Cd163 antibody or binding protein

Abstract

The present disclosure provides a monoclonal antibody that binds to porcine CD163 for use in the treatment or prevention of porcine reproductive and respiratory syndrome (PRRS) virus infection in a pig. Preferred antibodies comprise an antigen binding domain that binds to porcine CD163, the antigen binding domain comprising at least one heavy chain variable region comprising three complementarity determining regions (CDRs), wherein the heavy chain variable region comprises a variable heavy chain (VH) CDR2 comprising the amino acid sequence XYAD or XYAE or XYAN, wherein X can be any amino acid. Also provided are nucleic acid molecules, expression vectors, and compositions.

Description

Technical Field

[0001] This invention generally relates to binding proteins, particularly antibodies, that bind to CD163 (differentiation cluster 163), especially binding proteins and antibodies that bind to porcine CD163. Such anti-CD163 binding proteins and antibodies have therapeutic and protective uses, such as in the treatment or prevention of infections like porcine reproductive and respiratory syndrome (PRRS) virus infections, for example, reducing their morbidity and severity. Compositions, methods, and kits based on the binding proteins and antibodies are also provided. Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most devastating viral diseases of pigs worldwide, causing enormous economic losses to the swine industry. The pathogen is PRRS virus (PRRSV), an enveloped RNA virus classified under the family Arteriviridae in the order Nidovirales. PRRSV exhibits restricted host and cell tropism, with porcine alveolar macrophages (PAMs) as a key target cell. Clinical symptoms are diverse, but include respiratory distress and respiratory disease in piglets and piglets, late abortion and stillbirth in gilts and sows, fetal reabsorption in early pregnancy, and reduced growth in finishing pigs. Due to reduced or lost pregnancies, piglet mortality, and decreased growth rates in all infected pigs, it is estimated to cause more than $650 million in losses annually to pork producers in the United States alone.

[0003] All currently known PRRSV isolates belong to one of two genotypes (or species), type 1 (PRRSV-1) or type 2 (PRRSV-2), sharing only about 60% nucleotide identity, although both cause long-term infection and produce similar clinical signs. 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 Asian or American isolates or strains (see Stoian and Rowland, review, 2019, Veterinary Science, 6, 9). Within each genotype, a large number of strains have been found with significant diversity, including new highly pathogenic strains that have emerged since 2006, particularly in China and Vietnam. Similar highly pathogenic strains have also appeared elsewhere, from the Malay Peninsula to southern Russia, and these pose an increasing threat to swine populations (An et al., 2011, Emerging Infectious Diseases, 17(9):1782). In China alone, more than 20 million pigs were culled annually due to PRRS virus infection in 2006 and 2007 (An et al., 2010, Emerging Infectious Diseases 16(2):365). Recent reports of highly virulent strains in Europe indicate that the emergence of PRRS virus is becoming an increasing threat (Sinn et al., 2016, Porcine Health Management (2):28).

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

[0005] The exact mechanism of PRRSV infection remains unclear. However, as part of this mechanism, it is believed that PRRSV enters the endosome compartment of the cell, where the interaction between the CD163 and GP2-GP3-GP4 heterotrimers of PRRSV mediates viral uncoating and the release of the viral genome into the cytoplasm.

[0006] One proposed PRRSV treatment option involves some form of genetic knockout or gene editing of CD163 to make pigs resistant to PRRSV infection, and then breeding these pigs to reproduce the genetically modified organism (Burkard et al., 2017, PLOS Pathogens 13(2):e1006206). While this has proven very effective, such treatment would be complex and time-consuming in terms of being able to treat a substantial proportion of pig populations. Furthermore, importantly, there is significant resistance in many markets to technologies involving animal genetic modification, for example, when the expectation is to produce animal products from these animals.

[0007] The most common medical intervention used to limit the economic impact of PRRS is vaccination. Vaccines are typically used in all areas where the disease is prevalent. Two types of vaccines are usually used: inactivated virus vaccines (viruses) or (in most cases) modified live vaccines (MLVs). However, current vaccines are only partially effective and have the greatest value when used in a comprehensive approach to disease management that closely integrates biosafety and livestock decisions. The reasons for insufficient vaccine efficacy are complex, but the high genetic diversity of the PRRSV population, coupled with the virus's biology (tropism to alveolar macrophages and high variability), makes optimal results possible when vaccine strains and circulating strains are closely matched in terms of immunogenicity (review by Nan et al., 2017, Front. Immunol. 8:1635). Additionally, live vaccine strains can recombine with wild-type strains to produce new, potentially pathogenic wild-type strains.

[0008] There are currently no antiviral treatment options for PRRSV infection.

[0009] Therefore, for PRRSV infection (or other CD163-mediated infections), there is clearly a need for alternative, preferred, and improved treatment and prevention options that can be readily used to treat or prevent infection in large numbers of animals. Summary of the Invention

[0010] This invention provides such an alternative therapeutic or prophylactic option in the form of a binding protein and antibody against porcine CD163, which can be used to reduce or prevent PRRSV infection.

[0011] Surprisingly, a single type of antibody (monoclonal antibody) targeting the same epitope on porcine CD163, in contrast to polyclonal antibody preparations targeting multiple different epitopes on porcine CD163, has been shown to effectively and significantly reduce or prevent PRRSV infection. Furthermore, subgroups of these anti-CD163 antibodies have been identified that show differential inhibition against PRRSV type 1 and / or type 2 infection.

[0012] The inventors have therefore provided an anti-CD163 antibody capable of binding to and inhibiting the activity or function of CD163, particularly porcine CD163. Such an antibody (or other binding proteins, for example, comprising the CD163 antigen-binding domain described herein) can, for example, inhibit the ability of CD163 to interact with other proteins such as viral proteins, thereby inhibiting infection of cells such as porcine alveolar macrophages. Such an antibody (or other binding proteins, for example, comprising the CD163 antigen-binding domain described herein) can be conveniently and advantageously used for the treatment or prevention of porcine infections, particularly PRRSV infection.

[0013] In one embodiment, the present invention provides a binding protein, such as an antibody, or a monoclonal antibody, that binds to CD163, such as porcine CD163, for the treatment or prevention of infections in pigs, such as PRRS virus infection or CD163-mediated infection. Therefore, in particular, the present invention provides a monoclonal antibody that binds to porcine CD163 for the treatment or prevention of porcine PRRS virus infection. However, the antibody of the present invention can be used to treat or prevent any symptom in pigs that demonstrates the role of CD163, in which case the binding and inhibition of this protein may be a useful therapeutic tool.

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

[0015] The ability to suppress PRRSV type 1 and / or type 2 infection

[0016] Family 40

[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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0018] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence RYVMG (SEQ ID NO:2), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0019] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence GIAWSGRAPYADSVKG (SEQ ID NO:3), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0020] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence GEGAIRWTTLDAYDY (SEQ ID NO:4), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0021] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0022] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence RYVMG (SEQ ID NO: 10), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0023] (ii) A variable heavy chain (VH) CDR2 containing the amino acid sequence AISWSGRAPYADSVKG (SEQ ID NO: 11), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0024] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence GEGAIKWTTLDAYDY (SEQ ID NO:12), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0025] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0026] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence RYVMG (SEQ ID NO:18), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0027] (ii) A variable heavy chain (VH) CDR2 containing the amino acid sequence GIAWSGRAPYADSVKG (SEQ ID NO:19), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0028] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence GEGAILWTTPGAYNY (SEQ ID NO:20), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0029] 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 antigen-binding domain comprises at least one heavy chain variable region, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0030] (i) The variable heavy chain (VH)CDR1 containing the amino acid sequence RYVMG (SEQ ID NO:2),

[0031] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence GIAWSGRAPYADSVKG (SEQ ID NO:3), and

[0032] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence GEGAIRWTTLDAYDY (SEQ ID NO:4).

[0033] 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 antigen-binding domain comprises at least one heavy chain variable region, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0034] (i) The variable heavy chain (VH)CDR1 containing the amino acid sequence RYVMG (SEQ ID NO:10),

[0035] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence AISWSGRAPYADSVKG (SEQ ID NO:11), and

[0036] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence GEGAIKWTTLDAYDY (SEQ ID NO:12).

[0037] 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 antigen-binding domain comprises at least one heavy chain variable region, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0038] (i) The variable heavy chain (VH)CDR1 containing the amino acid sequence RYVMG (SEQ ID NO:18),

[0039] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence GIAWSGRAPYADSVKG (SEQ ID NO:19), and

[0040] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence GEGAILWTTPGAYNY (SEQ ID NO:20).

[0041] In a further embodiment of the invention, VH CDR2 has or comprises the amino acid sequence X1 I X3 WSGR AP YADSVKG (SEQ ID NO: 73). In these embodiments, X1 or X3 can be any amino acid. Preferably, one or more of these X residues, most preferably all of them, are selected from the following groups: X1 is G or A, and X3 is A or S. Therefore, a preferred VH CDR2 has or comprises the amino acid sequence G / AIA / SWSGRAPYADSVKG (SEQ ID NO: 74). For example, a preferred VH CDR2 sequence of this embodiment has or comprises SEQ ID NO: 3, 11, or 19.

[0042] In a further embodiment of the present invention, VH CDR3 has or includes the amino acid sequence GEG AI X6 WT TX 10 X 11 AYX 14 Y (SEQ ID NO:75). In these embodiments, X6, X 10 X 11 and X 14 It can be any amino acid. Preferably, one or more of these X residues, most preferably all of them, are selected from the following groups: X6 is R, K, or L; X 10 It is L or P; X 11 It is D or G, and X 14 It is D or N. Therefore, the preferred VH CDR3 has or contains the amino acid sequence GEGAIR / K / LWT TL / PD / GAYD / NY (SEQ ID NO:76). For example, the preferred VH CDR3 sequence of this embodiment has or contains SEQ ID NO:4, 12 or 20.

