, wherein said infectious disease is associated with CD163, or for which CD163 plays a role, e.g. a causative role (e.g. a wholly or partially causative role), or an essential role. Put another way, according to the invention, the anti-CD163 antibody (or binding protein) targets and can inhibit or reduce the function of CD163, in particular CD163 expressed on or within PAM or other CD163-positive cells. Thus, the anti-CD163 antibody (or binding protein) as defined herein can be used for the treatment or prevention of any disease or condition for which inhibition of CD163 or blockade or reduction of CD163 function is useful.

[0224] Preferred embodiments provide the anti-CD163 antibody (or binding protein) of the invention for use in the treatment or prevention of a porcine infectious disease, preferably a porcine viral infectious disease. Particularly preferred is the treatment or prevention of PRRSV infection. In embodiments where the pig is being treated, the anti-CD163 antibody (or binding protein) of the invention is typically an anti-porcine CD163 antibody (or binding protein).

[0225] CD163 is likely to be a receptor for all PRRS viruses. However, as described elsewhere in this document, there are two serotypes of PRRSV, type 1 and type 2 viruses. Type 1 and type 2 viruses are phenotypically similar at some levels but differ in viral genotype. The antibodies (or binding proteins) of the present invention can be used to treat or prevent type 1 and / or type 2 PRRS viruses, such as type 1 and type 2 PRRS viruses. In other embodiments, the present invention provides an antibody (or binding protein) having the ability to inhibit type 2 PRRSV infection, preferably specifically inhibit type 2 PRRSV infection, and thus can be used to treat or prevent type 2 PRRS virus.

[0226] Administration of the binding protein or antibody in the treatment methods and uses of the present invention is carried out in a pharmaceutically, therapeutically, or physiologically effective amount to a subject (animal, or mammal, such as a pig) in need of treatment. Thus, the foregoing methods and uses may include an additional step of identifying a subject in need of treatment.

[0227] Treatment of a disease or condition according to the present invention (e.g., treatment of an existing disease) includes cure of the foregoing disease or condition, or reduction or alleviation of the disease (e.g., reduction in the severity of the disease) or symptoms of the disease.

[0228] The treatment methods and uses of the present invention are suitable for the prevention of diseases as well as the active treatment of diseases (e.g., treatment of existing diseases). Thus, prophylactic and meta-infection protective (treatment in the face of the occurrence of a disease, e.g., for the purpose of preventing the spread of an infectious disease to animals in close contact and / or at significant risk, treatment of a group of subjects after diagnosis of an infectious disease, and / or treatment of clinical diseases in a part of the group) treatments are also encompassed by the present invention. For this reason in the methods and uses of the present invention, treatment also includes prevention of onset, meta-protection or prevention where appropriate.

[0229] Such prophylactic (or protective) aspects can be conveniently carried out on healthy or normal or at-risk subjects and can include both complete prophylaxis and significant prophylaxis. Similarly, significant prophylaxis includes scenarios where the severity or symptoms of the disease are reduced (e.g., measurably or substantially reduced) compared to the severity or symptoms expected if no treatment is given.

[0230] For example, the clinical symptoms of PRRS infection include fetal resorption, stillbirth and late abortion in pregnant or gilts, respiratory diseases and syndromes, such as dyspnea, in all pigs, especially in young and piglets. Other symptoms include anorexia (often leading to a decrease in growth rate), fever, lethargy, dyspnea, reproductive disorders, diarrhea (especially in piglets), and central nervous system (CNS) signs. Subjects with PRRSV infection are also reported to be prone to endemic diseases such as meningitis, Glasser's disease, exudative dermatitis, sarcoptic mange, and bacterial bronchopneumonia (Diseases of Swine, Eleventh Edition, Editor(s): Jeffrey J. Zimmerman, Locke A. Karriker, Alejandro Ramirez, Kent J. Schwartz, Gregory W. Stevenson, Jianqiang Zhang, First published: 29 March 2019), and such diseases are usually managed using antibacterial agents such as antibiotics. As a result, the present invention has a role in reducing the use of antibacterial products on farms.

[0231] Accordingly, the antibodies of the present invention can be used to treat or prevent clinical diseases or symptoms, such as those associated with PRRSV infection or downstream endemic diseases as outlined above, or to prevent the circulation (e.g., circulating virus particles) or infection (e.g., primary infection) or new infection (e.g., secondary or subsequent infection) of a virus, such as PRRSV, e.g., PRRSV infection (e.g., primary PRRSV infection) or new PRRSV infection (e.g., secondary or subsequent PRRSV infection).

[0232] Thus, preferred subjects for treatment according to the present invention include all types of pigs (sometimes also referred to as swine), for example, any pig, swine, or pig species, including all ages and breeds of pigs, provided that they are susceptible to, or prone to infection by, the pathogens defined herein, particularly PRRSV. Piglets, especially young piglets or piglets born to 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 finishing pigs (e.g., pigs up to slaughter age), especially growing pigs. Piglets before weaning, piglets up to 4 weeks of age (especially piglets of infected sows where infection can be transmitted through the mammary secretions of the infected sow), are also preferred subjects for treatment, as are sows and pregnant sows.

[0233] In some embodiments, for example, where prevention is concerned, the subject is at risk of being affected by the disease or condition in question, e.g., at risk of developing a disease upon infection with a pathogen or virus such as those described above (e.g., PRRSV). Such a subject can be a healthy subject, or a subject showing no symptoms of the disease, or any other suitable "at-risk" subject. In another embodiment, the subject is a subject having, or suspected of having (or developing), or potentially having (or developing), the disease or condition in question as described above.

[0234] Alternatively, the present invention provides a method for treating or preventing a disease or condition associated with CD163, or where CD163 plays a role, e.g., a causative role (e.g., wholly or in part causative) 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) of the present invention as defined herein. Suitable diseases or conditions are described elsewhere in this specification.

[0235] Treatment or prevention of infectious diseases in pigs, preferably treatment or prevention of viral infections in pigs, is preferred. Particularly preferred is the treatment or prevention of PRRSV infection, for example, treatment or prevention of type 1 and / or type 2 PRRS virus infection, such as treatment or prevention of type 1 and type 2 PRRS virus infection, or treatment or prevention of type 2 PRRS virus infection (e.g., specifically treating or preventing).

[0236] Accordingly, a further aspect provides a method for treating or preventing PRRSV infection in pigs, for example, treating or preventing type 1 and / or type 2 PRRS virus 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) for use in such methods are described herein.

[0237] The embodiments of the therapeutic use of the invention described herein apply, with the necessary modifications, to this aspect of the invention.

[0238] The therapeutically effective amount is determined based on clinical evaluation and can be easily monitored.