[0043] In a further embodiment, the present invention provides an antibody (or binding protein) comprising:

[0044] The VH region comprises VH CDR1 of SEQ ID NO:2 or a sequence substantially homologous to it, VHCDR2 of SEQ ID NO:73, and VH CDR3 of SEQ ID NO:75, wherein the substantially homologous sequence is a sequence containing one or two (preferably one) amino acid changes compared to a given CDR sequence. 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.

[0045] In one embodiment, the present invention provides an antibody (or binding protein) comprising:

[0046] The VH region comprises VH CDR1 of SEQ ID NO:2 or a sequence substantially homologous to it, VH CDR2 of SEQ ID NO:74, and VH CDR3 of SEQ ID NO:76, wherein the substantially homologous sequence is a sequence containing one or two (preferably one) amino acid changes compared to a given CDR sequence. 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.

[0047] In a further embodiment of the invention, the antibody (or binding protein) comprises:

[0048] The VH region comprises VH CDR1 of SEQ ID NO:2 or a sequence containing one or two (preferably one) amino acid changes compared to a given CDR sequence, VH CDR2 of SEQ ID NO:73 or a substantially homologous sequence thereto, and VH CDR3 of SEQ ID NO:75 or a substantially homologous sequence thereto. In such embodiments, the substantially homologous sequence is a sequence containing 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.

[0049] In a further embodiment of the invention, the antibody (or binding protein) comprises:

[0050] The VH region comprises VH CDR1 of SEQ ID NO:2 or a sequence containing one or two (preferably one) amino acid changes compared to a given CDR sequence, VH CDR2 of SEQ ID NO:74 or a substantially homologous sequence thereto, and VH CDR3 of SEQ ID NO:76 or a substantially homologous sequence thereto. In such embodiments, the substantially homologous sequence is a sequence containing 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.

[0051] Family 70

[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 antigen-binding domain comprises at least one heavy chain variable region, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0053] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence TYSMG (SEQ ID NO:26), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0054] (ii) A variable heavy chain (VH) CDR2 containing the amino acid sequence AHRWSGSAYYAEHADSVEG (SEQ ID NO:27), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0055] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence GVGSAAQYRY (SEQ ID NO:28), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0057] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence PGSMG (SEQ ID NO:34), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0058] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence AHRWSGSAYYADYADSVEG (SEQ ID NO:35), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0059] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence GVGSAAQYTY (SEQ ID NO:36), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0060] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0061] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence TYSMG (SEQ ID NO:42), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0062] (ii) A variable heavy chain (VH) CDR2 containing the amino acid sequence AHRWSGSAYYAEHADSVEG (SEQ ID NO:43), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0063] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence GVGSEAQYRY (SEQ ID NO:44), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0064] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0065] (i) The variable heavy chain (VH)CDR1 containing the amino acid sequence TYSMG (SEQ ID NO:26),

[0066] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence AHRWSGSAYYAEHADSVEG (SEQ ID NO:27), and

[0067] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence GVGSAAQYRY (SEQ ID NO:28).

[0068] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0069] (i) The variable heavy chain (VH)CDR1 containing the amino acid sequence PGSMG (SEQ ID NO:34),

[0070] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence AHRWSGSAYYADYADSVEG (SEQ ID NO:35), and

[0071] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence GVGSAAQYTY (SEQ ID NO:36).

[0072] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0073] (i) A variable heavy chain (VH)CDR1 containing the amino acid sequence TYSMG (SEQ ID NO:42),

[0074] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence AHRWSGSAYYAEHADSVEG (SEQ ID NO:43), and

[0075] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence GVGSEAQYRY (SEQ ID NO:44).

[0076] In a further embodiment of the invention, VH CDR1 has or comprises the amino acid sequence X1 X2 SMG (SEQ ID NO: 77). In these embodiments, X1 or X2 can be any amino acid. Preferably, one or more of these X residues, most preferably all of them, 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 T / PY / GSMG (SEQ ID NO: 78). For example, a preferred VH CDR1 sequence of this embodiment has or comprises SEQ ID NO: 26, 34, or 42.

[0077] In a further embodiment of the present invention, VH CDR2 has or includes the amino acid sequence AHRWSGSA YY AX 12 X 13 ADSVEG (SEQ ID NO:79). In these embodiments, X 12 or X 13 It can be any amino acid. Preferably, one or more of these X residues, most preferably all of them, are selected from the following groups: X 12 It is either E or D, and X 13 It is H or Y. Therefore, the preferred VH CDR2 has or contains the amino acid sequence AHRWSGSAYYAE / DH / YADSVEG (SEQ ID NO:80). For example, the preferred VH CDR2 sequence of this embodiment has or contains SEQ ID NO:27, 35 or 43.

[0078] In a further embodiment of the invention, VH CDR3 has or comprises the amino acid sequence GVGS X5 AQ YX9Y (SEQ ID NO: 81). In these embodiments, X5 and X9 can be any amino acid. Preferably, one or more of these X residues, most preferably all of them, are selected from the following groups: X5 is A or E, and X9 is R or T. Therefore, a preferred VH CDR3 has or comprises the amino acid sequence GVGSA / EAQYR / TY (SEQ ID NO: 82). For example, a preferred VHCDR3 sequence of this embodiment has or comprises SEQ ID NO: 28, 36, or 44.

[0079] In one embodiment, the present invention provides an antibody (or binding protein) comprising:

[0080] The VH region comprises 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.

[0081] In one embodiment, the present invention provides an antibody (or binding protein) comprising:

[0082] The VH region comprises 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.

[0083] In other embodiments of the invention, the antibody (or binding protein) comprises:

[0084] The VH region comprises VH CDR1 of SEQ ID NO:77 or a sequence containing one or two (preferably one) amino acid changes compared to a given CDR sequence, VH CDR2 of SEQ ID NO:79 or a substantially homologous sequence thereto, and VH CDR3 of SEQ ID NO:81 or a substantially homologous sequence thereto. In such embodiments, the substantially homologous sequence is a sequence containing 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.

[0085] In other embodiments of the invention, the antibody (or binding protein) comprises:

[0086] The VH region comprises VH CDR1 of SEQ ID NO:78 or a sequence containing one or two (preferably one) amino acid changes compared to a given CDR sequence, VH CDR2 of SEQ ID NO:80 or a substantially homologous sequence thereto, and VH CDR3 of SEQ ID NO:82 or a substantially homologous sequence thereto. In such embodiments, the substantially homologous sequence is a sequence containing 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.

[0087] In embodiments of the present invention, one or more CDR sequences contain X X If a residue (or another type of substitution residue as defined herein) is present, then the CDR and sequences substantially homologous to it containing one, two, three, or four, preferably one, two, or three (more preferably one, two, or one) different amino acids or amino acid substitutions compared to a given CDR sequence are also included in this invention. In some such embodiments, the alteration or substitution of the amino acid residue may include one or more X residues. X Residues may be present except for X XOn residues other than the specified residues. In other such embodiments, the change is on X. X residues and non-X X In the mixture of residues.

[0088] Clone 150 (#15)

[0089] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0090] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence SYSMG (SEQ ID NO:50), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0091] (ii) A variable heavy chain (VH) CDR2 containing the amino acid sequence AITWNGYITNYADSVKG (SEQ ID NO: 51), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0092] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence TTFSTTSPISRTYNY (SEQ ID NO:52), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0093] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0094] (i) The variable heavy chain (VH)CDR1 containing the amino acid sequence SYSMG (SEQ ID NO:50),

[0095] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence AITWNGYITNYADSVKG (SEQ ID NO:51), and

[0096] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence TTFSTTSPISRTYNY (SEQ ID NO:52).

[0097] Clones 70 (#23)

[0098] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0099] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence TYAMG (SEQ ID NO: 58), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0100] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence IISFGGTFYADSVKG (SEQ ID NO: 59), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0101] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence GRTLSKRADSYAS (SEQ ID NO:60), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0102] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0103] (i) A variable heavy chain (VH)CDR1 containing the amino acid sequence TYAMG (SEQ ID NO:58),

[0104] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence IISFGGTFYADSVKG (SEQ ID NO:59), and

[0105] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence GRTLSKRADSYAS (SEQ ID NO:60).

[0106] Clone 144 (#1)

[0107] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0108] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence MYAMS (SEQ ID NO: 66), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0109] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence AINTSGRYSRYADSVKG (SEQ ID NO: 67), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0110] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence TDKGNWALAMSYDY (SEQ ID NO:68), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0111] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0112] (i) The variable heavy chain (VH)CDR1 containing the amino acid sequence MYAMS (SEQ ID NO:66),

[0113] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence AINTSGRYSRYADSVKG (SEQ ID NO:67), and

[0114] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence TDKGNWALAMSYDY (SEQ ID NO:68).

[0115] All antibodies (or binding proteins) described in the above sections have the ability to inhibit PRRSV type 1 and type 2 infection and can therefore be used to treat or prevent PRRSV type 1 and / or type 2 infection.

[0116] The ability to suppress type 2 PRRSV infection

[0117] As described above, other anti-CD163 antibodies and binding proteins of the present invention have the ability to inhibit type 2 PRRSV infection, and preferably specifically (or only, or preferentially) inhibit type 2 PRRSV infection, for example, inhibiting type 2 PRRSV infection but not inhibiting (or not significantly inhibiting) type 1 PRRSV infection. Therefore, further embodiments of the present invention provide antibodies (or binding proteins) 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 aforementioned antibodies that can inhibit type 1 and / or type 2 PRRSV infection, preferably inhibiting type 1 or type 1 and type 2 PRRSV infection.

[0118] Clone 57 (#11)

[0119] Therefore, 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, said antigen-binding domain comprising at least one heavy chain variable region, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0120] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence VYGTG (SEQ ID NO: 84), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0121] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence GISGTTGSTLYADSVKG (SEQ ID NO: 85), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0122] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence GGRVYITTSSWAY (SEQ ID NO:86), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0123] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0124] (i) A variable heavy chain (VH)CDR1 containing the amino acid sequence VYGTG (SEQ ID NO:84),

[0125] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence GISGTTGSTLYADSVKG (SEQ ID NO:85), and

[0126] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence GGRVYITTSSWAY (SEQ ID NO:86).

[0127] Clone 41 (#12)

[0128] 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, said antigen-binding domain comprising at least one heavy chain variable region, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0129] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence RYAMG (SEQ ID NO: 92), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0130] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence AIAWSTGSTYYANSVKG (SEQ ID NO: 93), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0131] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence ETRYCSGFGCLDPRTYGS (SEQ ID NO:94), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0132] 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, said antigen-binding domain comprising at least one heavy chain variable region, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0133] (i) The variable heavy chain (VH)CDR1 containing the amino acid sequence RYAMG (SEQ ID NO:92),

[0134] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence AIAWSTGSTYYANSVKG (SEQ ID NO:93), and

[0135] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence ETRYCSGFGCLDPRTYGS (SEQ ID NO:94).

[0136] Clone 171 (#14)

[0137] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0138] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence TDTMA (SEQ ID NO: 100), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0139] (ii) A variable heavy chain (VH) CDR2 comprising the amino acid sequence GIGRSGGSIYYADAVKG (SEQ ID NO: 101), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0140] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence RQRIGLVVGALGYDY (SEQ ID NO:102), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0141] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0142] (i) A variable heavy chain (VH)CDR1 containing the amino acid sequence TDTMA (SEQ ID NO:100),

[0143] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence GIGRSGGSIYYADAVKG (SEQ ID NO:101), and

[0144] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence RQRIGLVVGALGYDY (SEQ ID NO:102).

[0145] Clone 29 (#17)

[0146] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0147] (i) A variable heavy chain (VH) CDR1 containing the amino acid sequence DYTIG (SEQ ID NO: 108), or a sequence substantially homologous to it, wherein the substantially homologous sequence is a sequence containing one or two amino acid substitutions compared to a given CDR sequence.

[0148] (ii) A variable heavy chain (VH) CDR2 containing the amino acid sequence CINSITSNTYYADSVKG (SEQ ID NO: 109), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three, or four amino acid substitutions compared to a given CDR sequence, and

[0149] (iii) A variable heavy chain (VH) CDR3 containing the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:110), or a sequence substantially homologous thereto, wherein the substantially homologous sequence is a sequence containing one, two, three or four amino acid substitutions compared to a given CDR sequence.

[0150] 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, the heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein the heavy chain variable region comprises:

[0151] (i) The variable heavy chain (VH)CDR1 containing the amino acid sequence DYTIG (SEQ ID NO:108),

[0152] (ii) The variable heavy chain (VH)CDR2 containing the amino acid sequence CINSITSNTYYADSVKG (SEQ ID NO:109), and

[0153] (iii) A variable heavy chain (VH)CDR3 containing the amino acid sequence DSGLFSGSSCLKYRAMRFGS (SEQ ID NO:110).

[0154] Other embodiments

[0155] Certain preferred embodiments of the present invention provide antibodies (or binding proteins) that bind to CD163, such as porcine CD163, comprising a VH domain having an 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 antibodies (or binding proteins) further comprise a VL domain comprising up to three light chain CDRs, and preferably three light chain CDRs.

[0156] 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 SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, or 65, or a sequence having at least 80% sequence identity with it (e.g., at least 85%, 90%, 95%, or 98% identity). In some embodiments, such an antibody (or binding protein) further comprises a VL domain containing up to three light chain CDRs, and preferably three light chain CDRs.

[0157] 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 SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, or 65. In some embodiments, such an antibody (or binding protein) further comprises a VL domain containing up to three light chain CDRs, and preferably three light chain CDRs.

[0158] Certain preferred embodiments of the present invention provide antibodies (or binding proteins) that bind to CD163, such as porcine CD163, comprising a VH domain having an amino acid sequence SEQ ID NO: 83, 91, 99, or 107, or a sequence substantially homologous thereto. In some embodiments, such antibodies (or binding proteins) further comprise a VL domain comprising up to three light chain CDRs, and preferably three light chain CDRs.

[0159] 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 SEQ ID NO: 83, 91, 99, or 107, or a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, or 98% identity). In some embodiments, such an antibody (or binding protein) further comprises a VL domain containing up to three light chain CDRs, preferably three light chain CDRs.

[0160] 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 an amino acid sequence SEQ ID NO: 83, 91, 99, or 107. In some embodiments, such an antibody (or binding protein) further comprises a VL domain comprising up to three light chain CDRs, and preferably three light chain CDRs.

[0161] Other preferred embodiments are immunoglobulin (Ig) forms of various antibodies (or binding proteins) as defined herein, such as IgG forms, or forms containing all or part of an immunoglobulin constant region, such as an IgG constant region, for example, full-length Ig or IgG forms. It is understood, of course, that full-length IgG antibodies typically comprise two substantially identical heavy chains and two substantially identical light chains. Preferred forms containing a portion of the immunoglobulin constant region are those containing an Fc region or domain, such as an Fc fusion. Such Fc regions or domains are known in the art and typically contain the CH2 and CH3 domains of the antibody heavy chain, which bind to form a homodimer. These regions can be derived from any suitable source or species, such as a source or species different from the host species used to generate antibodies, for example, through immunization, or different from the source or species from which the antibody is derived, but preferably correspond to or are derived from porcine Fc regions or domains. Because such Fc regions are homodimers (or form homodimers), they can be conveniently used to dimerize two polypeptide chains. Therefore, by linking or fusing one or more single-domain antibodies (e.g., VHH antibodies) of the present invention to each chain of the Fc region, they can be used to provide multiple copies of the single-domain antibody (e.g., VHH antibody) of the present invention in a single construct or molecule when the two chains of the Fc region dimerize. If more than one single-domain antibody (e.g., VHH antibody) of the present invention is 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 fusion can be used to provide constructs containing more than one copy of the same single-domain antibody of the present invention or more than one copy of different single-domain antibodies of the present invention. Since such constructs typically contain more than one copy of the same antibody of the present invention (e.g., more than one copy of a single-domain antibody or VHH antibody or more than one copy of multiple different single-domain antibodies or VHH antibodies), such constructs can exhibit improved CD163 binding, for example, due to affinity effects.

[0162] Binding proteins, such as 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) listed in Tables A, B, C, D, E, F, G, H, or I are preferred. This invention is illustrated by way of monoclonal antibodies, specifically VHH antibodies (single-domain antibodies), whose sequences are shown in Tables A, B, C, D, E, F, G, H, and I herein. The respective VHCDR domain and VH domain of these VHH antibodies are shown in Tables A through I herein. Antibodies (or binding proteins) comprising groups of these VH CDR domains, or VH domains, or IgG sequences comprising these domains (or sequences substantially homologous to them) are preferred embodiments of this invention.

[0163] Furthermore, binding proteins, such as antibodies, based on the antibody sequences 57(#11), 41(#12), 171(#14), and 29(#17) listed in Tables 1, 2, 3, or 4 are preferred. This invention is illustrated by way of monoclonal antibodies, specifically VHH antibodies (single-domain antibodies), whose sequences are shown in Tables 1, 2, 3, and 4 herein. The respective VH CDR domains and VH domains of these VHH antibodies are shown in Tables 1, 2, 3, and 4 herein. Antibodies (or binding proteins) comprising groups of these VH CDR domains, or VH domains, or IgG sequences (or sequences substantially homologous to them) comprising these domains are preferred embodiments of this invention.

[0164] Some examples of basic homologous sequences are sequences having at least 60% or 65% identity with a disclosed amino acid sequence. In some embodiments, the antibody (or binding protein) of the present invention comprises at least one heavy chain variable region, the 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%, and most preferably at least about 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, or 65.

[0165] Other examples of basic homologous sequences are sequences having at least 60% or 65% identity with a disclosed amino acid sequence. In some embodiments, the antibody (or binding protein) of the present invention comprises at least one heavy chain variable region, the 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%, and most preferably at least about 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence SEQ ID NO: 83, 91, 99, or 107.

[0166] Other preferred examples of basic homologous sequences are sequences with conserved amino acid substitutions containing a publicly disclosed amino acid sequence.

[0167] Other preferred examples of the basic homologous sequence are sequences containing one, two, three, or four, preferably one, two, or three, more preferably one or two (more preferably one) altered amino acid sequences in one or more disclosed CDR regions or one or more FR regions. Such alterations may be conserved or non-conserved amino acid substitutions, or a mixture thereof.

[0168] In these embodiments, the preferred change is the substitution of conserved amino acids.

[0169] In all embodiments, binding proteins, such as antibodies, containing substantially homologous sequences retain the ability to bind CD163, such as porcine CD163. Preferably, binding proteins, such as antibodies, containing substantially homologous sequences retain one or more (preferably all) other properties described herein associated with antibodies 49(#18), 47(#19), 48(#20), 76(#2), 77(#16), 78(#8), 150(#15), 70(#23), or 144(#1).

[0170] In all embodiments, binding proteins, such as antibodies, containing substantially homologous sequences retain the ability to bind CD163, such as porcine CD163. Preferably, binding proteins, such as antibodies, containing substantially homologous sequences retain one or more (preferably all) of the other properties described herein associated with antibodies 57(#11), 41(#12), 171(#14), or 29(#17).

[0171] Other examples of fundamentally homologous amino acid sequences according to the invention are described elsewhere herein.