[0239] Furthermore, alternatively, the present invention provides for the use of an anti-CD163 antibody (or binding protein) of the present invention, as defined herein, in the manufacture of a medicament for use in therapy, such as a monoclonal antibody of the present invention. Preferred therapeutic uses are, in particular, for use in the treatment or prevention of any disease or condition associated with CD163, or where CD163 plays a role, such as a causative role (e.g., a wholly or partially causative role) or an essential role, as described elsewhere herein. For example, the anti-CD163 antibody (or binding protein) of the present invention can be used in the treatment or prevention of any infectious disease caused by a virus or other pathogen, where the aforementioned infectious disease is associated with CD163, or where CD163 plays a role, such as a causative role (e.g., a wholly or partially causative role), or an essential role. Put another way, according to the present invention, the 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 antibody (or binding protein) as defined herein can be used in the treatment or prevention of any disease or condition where inhibition of CD163 or blockade or reduction of CD163 function is useful.

[0240] Preferred embodiments provide for the use of an anti-CD163 antibody (or binding protein) of the present invention in the manufacture of a medicament for use in the treatment or prevention of a porcine infectious disease, preferably a porcine viral infectious disease. Particularly preferred is the treatment or prevention of PRRSV infection, such as type 1 and / or type 2 PRRS virus infection, e.g., treating or preventing type 1 and type 2 PRRS virus infection, or treating or preventing (e.g., specifically treating or preventing) type 2 PRRS virus infection.

[0241] Accordingly, yet a further aspect provides for the use of a monoclonal antibody that binds to porcine CD163 in the manufacture of a medicament for use in the treatment or prevention of PRRS virus infection, preferably type 1 and / or type 2 PRRS virus infection in pigs. Suitable CD163 antibodies (or binding proteins) for such use are described herein.

[0242] The embodiments of the therapeutic use of the invention described herein apply, with the necessary modifications, to this aspect of the invention.

[0243] In some embodiments, the antibodies of the invention can be used in combination.

[0244] Respectively, as shown in Tables A, B, C, D, E, F, G, H, and I, any combination of VHH antibodies 49 (#18), 47 (#19), 48 (#20), 76 (#2), 77 (#16), 78 (#8), 150 (#15), 70 (#23) and 144 (#1) can be used. Thus, two, three, four, five, six, seven, eight or all nine of these can be combined, preferably two or three, more preferably two.

[0245] Preferred combinations include the following: 70 (#23) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 144 (#1) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 150 (#15) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), for example, 150 (#15) and 47 (#19), preferably one of them, 76 (#2) and one or more of 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 77 (#16) and one or more of 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 49 (#18) and one or more of 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 47 (#19) and one or more of 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 48 (#20) and one or more of 70 (#23), 144 (#1), or 150 (#15), preferably one of them, or 78 (#8) and one or more of 70 (#23), 144 (#1), or 150 (#15), preferably one of them.

[0246] Preferred combinations include the following: 76 (#2) and 150 (#15), 76 (#2) and 77 (#16), 76 (#2) and 48 (#20), 150 (#15) and 77 (#16), 150 (#15) and 48 (#20), 77 (#16) and 48 (#20), or 150 (#15) and 77 (#16) and 48 (#20).

[0247] As shown in Tables 1, 2, 3, and 4 respectively, any combination of VHH antibodies 57 (#11), 41 (#12), 171 (#14), and 29 (#17) can be used in the present invention. Thus, two, three, or all four of these can be combined, preferably two or three, more preferably two.

[0248] Preferred combinations include the following: 57 (#11) and one or more of 41 (#12) or 29 (#17), for example one or more of 57 (#11) and 29 (#17), preferably one of them, 171 (#14) and one or more of 41 (#12) or 29 (#17), preferably one of them, 41 (#12) and one or more of 57 (#11) or 171 (#14), preferably one of them, or 29 (#17) and one or more of 57 (#11) or 171 (#14), preferably one of them.

[0249] Other combinations include the following: Respectively, as shown in Tables A, B, C, D, E, F, G, H, and I, one or more of VHH antibodies 49 (#18), 47 (#19), 48 (#20), 76 (#2), 77 (#16), 78 (#8), 150 (#15), 70 (#23), or 144 (#1), preferably one of them, and respectively, as shown in Tables 1, 2, 3, and 4, one or more of VHH antibodies 57 (#11), 41 (#12), 171 (#14), or 29 (#17), preferably one of them, in any combination.

[0250] Preferred combinations include the following: 150 (#15) and 29 (#17), 47 (#19) and 29 (#17), or 144 (#1) and 29 (#17).

[0251] Other preferred combinations include the following: 57 (#11) and one or more of 41 (#12) or 29 (#17), preferably one of them, 57 (#11) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 171 (#14) and one or more of 41 (#12) or 29 (#17), preferably one of them, 171 (#14) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 70 (#23) and one or more of 41 (#12) or 29 (#17), preferably one of them, 70 (#23) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 144 (#1) and one or more of 41 (#12) or 29 (#17), preferably one of them, 144 (#1) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 150 (#15) and one or more of 41 (#12) or 29 (#17), preferably one of them, or 150 (#15) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them.

[0252] Alternative preferred combinations include: 41 (#12) and one or more of 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 41 (#12) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 29 (#17) and one or more of 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one of them, or 29 (#17) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them.

[0253] Preferred alternative combinations include the following: 76 (#2) and one or more of 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 76 (#2) and one or more of 41 (#12) or 29 (#17), preferably one of them, 77 (#16) and one or more of 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 77 (#16) and one or more of 41 (#12) or 29 (#17), preferably one of them, 49 (#18) and one or more of 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 49 (#18) and one or more of 41 (#12) or 29 (#17), preferably one of them, 47 (#19) and one or more of 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 47 (#19) and one or more of 41 (#12) or 29 (#17), preferably one of them, 48 (#20) and one or more of 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 48 (#20) and one or more of 41 (#12) or 29 (#17), preferably one of them, 78 (#8) and one or more of 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one of them, or 78 (#8) and one or more of 41 (#12) or 29 (#17), preferably one of them.

[0254] Preferred combinations include the following: 57 (#11) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 171 (#14) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 70 (#23) and one or more of 41 (#12) or 29 (#17), preferably one of them, 144 (#1) and one or more, preferably one of 41 (#12) or 29 (#17), 150 (#15) and one or more of 41 (#12) or 29 (#17), preferably one of them, 41 (#12) and one or more of 70 (#23), 144 (#1), or 150 (#15), preferably one of them, 41 (#12) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 29 (#17) and one or more of 70 (#23), 144 (#1) or 150 (#15), preferably one of them, 29 (#17) and one or more of 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19), or 48 (#20), preferably one of them, 76 (#2) and one or more of 57 (#11) or 171 (#14), preferably one of them, 76 (#2) and one or more of 41 (#12) or 29 (#17), preferably one of them, 77 (#16) and one or more of 57 (#11) or 171 (#14), preferably one of them, 77 (#16) and one or more of 41 (#12) or 29 (#17), preferably one of them. 49 (#18) and one or more of 57 (#11) or 171 (#14), preferably one of them, 49 (#18) and one or more of 41 (#12) or 29 (#17), preferably one of them, 47 (#19) and one or more of 57 (#11) or 171 (#14), preferably one of them, 47 (#19) and one or more of 41 (#12) or 29 (#17), preferably one of them, 48 (#20) and one or more of 57 (#11) or 171 (#14), preferably one of them, 48 (#20) and one or more of 41 (#12) or 29 (#17), preferably one of them, 78 (#8), and one or more of 57 (#11) or 171 (#14), preferably one of them, or 78 (#8), and one or more of 41 (#12) or 29 (#17), preferably one of them.

[0255] In all of the above combinations, if necessary, antibodies having three CDRs shown in Tables A - I and 1 - 4 can also be used.

[0256] Preferred combinations are those that result in an improvement or increase in the therapeutic effect, preferably a significant improvement or increase, compared to any of the single active agent (monotherapy), or a single antibody, or the antibody of the present invention (e.g., VHH) 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.

[0257] In such combination therapies using two or more antibodies (or binding proteins) of the invention, the second (or subsequent) anti-CD163 antibody can be administered to a subject substantially simultaneously with the first anti-CD163 antibody of the invention, from a single pharmaceutical composition, or from two pharmaceutical compositions administered closely together (simultaneously or at similar times). Alternatively, the second (or subsequent) anti-CD163 antibody of the invention can be administered to the subject before or subsequent to the administration of the first anti-CD163 antibody of the invention. As used herein, "before or subsequent to" means "alternating", and thus the second antibody is administered to the subject at a time different from the administration of the first anti-CD163 antibody component. Generally, two (or more) components can be administered either with an effective time interval between them or together in order to allow each component to exert its respective therapeutic effect, i.e., they are administered in a "biologically effective amount" at a "biologically effective time interval" and as part of the same treatment regimen.

[0258] Combinations of the anti-CD163 antibodies (or binding proteins) of the invention can, where appropriate, be conveniently administered as part of the same molecule or construct, for example, using an artificial linker, for example, conjugated or linked. This mode of administration can be particularly appropriate for VHH antibodies (or other types of antibody molecules composed of a single polypeptide chain), the individual antibodies of which are conveniently linked by an appropriate peptide (or other) linker, for example, a non-natural peptide, or an artificial linker in a single polypeptide chain comprising a plurality of VHH (or other) antibodies, either of the VHH antibodies of the invention or in combination with other VHHs or other antibodies. In such embodiments, the agents are generally linked together using appropriate techniques, such as spacing, such that each component can exert its respective effect, for example, binding to CD163. For example, in embodiments where the anti-CD163 antibodies of the invention bind to different epitopes on CD163, such combinations of antibodies are preferred and the construct is appropriately designed such that the individual antibodies can bind to CD163, for example, its CD163 epitope.

[0259] Thus, in some embodiments, the anti-CD163 antibody (or binding protein) of the invention can be used as a single active agent in a therapeutic regimen (monotherapy), or a plurality of anti-CD163 antibodies of the invention can be used, for example, in the combinations described above. In some embodiments, the anti-CD163 antibody (or binding protein) (or suitable combination) of the invention can be used as a single active anti-CD163 agent or a single active anti-CD163 antibody in a therapeutic regimen, or they can be a single effective anti-PRRSV agent in a therapeutic regimen. However, in some embodiments, additional anti-CD163 agents or anti-PRRSV agents can be used.

[0260] Thus, the anti-CD163 binding protein or antibody (or suitable combination) of the invention can be combined with one or more additional (additional CD163 target or non-CD163 target) active agents, for example, at least a second therapeutic or biological agent, where the first is the anti-CD163 binding protein or antibody (or combination of such binding proteins or antibodies) of the invention.

[0261] The anti-CD163 antibody (or binding protein) (or suitable combination) of the invention can be combined with, for example, any other therapeutic agent useful for treating the disease in question described elsewhere herein, such as PRRSV.

[0262] In the case of such combination therapy, generally speaking, the second (non-anti-CD163 antibody of the present invention) agent may be administered to a subject substantially simultaneously with the anti-CD163 antibody of the present invention (or a combination of such antibodies), for example, from a single pharmaceutical composition or from two pharmaceutical compositions administered closely together (simultaneously or at similar times). Alternatively, the second (non-anti-CD163 antibody of the present invention) agent may be administered to the subject before or subsequent to the administration of the anti-CD163 antibody component of the present invention. As used herein, "before or subsequent to" means "alternately", and thus the second (non-anti-CD163 antibody of the present invention) agent is administered to the subject at a time different from the administration of the anti-CD163 antibody component. Generally, two (or more) components may be administered with an effective time interval between them or together in order to enable each of the two components to exert its therapeutic effect, i.e., the components are administered in a "biologically effective amount" at a "biologically effective time interval" and as part of the same treatment regimen.

[0263] The present invention further includes a kit comprising one or more of the antibodies or compositions of the present invention, or one or more of the nucleic acid molecules encoding the antibodies of the present invention, or one or more recombinant expression vectors comprising the nucleic acid sequences of the present invention, or one or more host cells or viruses comprising the recombinant expression vectors or nucleic acid sequences of the present invention. Preferably, the aforementioned kit is for use in the methods and uses described herein, for example, for use in the treatment methods described herein. Preferably, the aforementioned kit includes instructions for use of the kit components. Preferably, the aforementioned kit is for treating a disease or condition described elsewhere herein and optionally includes instructions regarding the use of the kit components for treating such disease or condition. Equivalent embodiments with the binding proteins of the present invention are also provided.

[0264] The antibodies (or binding proteins) of the invention as defined herein can also be used as molecular tools for in vitro or in vivo applications and assays. Since antibodies (and some binding proteins) have antigen-binding sites, they can function as members of specific binding pairs, and these molecules can be used in any assay that requires a specific binding pair member.

[0265] Accordingly, yet another aspect of the invention provides a reagent comprising an antibody (or binding protein) of the invention as defined herein and the use of such an antibody (or binding protein) as a molecular tool in, for example, in vitro or in vivo assays.

[0266] Table of amino acid sequences and their sequence identifiers (SEQ ID NOs) disclosed herein In this specification, in accordance with the convention in this technical field, all amino acid sequences are described from the N-terminus to the C-terminus. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10]

Table 11

Table 12

Table 13

Table 14

[0267] Next, with reference to the following drawings, the present invention will be further described in the following non-limiting examples.

Brief Description of the Drawings

[0268]

Figure 1

[0269]

Figure 2

[0270]

Figure 3

[0271]

Figure 4

[0272]

Figure 5

[0273]

Figure 6

[0274] The right panel is an example of electron density (2m|Fo|-D|Fc|) contoured at 1 sigma level. The protein chain and individual amino acids are drawn as lines. The data shows clear interactions between tyrosine (Y59) and aspartic acid (D62) residues within the CDR2 domain of VHH14, and pairs of leucine residues (Leu526, Leu527) within the porcine SRCR5 domain. Additional interactions are also seen between leucine 104 of VHH14(CDR3), pairs of amino acids within SRCR5 serine 507 (S507), and glutamic acid 509 (E509). The crystal structure was refined to 2.0 - 2.3 Å. Leu(L)52 in this figure corresponds to Leu(L)527 of the full-length CD163 molecule shown in SEQ ID NO: 116. Leu(L)51 in this figure corresponds to Leu(L)526 of the full-length CD163 molecule shown in SEQ ID NO: 116. Ser(S)32 in this figure corresponds to Ser(S)507 of the full-length CD163 molecule shown in SEQ ID NO: 116. Glu(E)34 in this figure corresponds to Glu(E)509 of the full-length CD163 molecule shown in SEQ ID NO: 116. Asp(D)62, Tyr(Y)59 and Leu(L)104 of VHH02D01 are shown in SEQ ID NO: 17’ or 99 (Table 3).