[0172] The CDRs of the antibodies (or binding proteins) of the present invention are preferably separated by suitable structural regions, such as those found in naturally occurring antibodies and / or effective engineered antibodies. Therefore, the V of the present invention... H (e.g., VHH), V LThe individual CDR sequence is preferably set within or bound to a suitable frame or scaffold to enable binding of the antigen (here, CD163). Such a frame sequence or region may correspond to naturally occurring frame regions, FR1, FR2, FR3, and / or FR4, to appropriately form a suitable scaffold, or may correspond to a common frame region, for example, identified by comparing various naturally occurring frame regions. Alternatively, non-antibody scaffolds or frames, such as T-cell receptor frames, may be used.

[0173] Suitable sequences for use in the framework region are known and documented in the art, and any of these sequences may be used. Preferred sequences for the framework region are one or more framework regions constituting the VHH antibody of the present invention, preferably one or more framework regions of the 49(#18), 47(#19), 48(#20), 76(#2), 77(#16), 78(#8), 150(#15), 70(#23), or 144(#1) VHH antibodies, as disclosed in Tables A, B, C, D, E, F, G, H, and I, or framework regions substantially homologous to them, particularly framework regions that allow for maintenance of antigen specificity, such as framework regions that produce substantially the same or identical 3D structure as the antibody.

[0174] Other preferred sequences of the framework region, particularly for the “type 2” antibody of the present invention, are one or more framework regions constituting the VHH antibody of the present invention, preferably one or more framework regions of 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 to them, particularly framework regions that allow the maintenance of antigen specificity, such as framework regions that produce substantially the same or identical 3D structure as the antibody.

[0175] In some preferred embodiments, all four variable heavy chain (SEQ ID NO: 5, 6, 7 and 8) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0176] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 13, 14, 15 and 16) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0177] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 21, 22, 23 and 24) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0178] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 29, 30, 31 and 32) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0179] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 37, 38, 39 and 40) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0180] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 45, 46, 47 and 48) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0181] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 53, 54, 55 and 56) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0182] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 61, 62, 63 and 64) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0183] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 69, 70, 71 and 72) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0184] In some preferred embodiments, particularly for the “type 2” antibody of the present invention, all four variable heavy chain (SEQ ID NO: 87, 88, 89 and 90) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibody of the present invention.

[0185] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 95, 96, 97 and 98) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0186] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 103, 104, 105 and 106) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0187] In other preferred embodiments, all four variable heavy chain (SEQ ID NO: 111, 112, 113 and 114) framework regions (FRs), or substantially homologous FR regions, are suitably found in the antibodies of the present invention.

[0188] As described above, the present invention provides binding proteins, such as antibodies, that bind to (or specifically recognize or specifically bind to) CD163. CD163 is also known as M130, MM130, SCAR1, macrophage-associated antigen, hemoglobin scavenger receptor, or scavenger receptor cysteine-enriched type 1 protein M130. Preferred binding proteins of the present invention are antibodies, particularly VHH antibodies. However, the embodiments described herein involving antibodies such as VHH antibodies, with necessary modifications, are equally applicable to other types of binding proteins and vice versa.

[0189] Preferred binding proteins are any single polypeptide chain capable of binding (e.g., specifically binding) to porcine CD163. Suitable types of binding proteins for use in this invention are known in the art. For example, in some embodiments, immunoglobulin-based polypeptides are used, which typically contain a CDR region (and optionally a FR region or an immunoglobulin-based scaffold), such that the CDR region (and optionally the FR region) of the antibody of this invention can be grafted onto a suitable scaffold or framework, such as an immunoglobulin scaffold.

[0190] However, in other embodiments, non-immunoglobulin-based single-chain binding proteins / scaffold proteins can be used, which can be selected based on their ability to specifically bind to a particular target antigen (CD163 or porcine CD163). Such molecules are also known as antibody mimics (or antibody analogs). Suitable examples of non-immunoglobulin-based single-chain binding proteins are known and described in the art, and include fibronectin (or fibronectin-based molecules), such as module 10 based on the fibronectin type III domain, such as Adnectin (e.g., from Compound Therapeutics, Inc., Waltham, MA); affinity proteins (e.g., from Avacta); ankyrin repeats or DARPins (e.g., from Molecular Partners AG, Zurich, Switzerland); lipid transport proteins, such as antiicalin (e.g., from Pieris Proteolab AG, Freising, Germany); human A-domains (e.g., Avimer); staphylococcal protein A (e.g., from Affibody, Sweden). AG (Sweden); thioredoxin; and molecules based on γ-β-lens proteins or ubiquitin, such as affilin (e.g., from Scil Proteins GmbH, Halle, Germany). Such molecules can also be used as scaffolds on which suitable CDRs mediating target antigen binding can be grafted. For example, the CDR region (and optionally the FR region) of the antibody of the present invention can be grafted onto a suitable non-immunoglobulin scaffold.

[0191] In other embodiments of the invention, nucleic acid-based molecules, such as aptamers, may be used, provided that such molecules are selected based on their ability to bind to a specific target antigen (CD163 or porcine CD163) specifically. Therefore, while this text refers to binding proteins, these embodiments can be extended to other types of binding entities or portions, such as nucleic acid-based molecules.

[0192] The preferred non-antibody-binding protein (or binding portion) of the present invention has the ability to bind the same epitope as the anti-CD163 antibody of the present invention, and such a binding protein (or binding portion) can be selected, for example, by competitive assay, such as those described elsewhere herein, using, for example, the antibody of the present invention as a reference antibody.

[0193] CD163 is a 130 kDa type I transmembrane protein with a signal peptide followed by nine scavenger receptor cysteine ​​(SRCR)-rich domains, each approximately 100 amino acids long, and a 35-amino acid proline-serine-threonine (PST) enriched region separating SRCR domain 6 (SRCR6) and SRCR7. A second PST enriched region connects SRCR9 to the transmembrane domain and a short cytoplasmic tail containing a functional internalization motif. Surface expression of CD163 is restricted to monocyte-macrophage lineage cells.

[0194] Of particular relevance to this invention is that CD163 is expressed on the surface of porcine alveolar macrophages (PAM) and is believed to play an important role in the ability of various pathogens, including viral pathogens, particularly PRRSV, to cause disease in pigs.

[0195] Therefore, the binding protein or antibody of the present invention binds to or is capable of binding to CD163. According to the present invention, CD163 can be derived from any species, such as any mammalian species like pig, human, cattle, canine, cat, sheep, horse, mouse, and monkey. In a preferred embodiment, CD163 is porcine CD163, and the antibody binds to or is capable of binding to (or specifically recognizes or specifically binds to) porcine CD163.

[0196] In some embodiments, the antibody may cross-react (or also bind) to other species of CD163. Therefore, in some embodiments, the antibody may bind to porcine CD163, as well as one or more other species of CD163, such as one or more other mammalian species, such as those described above. In some embodiments, the antibody may bind to both porcine CD163 and human CD163. In other embodiments, the antibody may bind to porcine CD163 but not to (or not significantly bind to or cross-react with) human CD163.

[0197] The binding proteins and antibodies of the present invention can bind any suitable form of CD163, particularly forms of CD163 containing the SRCR5 domain. Thus, such forms can include full-length CD163, or non-full-length forms of CD163, such as truncated forms, or other variant forms of CD163 that, for example, contain a subset of the SRCR domain, but generally include the SRCR5 domain. Preferred and convenient forms of CD163 that the binding proteins and antibodies of the present invention can bind include recombinant CD163, such as recombinant porcine CD163, or CD163 when expressed on the cell surface (cell surface-expressed CD163). Therefore, these cell surface forms represent, in many cases, the original or native form of CD163, such as those found on cells that naturally express or overexpress CD163.

[0198] Suitable cell types for naturally expressing CD163 are well known to those skilled in the art, including monocytes and macrophages. Preferred cell types are PAMs. Alternatively, CD163 can be expressed or overexpressed in cell types that do not normally express CD163, for example, through recombinant means (or other engineering means); in other words, recombinant forms of CD163 can be expressed using cells.

[0199] Exemplary forms of CD163 that can be used herein to evaluate the binding ability of binding proteins and antibodies, such as recombinant CD163, are full-length CD163 or constructs containing subgroups 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 subgroups of different CD163 SRCR domains can also be used, provided that all or part (preferably all) of the SRCR5 domain is present. In some embodiments, the antibody does not bind (or does not significantly bind) CD163 molecules containing a deletion or internal or internal mutation of the SRCR5 domain. The porcine form is preferred for evaluating the antibodies of the present invention, although equivalent forms from other species, such as other mammalian species, can also be used, for example, to evaluate cross-reactivity.

[0200] The sequences of CD163 in various species are well known and documented in the art and can be obtained, for example, from various sequence databases, such as Uniprot. For ease of reference, porcine CD163 has the Uniprot number Q2VL90, and human CD163 has the Uniprot number Q86VB7.

[0201] Therefore, the preferred binding protein or antibody of the present invention has the ability to bind to the SRCR5 domain of CD163 or an epitope in the SRCR5 domain, preferably the porcine SRCR5 domain.

[0202] The sequence of the porcine SRCR5 domain is shown below and corresponds to residues 477-577 of Uniprot Q2VL90: PRLVGGDIPCSGRVEVQHGDTWGTVCDSDFSLEAASVLCRELQCGTVVSLLGGAHFGEGSGQIWAEEFQCEGHESHLSLCPVAPRPDGTCSHSRDVGVVCS (SEQ ID NO:115).

[0203] The sequence of porcine CD163 is shown below and corresponds to the full sequence of Uniprot Q2VL90:

[0204]

[0205] Methods for assessing binding (or binding ability) with appropriate forms of CD163 are well known to those skilled in the art, and any appropriate method may be used.