Example

[0275] Example 1: Immunization, library generation, screening and clone selection Materials and Methods Immunization Single-domain antibodies were obtained from llamas immunized with recombinant proteins. The llamas were injected with porcine CD163Fc fusion antigen preparations prepared with incomplete Freund's adjuvant (pCD163-SRCR1-9-huFc and pCD163-SRCR4-7-huFc). The animals were immunized with six subcutaneous injections at one-week intervals (two injections at 100 μg / dose, followed by four injections at 50 μg / dose). One week after the last booster immunization, sera were collected and antibody titers against pCD163-SRCR1-9-huFc and pCD163-SRCR4-7-huFc were determined by ELISA.

[0276] In this ELISA, 96-well plates (Maxisorp; Nunc) were coated with recombinant protein. After blocking and addition of diluted serum samples, the presence of anti-pCD163 antibodies was demonstrated using mouse anti-lama IgG2 / 3 (EMD millipore; catalog number MAC131), followed by anti-mouse immunoglobulin peroxidase conjugate (JIR, catalog number 715-035-150).

[0277] Library Construction RNA was extracted from PBMCs of three immunized llamas (400 ml each). 40 μg of RNA was used for cDNA synthesis using random primers. This cDNA was used in a primary PCR amplification using untagged primers annealing to the leader sequence and hinge CH1 region, followed by a secondary PCR amplification to introduce restriction endonuclease sites for cloning the VHH gene into the pDCL1 phagemid vector. The library was electroporated into TG1 E. coli cells and a cell glycerol stock of the immune library was stored at -80 °C (FL1158 and FL1159).

[0278] Selection Phage production from the llama VHH library pool was used in two consecutive rounds of phage display selection using the pCD163 recombinant protein or porcine alveolar macrophages (pPAM). The selection rounds for the recombinant protein were performed using 10 μg / ml of pCD163-SRCR1-9-huFc or pCD163-SRCR4-7-huFc at pH 7.4 (PBS buffer), non-specific phages were washed, followed by specific phage elution (total elution) with trypsin. The selection rounds for pPAM were performed using 5×10⁶ cells at pH 7.4 (PBS buffer), non-specific phages were washed, followed by specific phage elution (total elution) with trypsin. Serial dilutions of the eluted phages were made and used to infect exponentially growing TG1. The infected TG1 was plated on LBCarb100Glu2% plates and the enrichment value was calculated in the background (without the antigen for selection).

[0279] ELISA screening Individual clones from the second round of selection condition output were selected into a 96-well Master Plate and tested by binding ELISA for their ability to bind to pCD163-SRCR4-7-huFc or pCD163-SRCR5-6-huFc proteins at pH 7.0 as periplasmic extracts (PE). For the PE-binding ELISA, MaxiSorp™ high protein-binding capacity 96-well ELISA plates were coated overnight at 4 °C with 1 μg / ml of pCD163-SRCR4-7-huFc protein diluted in PBS. The next day, the plates were washed three times with PBS Tween 0.05% (pH 7.4) and blocked for 1 hour at room temperature with 250 μl / well of 4% Marvel / CPA or 4% Marvel / PBS. After blocking, the plates were washed three times with PBS Tween 0.05% (pH 7.4) and incubated for 1 hour at room temperature with shaking with 20 μl of PE + 80 μl in 1% Marvel / PBS (pH 7.4) per well. The plates were washed three times with PBS Tween 0.05% (pH 7.4) and incubated for 1 hour at room temperature with shaking with 100 μl of anti-c-Myc antibody (Roche; catalog number 11667203001), followed by secondary antibody DAM-HRP (JIR; catalog number 715-035-150) in 1% Marvel / PBS (pH 7.4). The plates were washed three times with PBS Tween 0.05% (pH 7.4), substrate solution (TMB solution) was added to the plates. The reaction was stopped with H2SO4 and the plates were read at 450 nm with a plate reader.

[0280] Cell screening (FACS): Periplasmic extracts (PE) from the selected clones were incubated for 30 minutes at room temperature (RT) with stirring with an anti-c-Myc antibody (Roche; catalog number 11667203001) specific for the c-myc tag present in the soluble VHH. The mixture (PE + anti-c-Myc antibody) was added to pPAM or pPAMΔ5 domain (cells lacking SRCR domain 5) and incubated for 60 minutes at 4 °C with gentle shaking.

[0281] The cells were washed three times with 150 μl / well of FACS buffer, incubated with 50 μl / well of secondary antibody GAM-APC for 30 minutes at 4°C with shaking, and protected from light.

[0282] The cells were washed three times with 150 μl / well of FACS buffer, resuspended in 75 μl / well of FACS buffer, and measured on a FACS machine (Attune(™) NxT) in the RL-1 channel (APC channel), and a total of 10,000 cells were acquired for each sample.

[0283] Sequencing Positive binders were sent for sequencing. Clones were classified by family according to different HCDR3 sequences.

[0284] Results and Discussion:

Table 15

Table 16

[0285] After immunizing llamas with recombinant CD163, phage clones were selected by two rounds of phage panning. Phage candidates were confirmed by binding to recombinant CD163 expression constructs (pCD163SRCR1-9-Fc or pCD163-SRCR4-7-Fc) by ELISA compared to control human IgG. As shown in Table 5, selective binding was demonstrated by several candidates to porcine CD163.

[0286] Clones were further selected for their ability to bind to native membrane-bound CD163 on isolated primary porcine alveolar macrophages prepared from cells recovered from bronchoalveolar lavage of donor animals (Burkard C., et al PLOS Pathogens 2017). Candidate phages demonstrated binding to native membrane-bound CD163 at both 4°C as well as at room temperature when the CD163 receptor is likely to be internalized by the endocytosis mechanism.

[0287] By selecting candidates that cannot bind to porcine alveolar macrophages isolated from pigs lacking this domain in the CD163 gene, all candidates were also evaluated for selective binding to the SRCR5 domain of CD163 (Burkard C., et al PLOS Pathogens 2017). All selected candidates showed preferential binding to the SRCR5 domain of porcine CD163 because they were unable to bind to PAM isolated from these animals.

[0288] Therefore, immunization of llamas with recombinant CD163 protein constructs led to the good isolation of phage antibody candidates that can bind to CD163 expressed in primary porcine alveolar macrophages and specifically target the SRCR5 domain, which is known to be essential for PRRS virus infection of these cells.

[0289] References: Burkard C, Lillico SG, Reid E, Jackson B, Mileham AJ, Ait-Ali T, et al. (2017) 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 Pathog 13(2):e1006206. doi:10.1371 / journal.ppat.1006206

[0290] Example 2: Determination of the affinity of anti-CD163 VHH antibodies for porcine CD163 Materials and methods: The affinity of binding of each of the identified VHH antibody candidates to porcine CD163 was determined by surface plasmon resonance.

[0291] Expression and purification of VHH candidate antibodies Synthetic genes encoding FLAG- and His-tagged VHH variable domains were purchased and reconstructed according to the manufacturer's instructions. Each DNA construct was digested with restriction enzymes, the inserts were gel-purified, and each variable domain insert was ligated to the mammalian expression vector pcDNA3.1. Twenty-three lead panel sequences were transfected into ExpiCHO-S cells using 40 μg of the total DNA plasmid construct. A total of 25 mL of cells were used for 8-day protein production (32 °C, 5% CO2). The produced VHH antibodies were captured from the clarified supernatant using a HisTrap HP 5 mL IMAC column (GE Healthcare, catalog number 17-5248-02) on an AKTA Pure 25F PLC system. The peak fractions of the eluted antibody were buffer-exchanged into 1× PBS pH 7.4 and concentrated using a 3 kDa MCO spin concentrator (Amicon, catalog number UFC900324). The purified protein was analyzed for the presence of the correct chain by analytical size exclusion chromatography (aSEC) and SDS-PAGE.

[0292] Determination of affinity To evaluate the affinity of the selected purified clones for pCD163, pCD163-SRCR1-9-huFc or pCD163-SRCR4-7-huFc proteins were coated on a CM5 sensor chip (GE Healthcare) by amine coupling. Surface plasmon resonance (SPR) (Biacore 3000, GE Healthcare) was used to determine the binding rate of the selected single-domain antibodies at pH 7.4. Approximately 2000 RU of pCD163-SRCR1-9-huFc or pCD163-SRCR4-7-huFc, 20 or 30 μg / ml of the protein in acetate buffer pH 5.0 or pH 5.5 were immobilized on the CM5 chip using standard amine coupling procedures. QC of the immobilization was performed using a commercially available anti-huFc antibody (JIR, catalog number 109-005-098) at a concentration of 30.0 μg / ml.

[0293] 1×HBS-EP pH 7.4 was used as the running buffer during the binding rate measurement. The purified VHH was injected at a flow rate of 30 μl / min for 120 seconds at 3-fold dilutions (300 nM, 100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.4 nM, 0 nM). The RU level was restored to the base level after regeneration with 10 μl of 10 mM NaOH / 1 M NaCl and 10 μl of 10 mM glycine pH 1.5 between samples. A 1:1 binding fitting with mass transfer was applied to the sample curves set using the simultaneous fitting option of BIAevaluation software to calculate the rate constants of the antibody-antigen interaction, including the association rate (ka), dissociation rate (kd), and affinity (KD). The residues were visually inspected, and the curves were removed from the fitting after considering the chi-squared value. In simultaneous fitting, at least four curves were considered.

[0294]

Table 17

[0295] As described above, phage candidates were expressed as VHH and purified. The characterization of each VHH that binds to either full-length or truncated CD163 was performed by surface plasmon resonance as described above. Both the association rate and dissociation rate were determined for each antibody, and the affinity determination was calculated. As seen in Table 7, the KD values of the antibody candidates were in the range of 1 - 75 nM. The relative affinities for either the full-length or truncated pCD163-SRCR4-7 constructs were approximately the same.

[0296] Example 3: Inhibition of Porcine Reproductive and Respiratory Syndrome (PRRS) Virus Infection of Primary Porcine Alveolar Macrophage Cells by Candidate CD163-Specific Candidate Antibodies Materials and Methods PRRS Virus Infection Protocol Reagents VHH candidate antibodies were aliquoted at a concentration of 1 mg / mL Control antibody: Primary antibody: anti-PRRS 1AC7, Ingenasa Secondary antibody: Goat anti-mouse IgG (H+L) Alexa Fluor Plus 488, ThermoFisher, A32723 Medium: Complete RPMI (10% FBS or 80% (high) pig serum, ultraglutamine, penicillin / streptomycin) PAM isolation: Porcine alveolar macrophage isolation was performed as described in Burkard et al PLoS Pathogens 2017. Virus isolate: Type 1 virus: BOR57 isolate (Roslin Institute, Edinburgh, UK) Type 2 virus: MN184 US strain (Han et al 2006) Infection protocol Day 1 - Cell seeding Porcine alveolar macrophage cells were seeded at 20 million cells per well in complete RPMI in 48-well plates and left overnight in a CO2 incubator. Day 2 - VHH treatment and infection challenge 1. Pretreatment (30 minutes before infection) a. Aspirate the medium from the cells b. Add 100 μL of medium to untreated non-infected controls and untreated infected controls c. Add 20 μL of PBS in 100 μL of medium to sham-treated infected controls d. Add the appropriate amount of VHH stock in 100 μL of medium to treated samples and infected samples e. Return the plates to the CO2 incubator for 30 minutes 2. Thaw the virus stock and sonicate for 15 seconds before use 3. Infection challenge (2 hours) a. Remove the medium from the cells and keep the VHH-containing medium for the overnight incubation step b. Add 100 μL of medium to untreated non-infected controls c. Add 10 μL of virus in 100 μL of medium to untreated infected controls d. Add 10 μL of virus and 20 μL of PBS to 100 μL of medium of the sham-treated infection control. e. Add an appropriate amount of VHH stock in 100 μL of medium and 10 μL of virus to the treated samples and the infected samples. f. Gently agitate the plate and return it to the CO2 incubator. g. Gently agitate the plate every 15 minutes for 2 hours. 4. Incubate overnight (15 hours) a. Aspirate the medium from the cells. b. Add 100 μL of medium to the untreated non-infected control and the untreated infected control. c. Add 20 μL of PBS in 100 μL of medium to the sham-treated infected control. d. Add the VHH-containing medium retained from the pretreatment step to the appropriate samples. e. Return the plate to the CO2 incubator for 15 hours. Day 3 - In-well fixation and staining protocol 5. Aspirate the medium from the cells. 6. Fix the cells with 4% formaldehyde / PBS++ (containing calcium and magnesium) solution at room temperature for 30 minutes. 7. Wash once with PBS++. 8. Permeabilize with Triton-X (1% in PBS++) at room temperature for 5 minutes. 9. Wash once with PBS++ or blocking solution (PBS++ / 5% FBS). 10. Block with blocking solution (PBS++ / 5% FBS) at room temperature for 20 minutes. 11. Add primary antibody anti-PRRS1AC7 at 1:5000 to all wells except the non-staining control and the secondary antibody-only control. 12. Incubate at room temperature for 1 hour. 13. Wash 3 times with PBS++. 14. Add secondary antibody goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 at 1:5000 to all wells except the non-staining control. 15. Incubate at room temperature for 45 minutes. 16. Wash three times with PBS++. 17. Add 300 μL of PBS++. 18. Use a wide-bore p200 pipette tip to scrape the cells, then use a normal p200 tip to scrape around the edge of the well, then use a p1000 to wash the surface of the well three times, and collect the cells and transfer them to a FAC tube. 19. Measurement was performed with Fortessa×20 (voltage: FSC = 418, SSC = 308 & 530 - 30 = 386)

[0297] Results and discussion: The ability of individual VHHs to block the infection of porcine alveolar macrophage (PAM) host cells by PRRS virus family members is described below. Using the above assay, the degree of virus infection, quantified by the ability to grow the virus, measured by FACS following a 17-hour infection cycle, was determined. The data presented are shown in Figures 1 and 2 and summarized in Table 8 below.