[0206] Convenient and suitable methods for assessing binding include in vitro binding assays, such as ELISA assays, to evaluate the binding of antibodies to immobilized antigens, such as CD163 as described above. Those skilled in the art are familiar with ELISA assays and can readily establish suitable conditions to assess the ability of binding proteins or antibodies to bind CD163 in such assays. A particularly preferred ELISA assay is described in the Examples section. Alternatively or additionally, the binding of antibodies to CD163 expressed on the cell surface can be assessed by any suitable method, including by flow cytometry analysis (e.g., FACS analysis), such as using PAM or cells expressing recombinant forms of CD163, such as those described elsewhere herein. A particularly preferred flow cytometry analysis is described in the Examples section. Another method for testing the ability of antibodies to bind to CD163 on the cell surface is immunohistochemistry.

[0207] In some embodiments, the binding protein or antibody of the present invention binds CD163 (e.g., porcine CD163 or human CD163) in a surface plasmon resonance (SPR) assay (e.g., a BIACore assay), as determined. Suitable SPR assays are known in the art. In some preferred SPR assays, CD163 in an appropriate form is captured (or immobilized) on a solid support (e.g., a sensor chip), for example by amine coupling (e.g., 2000 response units (RU) of CD163 are immobilized), and then various concentrations (e.g., dilution series, such as double or triple 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 herein.

[0208] Such SPR assays can also be conveniently used to measure the binding kinetics of antibody-antigen interactions, such as the association rate (ka), dissociation rate (kd), and affinity (KD). In some embodiments, measurements can be performed at 25°C in a suitable buffer at pH 7.4, such as a standard HEPES-EDTA buffer, like HBS-EP (sold by GE Healthcare Life Sciences, 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.0005% surfactant P20). Kinetic parameters can be determined or calculated using any suitable model or software, for example by assuming a 1:1 interaction, such as by fitting sensogram experimental data using BIAevaluation software. Particularly preferred SPR assays are described in the Examples section of this document.

[0209] Therefore, in a particularly preferred embodiment, the binding protein or antibody of the present invention binds CD163 (e.g., porcine or human CD163, preferably porcine CD163) in a surface plasmon resonance (SPR) assay (e.g., a BIACore assay) (as measured therein, when evaluated therein).

[0210] In some preferred embodiments, the antibody of the present invention, when in VHH form, has a high binding affinity for CD163 (e.g., porcine CD163), for example having a K+ level in the range of 50 nM or lower (preferably). D (Equilibrium dissociation constant).

[0211] Therefore, preferably, the antibody of the present invention, when in VHH form, has a Kc 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 against CD163 (e.g., porcine CD163), 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. DCorresponding binding affinity. Specific exemplary binding affinities are disclosed in the Examples section. Exemplary forms of CD163 that can be used to evaluate such binding affinity are recombinant porcine CD163 containing SRCR4-7 or recombinant porcine CD163 containing SRCR1-9. Suitable exemplary forms, such as the constructs pCD163-SRCR4-7huFc or pCD163-SRCR1-9huFc, are described in the Examples section. Therefore, the above-described binding affinity can be observed when the antibodies of the present invention are measured using these constructs, or if the antibodies of the present invention are measured using these constructs, for example, in an SPR assay.

[0212] As described above, in some embodiments of the invention, the antibodies may bind to porcine CD163 but not (or not significantly bind to) human CD163. Alternatively, they may be considered to preferentially bind to porcine CD163, unlike human CD163.

[0213] The preferred use of the binding protein or antibody of the present invention is for the treatment or prevention of infection by pathogens involving CD163, most notably PRRSV infection. Typically, the binding protein or antibody of the present invention inhibits (or blocks or reduces) pathogen infection (e.g., PRRSV), for example, inhibiting (or blocking or reducing) the ability of a pathogen (e.g., PRRSV) to cause infection (e.g., infect suitable host cells). Preferably, the inhibition or reduction is measurable, more preferably significant, such as statistically significant inhibition or reduction, for example, a probability value ≤0.05 or <0.05. In some embodiments, the binding protein or antibody of the present invention can inhibit (or block or reduce) the ability of a pathogen, such as PRRSV, to infect host cells 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 percentage inhibition (and other percentage inhibition levels as described herein) is compared (or relative to) an appropriate control assay or control level, such as 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). Therefore, a 0% inhibition (control) level (or conversely 100% or maximum infection level) is typically the level in the absence of the binding protein or antibody (anti-CD163 antibody).

[0214] This ability to inhibit infection can be determined or tested in any suitable assay, examples of which are readily available to those skilled in the art. Suitable assays can be, for example, in vitro or ex vivo assays, and involve, for example, the use of CD163-expressing host cells such as PAM or recombinant CD163-expressing host cells, as discussed elsewhere herein. Such cells can be exposed to PRRSV or other suitable pathogens at levels that induce cellular infection. Suitable assays can typically be performed in the presence of serum, such as porcine serum or fetal bovine serum (FBS). Those skilled in the art can readily determine suitable percentages of serum used, such as the level of 10% FBS and 80% porcine serum used in the assay described in the Examples section. The ability of the binding protein or antibody of the present invention to inhibit or reduce such infection can then be readily analyzed, for example, compared to a 100% infection level set in a control assay. Suitable exemplary infection assays are described in the Examples section.

[0215] Any appropriate concentration of binding protein or antibody can be used to inhibit or reduce infection. 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 at concentrations up to 200, 300, or 400 μg / ml, for example at concentrations from 50 or 100 to 200, 300, or 400 μg / ml, the exemplary antibodies of the present invention have the ability to induce inhibition of antibodies, particularly VHH, such as the levels of inhibition outlined herein. If a combination of antibodies (e.g., VHH antibodies) is used, in some embodiments these levels may refer to the total amount of antibodies (e.g., VHH) present, i.e., the sum of the individual concentrations of the present antibodies.

[0216] In some embodiments, the binding protein or antibody of the present invention can inhibit (or block or reduce) the ability of type 1 PRRSV 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 can be noted that, contrary to PRRSV (or other pathogenic entities) themselves, the binding protein or antibody of the present invention targets host cell CD163. This provides the important advantage of being able to inhibit infection by any virus, such as PRRSV, which uses the same binding region on CD163 for infection or pathogenesis. Thus, the antibodies of the present invention, etc., can provide a method for blocking many strains or isolates of PRRSV (including highly pathogenic strains or isolates), provided that they use CD163 to infect cells. It is believed that the use of CD163 for infection by a variety of PRRSV strains is common. Therefore, the antibodies of the present invention have broad applications. This contrasts with some known methods used for PRRSV, such as vaccination, which can be strain-specific and whose efficacy (or whether they are fully effective) can vary depending on the strain. Therefore, the antibodies of the present invention offer significant advantages and flexibility over these existing methods.

[0217] The 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 against type 1 PRRSV infection, more preferably at least 85%, 90%, or 95%, are preferred.

[0218] The preferred antibody of the present invention has 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%.

[0219] Some preferred antibodies of the present invention have the ability to inhibit both PRRSV type 1 and type 2 infection, for example at levels described above and elsewhere herein. These antibodies are sometimes referred to herein as “dual” antibodies. Thus, exemplary antibodies are capable of inhibiting PRRSV type 2 by at least 50%, in combination with inhibition of PRRSV type 1 by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. Alternative exemplary antibodies are capable of inhibiting PRRSV type 2 by at least 55% or 60%, in combination with inhibition of PRRSV type 1 by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. Alternative exemplary antibodies are capable of inhibiting PRRSV type 2 by at least 65%, 70%, or 75%, in combination with inhibition of PRRSV type 1 by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%. In some embodiments, the preferred antibody of the present invention is capable of inhibiting type 2 PRRSV by at least 65%, 70%, or 75%, and is combined with inhibition of type 1 PRRSV by at least 90% or 95%.

[0220] 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), as shown in Tables A, B, C, D, E, F, G, H, and I, respectively.

[0221] 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) type 2 PRRSV-induced infection (e.g., infection of CD163-expressing host cells or specific inhibition of type 2 PRRSV infection). In contrast to type 1 PRRSV infection, such a binding protein or antibody preferentially inhibits or reduces type 2 PRRSV infection. Thus, exemplary antibodies are capable of inhibiting type 2 PRRSV infection by at least 40%, 45%, or 50% (e.g., inhibiting type 2 PRRSV infection of host cells by at least 40%, 45%, or 50%).

[0222] In other embodiments, such binding proteins or antibodies do not inhibit or reduce (e.g., do not significantly inhibit or reduce) type 1 PRRSV infection (e.g., do not inhibit or reduce, or do not significantly inhibit or reduce, the ability of type 1 PRRSV to infect host cells). By way of example only, such antibodies that do not significantly inhibit or reduce type 1 PRRSV infection may reduce such infection by less than 10%, or less than 5%, or less than 2%, and preferably not at all (0% reduction). These antibodies are sometimes referred to herein as “type 2 specific” or “type 2 only” antibodies. Exemplary examples of 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.

[0223] It is easy to compare type 1 and type 2 PRRSV inhibition using assays where the assay conditions are kept identical, such as using the same concentration of the test antibody or binding protein, but one assay is performed with type 1 PRRSV and the other with type 2 PRRSV. Suitable controls for assessing such inhibition are also described elsewhere in this article.

[0224] In some embodiments, the IC50 of the antibody of the present invention 50 (e.g., for inhibiting PRRSV1 infection in host cells, such as PAM) at concentrations 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. In some embodiments, IC 50 The concentrations are 80 to 350, 300, 250, or 200 μg / ml, or 80 to 160 μg / ml, or 80 to 120 μg / ml, or 100 to 200 μg / ml, or 100 to 160 μg / ml, or 100 to 120 μg / ml. Specific exemplary ICs are provided. 50 The value is also shown in the instance.

[0225] In some embodiments, the antibodies of the present invention have IC 50(e.g., for inhibiting PRRSV2 infection in host cells, such as PAM) at concentrations 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. In some embodiments, IC 50 The concentrations are 100, 150, 200 to 300 μg / ml, 200 to 260 μg / ml, 200 to 220 μg / ml, 220 to 300 μg / ml, 220 to 260 μg / ml, or 220 to 240 μg / ml. Specific exemplary ICs are provided. 50 The value is also shown in the instance.

[0226] The above preferred IC 50 The value is preferably determined as in a suitable viral infectivity assay, such as as described above or in the Examples section.

[0227] Although the dual antibodies described herein exhibit good ability to inhibit PRRSV type 2 infection, inhibition of PRRSV type 2 infection is generally observed to be less complete or not as high as the inhibition observed against PRRSV type 1 infection. While not wishing to be bound by theory, it is possible that more than one epitope exists on CD163 that relates to type 2 infection. Therefore, in preferred embodiments of the invention, for example, the dual antibodies and type 2 specific antibodies described herein may be used in combination. Such combinations can be particularly useful when treatment or prevention of PRRSV type 2 infection is required or desired.

[0228] In alternative embodiments of the invention, the binding protein or antibody of the invention may be used to reduce the risk of PRRSV infection or to prevent PRRSV infection.

[0229] Preferably, the aforementioned abilities and properties are observed at a measurable or significant level, more preferably at a statistically significant level, when compared to an appropriate control level. Appropriate levels of significance are discussed elsewhere herein. More preferably, one or more of the aforementioned abilities and properties are observed at a level that is measurably better or more preferably significantly better (preferably statistically significantly better) when compared to the abilities observed with prior art antibodies.

[0230] In any statistical analysis mentioned herein, preferably, the probability of a statistically significant difference relative to a relevant control or other comparative entity or measurement is ≤0.1 or <0.1, preferably ≤0.05 or <0.05. Appropriate methods for determining statistical significance are well known and documented in the art, and any of these methods may be used.

[0231] In some embodiments, the binding protein or antibody of the present invention has one or more, preferably two or more, or three or more, most preferably all of the functional properties, especially the preferred functional properties described herein.

[0232] As used throughout the application, the terms “a” and “an” are used in the sense that they refer to “at least one,” “at least first,” “one or more,” or “multiple” of the mentioned components or steps, except where the upper limit is specifically stated thereafter. Thus, as used herein, “antibody” means “at least a first antibody.”

[0233] Furthermore, when the terms “comprising,” “including,” “having,” or “containing” or other equivalent terms are used herein, in some more specific embodiments, such as in the definition of a CDR or FR sequence herein, these terms include the terms “consisting of” or “substantially consisting of” or other equivalent terms.

[0234] Other aspects of the invention comprise nucleic acid molecules that encode, or substantially homologous to, the binding proteins or antibodies of the invention as defined herein, or portions thereof.

[0235] Preferred nucleic acid molecules are those encoding the VHH antibody or VH region or domain of the present invention (e.g., nucleic acid molecules encoding SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, or 65). Other preferred nucleic acid molecules are those encoding groups of three CDR sequences as defined in any of Tables A, B, C, D, E, F, G, H, or I. Such preferred nucleic acid molecules also encode suitable framework regions, such as FR1, FR2, FR3, and FR4 regions, preferably groups of FR sequences as defined in any of Tables A, B, C, D, E, F, G, H, or I.

[0236] In other embodiments, preferred nucleic acid molecules are those encoding the VHH antibody or VH region or domain of the present invention (e.g., nucleic acid molecules encoding SEQ ID NO: 83, 91, 99, or 107). Other preferred nucleic acid molecules are those encoding groups of three CDR sequences as defined in any of Tables 1, 2, 3, or 4. Preferred such nucleic acid molecules also encode suitable framework regions, such as FR1, FR2, FR3, and FR4 regions, preferably groups of FR sequences as defined in any of Tables 1, 2, 3, or 4 (e.g., type 2 specific antibodies of the present invention).

[0237] As used herein, the term "fundamentally homologous" in relation to amino acid or nucleic acid sequences includes sequences having at least 60%, 65%, 70%, or 75%, preferably at least 80%, and even more preferably at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the disclosed amino acid or nucleic acid sequence. Therefore, fundamentally homologous sequences of the present invention include single or multiple base or amino acid alterations (additions, substitutions, insertions, or deletions) of the sequences of the present invention. At the amino acid level, preferred fundamentally homologous sequences contain up to five, for example only one, two, three, four, or five, preferably one, two, three, or four, more preferably one, two, or three, and even more preferably one or two altered amino acids, in one or more framework regions and / or one or more CDRs constituting the sequences of the present invention. The alterations can be conserved or non-conserved amino acids. Preferably, the alterations are substitutions, preferably substitutions of conserved amino acids.

[0238] In some embodiments, if the given starting sequence is relatively short (e.g., 5 amino acids in length), fewer amino acid substitutions may be present in sequences substantially homologous to a longer starting sequence compared to the number of amino acid substitutions that may optionally be made in sequences substantially homologous to it. For example, in some embodiments, sequences substantially homologous to the starting VHCDR1 sequence of the present invention, such as the starting VHCDR1 sequence, may be 5 amino acid residues in length, preferably having 1 or 2 (more preferably 1) changed amino acids compared to the starting sequence. Therefore, in some embodiments, the number of changed amino acids in a substantially homologous sequence (e.g., a substantially homologous CDR sequence) can be tailored to the length of a given starting CDR sequence. For example, depending on the length of a given starting CDR sequence, different numbers of changed amino acids may be present to achieve a specific percentage of sequence identity in the CDR, such as at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0239] Conventional methods in the art, such as alanine scanning mutagenesis and / or analysis of the crystal structure of the antigen-antibody complex, can be used to determine which amino acid residues of the CDR do not contribute or do not significantly contribute to antigen binding, and are therefore good candidates for alteration or substitution in embodiments of the invention involving substantially homologous sequences.

[0240] Once determined, one or more amino acids in the amino acid sequence of the parent antibody can be added, deleted, substituted, or inserted using techniques conventional in the art to form a new antibody, wherein the parent antibody is one of the antibodies of the present invention as defined elsewhere herein, and the resulting new antibody is tested to identify an antibody that binds to CD163 according to the present invention. These methods can be used to form a variety of new antibodies, all of which can be tested for their ability to bind to CD163. Preferably, the addition, deletion, substitution, or insertion of one or more amino acids occurs in one or more CDR domains.

[0241] For example, the operation can be conveniently performed at the nucleic acid level via genetic engineering, wherein nucleic acid molecules encoding suitable binding proteins and their domains are modified such that the amino acid sequence of the resulting expressed protein is subsequently modified in a suitable manner. The ability of one or more modified antibodies to bind CD163 can be tested by any suitable method known and described in the art. Suitable methods are also described elsewhere herein and in the Examples section.

[0242] The generation, acquisition, or availability of new antibodies through these methods constitutes another aspect of the present invention.

[0243] The term "fundamentally homologous" also includes modifications or chemical equivalents of the amino acid and nucleotide sequences of the present invention that perform substantially the same function as the protein or nucleic acid molecules of the present invention in substantially the same manner. For example, any substantially homologous antibody should retain the ability to bind CD163 as described above. Preferably, any substantially homologous antibody should retain one or more (or all) of the functional capabilities of the starting antibody.

[0244] Preferably, any substantially homologous antibody should retain the ability to specifically bind to the same epitope of CD163 recognized by the starting antibody in question, for example, the same epitope recognized by the CDR domain of one or more antibodies of the present invention or the VH (VHH) domain of the present invention as described herein, for example, binding to one or more of the same epitopes as a variety of antibodies of the present invention (e.g., 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), as shown in Tables A, B, C, D, E, F, G, H and I, respectively). Therefore, preferably, any substantially homologous antibody should retain, in a suitable assay, the ability to competitively bind to CD163 with one or more of the various antibodies of the present invention (e.g., VHH antibodies 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).

[0245] In other embodiments, any substantially homologous antibody should retain the ability to specifically bind to the same epitope of CD163 recognized by the starting antibody in question, for example, the same epitope recognized by the CDR domain of one or more antibodies of the present invention or the VH (VHH) domain of the present invention as described herein, for example, binding to the same epitope as one or more of the various type 2 antibodies of the present invention (e.g., one or more of VHH antibodies 57(#11), 41(#12), 171(#14), or 29(#17), as shown in Tables 1, 2, 3, and 4, respectively). Therefore, preferably, any substantially homologous antibody should retain the ability to competitively bind to CD163 with one or more of the various type 2 antibodies of the present invention (e.g., VHH antibodies 57(#11), 41(#12), 171(#14), or 29(#17), as shown in Tables 1, 2, 3, and 4, respectively, such as type 2 specific antibodies of the present invention).

[0246] Binding to the same epitope / antigen can be readily tested using methods known and described in the art, such as binding assays, competitive assays, or by analyzing the crystal structure of the antigen-antibody complex. Retention of other functional properties can also be readily tested using methods known and described in the art or herein.

[0247] Therefore, those skilled in the art will understand that binding assays can be used to test whether any antibody (e.g., a "fundamentally homologous" antibody) has the same binding specificity as the antibodies and antibody fragments of the present invention, such as binding to the same epitopes, or having the same or equivalent affinity, such as binding assays, competitive assays, or ELISA assays as described elsewhere herein. The BIAcore assay can also be readily used to determine whether an antibody, such as a "fundamentally homologous" antibody, can bind to CD163. Those skilled in the art will recognize other suitable methods and variations.