[0298]

Table 18

[0299] The data clearly show that VHHs can inhibit the productive infection of porcine alveolar macrophage cells by both PRRS type 1 (see also Figure 1) and type 2 (see also Figure 2) isotypes. VHHs showing activity in the infection assay can be divided into those that were effective against both type 1 and type 2 virus infections (VHHs 001, 002, 008, 015, 016, 018, 019, 020, and 023) and those that did not show inhibitory activity against type 1 PRRS virus infection but showed selective inhibitory activity against type 2 PRRS virus infection (VHHs 011, 012, 014, 017). The data clearly show that VHHs can inhibit the productive infection of porcine alveolar macrophage cells by both PRRS type 1 and type 2 isotypes.

[0300] Some VHHs showed inhibitory activity only in type 2 virus infection (VHHs 011, 012, 014, 017) in the concentration range up to 300 μg / mL (Figure 2). These candidate VHHs were specific to PRRS type 2 virus and showed no inhibitory activity in the same dose-response concentration range up to 100 μg / mL in the type 1 virus infection assay (Figure 3).

[0301] 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 J., Y. Wang, K. S. Faaberg. Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus Virus Research, 122(2006), pp. 175.

[0302] Example 4: Inhibition of porcine reproductive and respiratory (PRRS) virus infection of primary porcine alveolar macrophage cells by combinations of candidate CD163-specific candidate antibodies Materials and methods PRRS virus infection protocol Reagents VHH candidate antibodies were aliquoted at a concentration of 1 mg / mL Control antibody: Primary antibody: anti-PRRS 1AC7, Ingenasa Secondary antibody: goat anti-mouse IgG(H+L) Alexa Fluor Plus 488, ThermoFisher, A32723 Medium: Complete RPMI (10% FBS or 80% high pig serum, ultraglutamine, penicillin / streptomycin) PAM isolation: Porcine alveolar macrophage isolation was performed as described in Burkard et al PLoS Pathogens 2017. Virus isolates: Type 1 virus: BOR57 isolate (Roslin Institute, Edinburgh, UK) Type 2 virus: MN184 US strain (Han et al 2006) Infection protocol Day 1 - Seed cells Porcine alveolar macrophage cells were seeded at 20 million cells per well in complete RPMI in 48-well plates and left overnight in a CO2 incubator. Day 2 - VHH treatment and infection challenge 1. Pretreatment (30 minutes before infection) a. Aspirate the medium from the cells b. Add 100 μL of medium to untreated non-infected and untreated infected controls c. Add 20 μL of PBS to 100 μL of medium and add to sham-treated infected controls d. Add the appropriate amount of VHH stock in 100 μL of medium to treated samples and infected samples e. Return the plates to the CO2 incubator for 30 minutes 2. Thaw the virus stock and sonicate for 15 seconds before use 3. Infection challenge (2 hours) a. Remove the medium from the cells and keep the VHH-containing medium for the overnight incubation step b. Add 100 μL of medium to untreated non-infected controls c. Add 10 μL of virus to 100 μL of medium to untreated infected controls d. Add 10 μL of virus and 20 μL of PBS in 100 μL of medium to sham-treated infected controls e. Add an appropriate amount of the individual VHH stock solution (or combination of VHHs) and 10 μL of virus in 100 μL of medium to the treatment samples and the infected samples. f. Gently agitate the plate and return it to the CO2 incubator. g. Gently agitate the plate every 15 minutes for 2 hours. 4. Incubate overnight (15 hours). a. Aspirate the medium from the cells. b. Add 100 μL of medium to the untreated non-infected control and the untreated infected control. c. Add 20 μL of PBS in 100 μL of medium to the sham-treated infected control. d. Add the VHH-containing medium retained from the pretreatment step to the appropriate samples. e. Return the plate to the CO2 incubator for 15 hours. Day 3 - In-well fixation and staining protocol 5. Aspirate the medium from the cells. 6. Fix the cells in a 4% formaldehyde / PBS++ (containing calcium and magnesium) solution at room temperature for 30 minutes. 7. Wash once with PBS++. 8. Permeabilize with Triton-X (1% in PBS++) at room temperature for 5 minutes. 9. Wash once with PBS++ or a blocking solution (PBS++ / 5% FBS). 10. Block with a blocking solution (PBS++ / 5% FBS) at room temperature for 20 minutes. 11. Add the primary antibody anti-PRRS1AC7 at 1:5000 to all wells except the non-staining control and the secondary antibody-only control. 12. Incubate at room temperature for 1 hour. 13. Wash three times with PBS++. 14. Add the secondary antibody goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 at 1:5000 to all wells except the non-staining control. 15. Incubate at room temperature for 45 minutes. 16. Wash three times with PBS++. Add 17.300 μL of PBS++ 18. Use a wide-bore p200 pipette tip to scrape the cells, then use a normal p200 tip to scrape around the edge of the well, then use a p1000 to wash the surface of the well three times, and collect the cells and transfer them to a FAC tube. 19. Measurements were performed on a Fortessa × 20 (voltage: FSC = 418, SSC = 308 & B530 - 30 = 386)

[0303] Results and discussion: The ability of individual VHHs to block the infection of PAM host cells by PRRS virus family members is described below. Using the above assay, the degree of virus infection quantified by the ability to grow the virus, measured by FACS following a 17-hour infection cycle, was determined. The presented data are shown in Figures 4 and 5.

[0304] The combination of VHH15, 16, and 20 was used in an infection assay using BOR57 type 1 PRRS virus in the presence of 10% FBS-containing medium. Specific combinations of 100 μg each of VHH15 and VHH16, VHH15 and VHH20, VHH16 and VHH20, and a triple combination of 50 μg each of VHH15 + VHH16 + VHH20 were shown to reduce the likelihood of infection with type 1 PRRS virus to a very low level of infectivity (see Figure 4).

[0305] The potential of various pairwise combinations of VHHs was examined in an infection assay using BOR57 type 1 PRRS virus in the presence of medium containing 10% FBS. VHH02 (01B04) was used as a partner for VHH15 (02G01), VHH16 (02H11), and VHH20 (03H11) to evaluate the potential to block infection. Combinations were tested by increasing the concentration of each partner VHH. The data indicate that combinations of VHHs can block type 1 PRRS virus infection (see Figure 5).

[0306] The data indicate that, through combinations of VHHs, the inhibition of productive infection of porcine alveolar macrophage cells by PRRS type 1 isotypes can potentially be significantly enhanced compared to individual VHH candidates. The data suggest that combining individual VHHs may be able to enhance the potential for blocking infection by PRRS virus family members.

[0307] References: Burkard C., et al, supra; Han J., Y. Wang, K. S. Faaberg, supra.

[0308] Example 5: Determination of anti-CD163 VHH antibodies that bind to porcine CD163 SRCR5 Materials and Methods: The X-ray crystal structure of the porcine CD163 SRCR5 domain that binds to the VHH antibody candidate 2D01 (VHH14) was determined.

[0309] Expression and purification of the VHH14 antibody Synthetic genes encoding FLAG- and His-tagged VHH variable domains were purchased and reconstituted according to the manufacturer's instructions. 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 40 μg of the total DNA plasmid construct for 23 lead panel sequences. A total of 25 mL of cells were used for 8 days of protein production (32 °C, 5% CO2). The VHH antibodies produced were captured from the clarified supernatant using a HisTrap HP 5 mL IMAC column (GE Healthcare, catalog number 17-5248-02) on an AKTA Pure 25F PLC system. The peak fractions of the eluted antibody were buffer-exchanged into 1× PBS pH 7.4 and concentrated using a 3 kDa MCO spin concentrator (Amicon, catalog number UFC900324). The purified protein was analyzed for the presence of the correct chain by analytical size exclusion chromatography (aSEC) and SDS-PAGE.

[0310] Expression and Purification of Porcine SRCR5 Before transforming E. coli DH10Bac to produce recombinant Bacmid, a synthetic gene encoding the porcine CD163 SRCR5 region with a ^xHIS tag was subcloned into pTXBac1 (a proprietary vector). Recombinant Bacmid was used to transform Spodoptera frugiperda (Sf) cells to generate P1 virus clones. Cell and media samples from the P1 clones were checked periodically to examine protein expression and identify high-expression clones. High-expression clones were amplified by infecting Sf cells with the P1 virus stock to generate P2 virus stock, which was then used to express recombinant SRCR5 protein in Sf1 cells for 72 hours at a multiplicity of infection (MOI) of approximately 1.

[0311] Scale-up production was carried out in a 10 L culture of Sf cells. Optimal expression conditions were used as described above. After equilibrating the culture supernatant with phosphate-buffered saline (PBS) pH 7.5, it was purified using the HIS tag on IMAC according to the standard protocol. Samples were washed with PBS pH 7.5 and 0.1% Triton X-114 buffer. The protein was eluted with imidazole according to the manufacturer's instructions. Before further purification with cobalt resin, the eluted sample was buffer-exchanged with PBS pH 7.5. After washing using the imidazole shift (as described above), the sample was eluted. The resulting eluted sample was buffer-exchanged into 20 mM Tris-HCl, pH 7.4, 150 mM NaCl.

[0312] [Table 19]

[0313] Crystallization and X-ray Structure Determination Studies The CD163 SRCR5:VHH14 complex was prepared at a concentration of 11.84 mg / mL in PBS pH 7.4 buffer. Plate-like crystals were grown in 0.2 M NaCl, 0.1 M phosphate / citrate pH 4.5, 20% w / v PEG8000. After adding a cryo solution containing 0.14 M NaCl, 0.07 M phosphate / citrate buffer pH 4.5, 13.9% PEG8000 and 46% ethylene glycol, individual crystals were flash-frozen in liquid nitrogen.

[0314] Data were collected at 100 K on the BioMAX beamline (l = 0.97625 Å) at MAX IV in Sweden. The beamline was equipped with an Eiger16M hybrid pixel detector.

[0315] The structure was determined using Phaser software and two homologous proteins (PDB codes: 5DA4 and 5JFB), which were determined to be 2.4 Å and 2.0 Å respectively. 103 amino acids of the CD163 SRCR5 domain (bold in Table 9) and 122 amino acids of VHH014 (02D01) from residue 4 onwards marked in bold could be modeled (Table 9).

[0316] Results and discussion: From the structure of the CD163 SRCR5:VHH014 complex, as shown in Figure 6, a single monomeric SRCR5 domain (left side) interacting with a single monomeric VHH014 antibody (right side) is revealed. From the electron density map, specific interactions between residues of VHH014 and the identified amino acids within the CD163:SRCR5 domain become apparent. In particular, leucine 526 and leucine 527 within the porcine CD163 SRCR5 domain appear to form interactions with specific conserved residues according to the XYAD / E / N motif within the CDR2 of VHH14, a motif shared among all identified VHH candidates. VHH14 (02D01) is interesting because it does not inhibit infection by type 1 PRRS virus but can reduce infection by type 2 PRRS family viruses. The di-leucine motif identified in porcine CD163 SRCR5 may represent an important feature in the interaction between type 2 PRRS virus and SRCR5, as well as in the productive infection of porcine alveolar macrophages.

Claims

**Claim 1** A medicament for use in the treatment or prevention of PRRS virus infection in pigs, comprising a monoclonal antibody that binds to porcine CD163. **Claim 2** The medicament according to claim 1, wherein the antibody has the ability to bind to the SRCR5 domain of porcine CD163. **Claim 3** The medicament according to claim 1 or 2, wherein the antibody has the ability to inhibit type 1 and / or type 2 PRRSV infection. **Claim 4** A monoclonal antibody suitable for use according to any one of claims 1 to 3, comprising an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one variable heavy chain region comprising three complementarity-determining regions (CDRs), and the variable heavy chain 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), (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GEGAIKWTTLDAYDY (SEQ ID NO: 12). **Claim 5** A monoclonal antibody suitable for use according to any one of claims 1 to 3, comprising an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one variable heavy chain region comprising three complementarity-determining regions (CDRs), and the variable heavy chain region comprises (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of RYVMG (SEQ ID NO: 2), (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of GIAWSGRAPYADSVKG (SEQ ID NO: 3), (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GEGAIRWTTLDAYDY (SEQ ID NO: 4). **Claim 6** A monoclonal antibody suitable for use according to any one of claims 1 to 3, comprising an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one variable heavy chain region comprising three complementarity-determining regions (CDRs), and the variable heavy chain region comprises (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of RYVMG (SEQ ID NO: 18), (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of GIAWSGRAPYADSVKG (SEQ ID NO: 19), An antibody comprising a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GEGAILWTTPGAYNY (SEQ ID NO: 20). **Claim 7**: A monoclonal antibody suitable for use according to any one of claims 1 to 3, comprising an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the heavy chain variable region comprises: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of TYSMSG (SEQ ID NO: 26); (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AHRWSGSAYYAEDSVEG (SEQ ID NO: 27); (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GVGSAAQYRY (SEQ ID NO: 28). **Claim 8**: A monoclonal antibody suitable for use according to any one of claims 1 to 3, comprising an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the heavy chain variable region comprises: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of PGSMG (SEQ ID NO: 34); (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AHRWSGSAYYADYADSVEG (SEQ ID NO: 35); (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GVGSAAQYTY (SEQ ID NO: 36). **Claim 9**: A monoclonal antibody suitable for use according to any one of claims 1 to 3, comprising an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one heavy chain variable region comprising three complementarity-determining regions (CDRs), and the heavy chain variable region comprises: (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of TYSMSG (SEQ ID NO: 42); (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AHRWSGSAYYAEDSVEG (SEQ ID NO: 43); (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GVGSEAQYRY (SEQ ID NO: 44). **Claim 10** A monoclonal antibody suitable for use according to any one of claims 1 to 3, comprising an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region is (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of SYSNG (SEQ ID NO: 50), (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AITWNGYITNYADSVKG (SEQ ID NO: 51), (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of TTFSSTTSPI-SRTYNY (SEQ ID NO: 52), and the antibody.