[0248] As described below, competitive binding assays can be used to test whether antibodies, for example, “substantially homologous” antibodies, retain the ability to specifically bind to substantially the same epitopes of CD163 recognized by one or more antibodies of the present invention as shown in the sequence listings herein, or whether they have the ability to compete with one or more antibodies of the present invention as shown in the sequence listings herein. The methods described below are merely one example of suitable competitive assays. Other suitable methods and variations will be appreciated by those skilled in the art.

[0249] An exemplary competitive assay involves evaluating the binding of various effective concentrations of the present invention's antibody to CD163 in the presence of different concentrations of the test antibody (e.g., substantially homologous antibodies). The amount of binding inhibition induced by the test antibody can then be evaluated. At increasing concentrations (i.e., an increased test antibody concentration resulting in a corresponding decrease in the amount of the present invention's antibody binding to CD163), evidence is shown that the test antibody competing with the present invention's antibody for increased binding is binding to substantially the same epitope. Preferably, the test antibody significantly reduces the amount of the present invention's antibody binding to CD163. Preferably, the test antibody reduces the amount of the present invention's antibody binding to CD163 by at least about 95%. ELISA and flow cytometry analyses can be used to evaluate binding inhibition in such competitive assays, but other suitable techniques are well known to those skilled in the art.

[0250] Such antibodies (monoclonal antibodies) specifically bind to substantially the same (or identical) CD163 epitopes recognized by the antibodies of the present invention (e.g., VHH antibodies 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). The ability of CD163 to overlap epitopes, or the ability to compete with one or more of the various antibodies of the present invention (e.g., VHH antibodies 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.

[0251] In another embodiment, an antibody (monoclonal antibody) having the ability to specifically bind to substantially the same (or identical) CD163 epitopes or CD163 overlapping epitopes recognized by the antibodies of the present invention (e.g., VHH antibodies 57(#11), 41(#12), 171(#14), or 29(#17), as shown in Tables 1, 2, 3, and 4, respectively), or having the ability to compete with one or more of the various antibodies of the present invention (e.g., VHH antibodies 57(#11), 41(#12), 171(#14), or 29(#17), as shown in Tables 1, 2, 3, and 4, respectively, such as the type 2 specific antibodies of the present invention), is a further embodiment of the present invention. In some embodiments, a preferred such antibody is VHH antibody 171(#14) comprising SEQ ID NO:99 as outlined in Table 3 (or the three related CDR sequences of the sequence).

[0252] As used herein, the term "competitive antibody" refers to an antibody that binds to approximately, substantially, or substantially the same, or even the same, epitope as a "reference antibody." "Competitive antibodies" include antibodies with overlapping epitope specificity. Therefore, a competitive antibody can effectively compete with a reference antibody for binding to CD163. Preferably, the competitive antibody may bind to the same epitope as the reference antibody. Alternatively, the competitive antibody preferably has the same epitope specificity as the reference antibody.

[0253] As used herein, a “reference antibody” is an antibody according to the invention capable of binding CD163, which preferably has a VH domain as defined herein, more preferably has a VH domain or comprises a VHH antibody as outlined in Tables A, B, C, D, E, F, G, H or I, namely SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57 or 65 (or the three related CDR sequences of the said sequences).

[0254] As used herein, a “reference antibody” is an antibody according to the invention capable of binding to CD163, preferably having a VH domain as defined herein, more preferably having a VH domain or comprising a VHH antibody (e.g., the type 2 specific antibody of the invention) as outlined in Tables 1, 2, 3, or 4, namely SEQ ID NO: 83, 91, 99, or 107 (or the three related CDR sequences of the sequence). In some embodiments, a preferred reference antibody is a VHH antibody comprising SEQ ID NO: 99 (or the three related CDR sequences of the sequence) as outlined in Table 3.

[0255] Since reference antibodies, such as those outlined in this sequence listing, have been provided, identifying one or more competitive antibodies or antibodies that bind to the same epitope is a straightforward technical problem. Because the identification of competitive antibodies or antibodies that bind to the same epitope can be determined by comparison with reference antibodies, it should be understood that practically determining the epitope bound by any one or two antibodies is not necessary for identifying competitive antibodies or antibodies that bind to the same epitope. However, epitope mapping can be performed using standard techniques if desired.

[0256] The crystal structure of the antigen-antibody complex between the SRCR5 domain of porcine CD163 shown in SEQ ID NO:115 and VHH antibody 171(#14) (i.e., VHH 014(2D01), which has the amino acid sequence shown in Table 3 (SEQ ID NO:17' or 99)) was analyzed to determine the region (epitope) in CD163 to which the antibody binds (see...). Figure 6 Residues on porcine CD163 that facilitate antigen binding have been identified as S507, E509, L526, and L527 of porcine CD163 (refer to the Uniprot Q2VL90 sequence shown in SEQ ID NO: 116). Crystal structures show 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).

[0257] Tablets on CD163

[0258] Therefore, a further aspect provides an antibody (or binding protein) comprising an antigen-binding domain that binds to or specifically binds to porcine CD163, wherein the antibody (antigen-binding domain) binds to an epitope in the SRCR5 domain of porcine CD163, the epitope comprising (or defined therein) amino acids S507, E509, L526, and L527 of SEQ ID NO:116, or alternative CD163 sequences, such as corresponding residues in CD163 sequences from another species.

[0259] Alternatively, a further aspect provides an antibody (or binding protein) comprising an antigen-binding domain that binds to or specifically binds to porcine CD163, wherein the antibody (antigen-binding domain) binds to an epitope in the SRCR5 domain of porcine CD163, the epitope comprising (or defined by) amino acids S32, E34, L51, and L52 of SEQ ID NO:115, or corresponding residues in an alternative CD163 sequence, such as a CD163 sequence from another species. The relevant residues are underlined in SEQ ID NO:115 below.

[0260] PRLVGGDIPCSGRVEVQHGDTWGTVCDSDF S L E AASLVLCRELQCGTVVS LL GGAHFGEGSGQIWAEEFQCEGHESHLSLCPVAPRPDGTCSHSRDVGVVCS (SEQ ID NO: 115).

[0261] In particular, the interaction between VHH's CDR2 and residues L526 and L527 of porcine CD163 (SEQ ID NO: 116) is important for antigen-antibody (antigen-binding domain) interactions. Therefore, a further aspect of the invention provides antibodies (or binding proteins) comprising an antigen-binding domain that binds to or specifically binds to porcine CD163, wherein the antibody (antigen-binding domain) binds an epitope in the SRCR5 domain of porcine CD163 containing (or defined by) amino acids L526 and L527 of SEQ ID NO: 116, or alternative CD163 sequences, such as corresponding residues in CD163 sequences from another species.

[0262] In other embodiments, the present invention provides an antibody (or binding protein) comprising an antigen-binding domain that binds to or specifically binds to porcine CD163, wherein the antibody (antigen-binding domain) binds to an epitope in the SRCR5 domain of porcine CD163, the epitope comprising (or defined thereas) amino acids L526, L527, and S507 of SEQ ID NO:116, or L526, L527, and E509, or amino acids L526, L527, S507, and E509, or alternative CD163 sequences, such as corresponding residues in CD163 sequences from another species.

[0263] In other embodiments, the antibody (or binding protein) binds to an epitope in the SRCR5 domain of porcine CD163, which contains (or is defined by) one, two, three, or all of residues S507, E509, L526, and L527 of SEQ ID NO:116, or alternative CD163 sequences, such as corresponding residues from a CD163 sequence of another species. 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 alternative CD163 sequences, such as corresponding residues from a CD163 sequence of another species. Such antibodies can be considered examples of antibodies that bind overlapping epitopes.

[0264] Alternatively, the antibody (or binding protein) may bind to an epitope in the SRCR5 domain of porcine CD163 that contains (or is defined by) one, two, three, or all of residues S32, E34, L51, and L52 of SEQ ID NO:115, or alternative CD163 sequences, such as corresponding residues from a CD163 sequence of another species. In other words, at least one amino acid of the epitope on CD163 bound by the antibody (or binding protein) of the present invention contains S32, E34, L51, or L52 of SEQ ID NO:115, or alternative CD163 sequences, such as corresponding residues from a CD163 sequence of another species. Such antibodies may be considered examples of antibodies that bind overlapping epitopes.

[0265] To the knowledge of the inventors, no monoclonal antibody has been described in the art that can bind or specifically bind porcine CD163, particularly binding to epitopes in the SRCR5 domain of porcine CD163, and that can inhibit or reduce type 2 PRRSV infection, either in a form that inhibits or reduces type 2 PRRSV infection only, or in a form that can inhibit or reduce both type 1 and type 2 PRRSV infection.

[0266] Therefore, the single monoclonal antibody described herein is unusual and advantageous. Furthermore, as stated above, the inventors believe they have identified an epitope on porcine CD163 that is important for type 2 PRRSV infection and is therefore typically a target for antibodies and binding proteins to reduce or inhibit PRRSV infection. It is also noted that the residues on porcine CD163 identified herein as part of an epitope are located in a different region of CD163 than those previously identified as potentially important for PRRSV infection. For example, previous reports, such as 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 present in loop 5-6 of porcine CD163, located between residues Phe 544 and Arg 570 of CD163. Other reports have speculated that the ligand-binding pocket (LBP) between residues S487 and G499 in CD163 may also be an important region for PRRSV infection. In this study, the four residues identified as part of epitopes were not located in these regions.

[0267] Therefore, it is believed that the present invention has identified novel epitopes important for PRRSV infection, particularly type 2 PRRSV infection, in different portions of the SRCR5 region of porcine CD163, and antibodies (or binding proteins) or overlapping epitopes binding to these epitopes are particularly preferred. As mentioned above, antibody 171 (#14) shown in Table 3 has been shown to bind to this epitope. Initial experiments using competitive binding studies have shown that at least antibodies 57 (#11), 70 (#23), 144 (#1), and 150 (#15) can bind to the same or overlapping epitopes.