11. A monoclonal antibody suitable for use according to any one of claims 1 to 3, comprising an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region is (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of TYAMG (SEQ ID NO: 58), (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of IISFGGTFYADSVKG (SEQ ID NO: 59), (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GRTLSKRADSYAS (SEQ ID NO: 60), and the antibody.

12. A monoclonal antibody suitable for use according to any one of claims 1 to 3, comprising an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region is (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of MYAMS (SEQ ID NO: 66), (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AINTSGRYSRYADSVKG (SEQ ID NO: 67), (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of TDKGNWALAMSYDY (SEQ ID NO: 68), and the antibody.

13. A monoclonal antibody suitable for use according to any one of claims 1 to 3, wherein the antibody is capable of specifically inhibiting type 2 PRRSV infection.

14. The antibody can specifically inhibit type 2 PRRSV infection, and comprises an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of VYGTG (SEQ ID NO: 84), and (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of GISGTTGSTLYADSVKG (SEQ ID NO: 85), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of GGRVYITTSWAY (SEQ ID NO: 86), and the antibody according to claim 13.

15. The antibody can specifically inhibit type 2 PRRSV infection, and comprises an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of RYAMG (SEQ ID NO: 92), and (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of AIAWSTGSTYYANSVKG (SEQ ID NO: 93), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of ETRYCSGFGCLDPRTYGS (SEQ ID NO: 94), and the antibody according to claim 13.

16. The antibody can specifically inhibit type 2 PRRSV infection, and comprises an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of TDTMA (SEQ ID NO: 100), and (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of GIGRSGGSIYYADA VKG (SEQ ID NO: 101), and (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of RQRIGLVVGALGYDY (SEQ ID NO: 102), and the antibody according to claim 13.

17. The antibody can specifically inhibit type 2 PRRSV infection, and comprises an antigen-binding domain that binds to the SRCR5 domain of porcine CD163, wherein the antigen-binding domain comprises at least one heavy-chain variable region comprising three complementarity-determining regions (CDRs), and the heavy-chain variable region (i) a variable heavy chain (VH) CDR1 comprising the amino acid sequence of DYTIG (SEQ ID NO: 108), (ii) a variable heavy chain (VH) CDR2 comprising the amino acid sequence of CINSITSYYADSVRKG (SEQ ID NO: 109), (iii) a variable heavy chain (VH) CDR3 comprising the amino acid sequence of DSGLFSSGSSCLKYRAMRFGGS (SEQ ID NO: 110), the antibody according to claim 13.

18. The monoclonal antibody suitable for use according to any one of claims 1 to 3, or the monoclonal antibody according to any one of claims 4 to 17, wherein the antibody is a single-domain antibody.

19. The monoclonal antibody suitable for use according to any one of claims 1 to 3, or the monoclonal antibody according to any one of claims 4 to 12 and 18, wherein the antibody can inhibit type 1 and type 2 PRRSV infections.

20. The antibody according to claim 19, wherein the antibody can inhibit the ability of type 2 PRRSV to infect host cells by at least 50%, and / or can inhibit the ability of type 1 PRRSV to infect host cells by at least 50%, at least 80% or at least 90%.

21. The monoclonal antibody suitable for use according to any one of claims 1 to 3, or the monoclonal antibody according to any one of claims 13 to 18, wherein the antibody can specifically inhibit type 2 PRRSV infection.

22. The antibody according to claim 21, wherein the antibody can inhibit the ability of type 2 PRRSV to infect host cells by at least 40%.

23. The antibody according to claim 21 or 22, wherein the antibody does not significantly inhibit the ability of type 1 PRRSV to infect host cells.

24. A monoclonal antibody that binds to the same epitope of porcine CD163 as the antibody according to any one of claims 4 to 17.

25. A combination of two or more antibodies according to any one of claims 4 to 17, or a combination of an antibody according to any one of claims 4 to 12 and an antibody according to any one of claims 14 to 17.

26. One or more nucleic acid molecules comprising a nucleotide sequence encoding the antibody according to any one of claims 4 to 24, or One or more expression vectors containing such nucleic acid molecules, or one or more host cells that contain or express the expression vector or nucleic acid molecule, or the antibody according to any one of claims 4 to 24.

27. A method for producing an antibody according to any one of claims 4 to 24, the method comprising: (i) culturing a host cell containing one or more of the expression vectors or one or more of the nucleic acid sequences as defined in claim 26 under conditions suitable for the expression of the encoded antibody; and optionally, (ii) isolating or obtaining the antibody expressed from the host cell or the growth medium / supernatant.

28. A composition comprising an antibody according to any one of claims 4 to 24, or a combination of antibodies according to claim 25, or one or more nucleic acid molecules or expression vectors according to claim 26.

29. The composition according to claim 28, wherein the composition is a pharmaceutically acceptable composition.

30. A medicament for use in therapy, the medicament comprising an antibody according to any one of claims 4 to 24, or a combination of antibodies according to claim 25, or one or more nucleic acid molecules or expression vectors according to claim 26.

31. Use of an antibody according to any one of claims 4 to 24, or a combination of antibodies according to claim 25, or one or more nucleic acid molecules or expression vectors according to claim 26, in the manufacture of a medicament or composition for use in the treatment or prevention of PRRS virus infection in pigs.

32. A medicament for use in a method for the treatment or prevention of PRRS virus infection in pigs, the medicament comprising an antibody according to any one of claims 4 to 24, or a combination of antibodies according to claim 25, or one or more nucleic acid molecules or expression vectors according to claim 26, the method comprising administering to a subject in need thereof a therapeutically effective amount of an antibody according to any one of claims 4 to 24, or a combination of antibodies according to claim 25, or one or more nucleic acid molecules or expression vectors according to claim 26.

33. The medicament according to claim 30 or 32, wherein the medicament is used for the treatment or prevention of type 1 and / or type 2 PRRSV infection.

34. The use according to claim 31, wherein the PRRS virus infection is type 1 and / or type 2 PRRSV infection.

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