[0268] complementary sites on antibodies

[0269] Two L residues, L526 and L527, in CD163 have been shown to interact with Y59 and D62 in the YYAD motif found in the CDR2 of VHH antibody 171 (#14), namely VHH 014 (2D01). This VHH antibody has been shown to inhibit or reduce type 2 PRRSV infection. It can be noted that the sequence YYAD, or a sequence highly similar to YYAD, is present in the equivalent or corresponding region of the CDR2 of all VHH antibodies described herein. All VHH antibodies described herein have been shown to inhibit or reduce type 2 PRRSV infection. Therefore, this VH CDR2 region appears to be an important feature of antibodies (e.g., VHH antibodies) with the ability to inhibit or reduce type 2 PRRSV infection.

[0270] Therefore, the preferred antibody (or binding protein) of the present invention comprises CDR2, particularly VH CDR2, which comprises the 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 may comprise YAN or XYAN as a substitute for YAD or YAE.

[0271] In the embodiments, the X residue is located at position 59 of SEQ ID NO: 17' or 99 of VHH antibody 171 (#14), i.e., VHH 014 (2D01) shown in Table 3, or at the corresponding position in the VH CDR2 of an alternative antibody (or VHH). Alternatively, the X residue may be located at position 10 of SEQ ID NO: 19' or 101 (CDR2) of VHH antibody 171 (#14), i.e., VHH 014 (2D01) shown in Table 3, or at the corresponding position in the VH CDR2 of an alternative antibody (or VHH), which may be, for example, located at position 8 or 9 in the CDR2 region of other VHH antibodies as described herein. The positions of other residues in the XYAD, XYAE, or XYAN motifs can be determined based on these positions.

[0272] Two residues, S507 and E509, in CD163 have been shown to interact with L104 in the CDR3 of VHH antibody 171 (#14), namely VHH 014 (2D01). This VHH antibody has been shown to inhibit or reduce type 2 PRRSV infection. Therefore, this VH CDR3 residue (or the corresponding residue in other antibodies such as VHH antibody) can be an important residue in antibodies (e.g., VHH antibody) with the ability to inhibit or reduce type 2 PRRSV infection.

[0273] Therefore, in some embodiments, the antibody (or binding protein) of the present invention comprises CDR3, particularly VH CDR3, which comprises amino acid residue L at the corresponding position in SEQ ID NO: 17' or 99 of VHH antibody 171 (#14), i.e., VHH 014 (2D01) shown in Table 3, or in the VH CDR3 of an alternative antibody (or VHH). Alternatively, the L residue may be located at position 6 in SEQ ID NO: 20' or 102 of VHH antibody 171 (#14), i.e., VHH 014 (2D01) shown in Table 3, or in the VH CDR3 of an alternative antibody (or VHH).

[0274] In some embodiments, the L residues mentioned above are present not only in the YAD, YAE, or YAN sequence in CDR2, but also in CDR3, preferably XYAD, XYAE, or XYAN, 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, and most preferably Y.

[0275] In embodiments of the present invention that provide a basic homologous sequence, in some embodiments, residues YAD, YAE, or YAN, or XYAD, XYAE, or XYAN as defined above are maintained or present, and variations occur outside of these residues.

[0276] The basic homologous sequences of the proteins of the present invention include, but are not limited to, conserved amino acid substitutions, or alterations that do not affect the VH, VL, or CDR domains of antibodies, such as the addition of a tag sequence, toxin, or other component of the antibody that does not contribute to antigen binding, or alterations that convert one type or form of binding protein, antibody molecule, or fragment into another type or form of binding protein, antibody molecule, or fragment (e.g., from VHH to Fab or scFv or a whole antibody, or vice versa), or antibody molecules that convert to a specific class or subclass of antibody molecules (e.g., antibody molecules that convert to IgG or its subclass, such as IgG2).

[0277] As used herein, "conservative amino acid substitution" refers to the substitution of an amino acid residue by another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In other instances, amino acid residue families may be grouped based on hydrophobic or hydrophilic side groups.

[0278] Homology can be assessed by any convenient method. However, computer programs for multiple sequence alignment are useful for determining the degree of homology between sequences, such as Clustal W (Thompson, Higgins, Gibson, Nucleic Acids Res., 22:4673-4680, 1994). If desired, the Clustal W algorithm can be used with the BLOSUM 62 scoring matrix (Henikoff and Henikoff, Proceedings of the National Academy of Sciences, 89:10915-10919, 1992) with a gap opening penalty of 10 and a gap extension penalty of 0.1, resulting in a highest-order match between the two sequences, where at least 50% of the total length of one of the sequences is involved in the alignment. Other methods that can be used for sequence alignment are the Needleman and Wunsch alignment method (Needleman and Wunsch, Journal of Molecular Biology, 48:443, 1970), revised by Smith and Waterman (Smith and Waterman, Advances in Applied Mathematics, 2:482, 1981), which achieves the highest order of matching between two sequences and determines the number of identical amino acids between the two sequences. Other methods for calculating the percentage of identity between two amino acid sequences are generally well known in the art, including, for example, those described by Carillo and Lipton (Carillo and Lipton, SIAM J. Applied Math, 48:1073, 1988) and those described in Computational Molecular Biology, edited by Lesk, Oxford University Press, New York, 1988, “Biocomputing: Informatics and Genomics Projects”.

[0279] Typically, computer programs are used to perform such calculations. Programs for comparing and aligning sequences, such as ALIGN (Myers and Miller, CABIOS, 4:11-17, 1988), FASTA (Pearson and Lipman, Proceedings of the National Academy of Sciences, 85:2444-2448, 1988; Pearson, Methods in Enzymology, 183:63-98, 1990), and nicked BLAST (Altschul et al., Nucleic Acid Research, 25:3389-3402, 1997), BLASTP, BLASTN, or GCG (Devereux, Haeberli, Smithesies, Nucleic Acid Research, 12:387, 1984), can also be used for this purpose. In addition, the Dali server of the European Institute of Bioinformatics provides structure-based protein sequence alignment (Holm, Trends in Biochemical Sciences, 20:478-480, 1995; Holm, Journal of Molecular Biology, 233:123-38, 1993; Holm, Nucleic Acid Research, 26:316-9, 1998).

[0280] By providing reference points, sequences according to the invention having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology, sequence identity, etc., can be determined using the ALIGN program with default parameters (e.g., available on the Internet via the GENESTREAM web server, IGH (IGH, Montpellier, France)).

[0281] As used herein, the terms "antibody" and "immunoglobulin" broadly refer to any immunobinding agent containing an antigen-binding domain, including polyclonal and monoclonal antibodies. Monoclonal antibodies are preferred, however. In other words, in some embodiments, the antibodies of the present invention are not polyclonal antibodies. Whole antibodies are classified into one of five classes based on the type of constant domain in the heavy chain: IgA, IgD, IgE, IgG, and IgM, and the antibodies of the present invention can be any of these classes. Some of these are further subdivided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different types of immunoglobulins are well known.

[0282] Generally, when using whole antibodies instead of antigen-binding regions in this invention, IgG is preferred because they are the most common antibodies under physiological conditions and because they are the easiest to prepare in a laboratory setting.

[0283] The “light chain” of mammalian antibodies refers to one of two distinct types: kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains and some amino acids in the framework region of their variable domains.

[0284] As used herein, the term "heavy chain complementarity-determining region" ("heavy chain CDR") refers to the variable region (V) of the heavy chain in an antibody molecule. H The heavy chain variable region is a highly variable region within the structural domain or VHH antibody molecule. The heavy chain variable region has three CDRs (CDR1, CDR2, and CDR3) extending from the amino terminus to the carboxyl terminus. The heavy chain variable region also has four scaffold regions (FR1, FR2, FR3, and FR4, extending from the amino terminus to the carboxyl terminus). These scaffold regions space the CDRs apart.

[0285] As used in this article, the term "heavy chain variable region" (V) H A structural domain refers to a variable region of the heavy chain of an antibody molecule.

[0286] As used herein, the term "light chain complementarity-determining region" ("light chain CDR") refers to the variable region (V) of the light chain in an antibody molecule. L The light chain variable region is a highly variable region within the structural domain. It has three CDRs, called light chain CDR1, light chain CDR2, and light chain CDR3, extending from the amino terminus to the carboxyl terminus. The light chain variable region also has four framework regions (FR1, FR2, FR3, and FR4, extending from the amino terminus to the carboxyl terminus). These framework regions space the CDRs apart.

[0287] As used in this article, the term "light chain variable region" (V) L A structural domain refers to a variable region of the light chain of an antibody molecule.

[0288] As understood by those skilled in the art, the term "antibody" covers immune-binding reagents including or extending to all antibodies and their antigen-binding fragments, including whole antibodies, dimers, trimers and multimers; bispecific antibodies; chimeric antibodies; recombinant antibodies and engineered antibodies and their fragments.

[0289] Therefore, the term "antibody" is used to refer to any antibody-like molecule having an antigen-binding domain, and the term includes antibody fragments containing an antigen-binding domain (e.g., Fab', Fab, F(ab')2), single-domain antibodies (DAB), TandAbs dimers, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, linear antibodies, microantibodies, double-chain antibodies, bispecific antibody fragments, biantibodies, triantibodies (scFv-fab fusions, bispecific or trispecific, respectively); sc-double-chain antibodies; kappa (lambda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandem for attracting T cells); DVD-Ig (double variable domain antibody, bispecific format); SIP (small immunoprotein, a type of microantibody); SMIP (small modular immunopharmaceutical). Modular immunoopharmaceutical (SCFv-Fc dimer); DART (ds-stable double-chain antibody "Dual Affinity ReTargeting"); small antibody mimics containing one or more CDRs, etc.

[0290] Techniques for preparing and using various antibody-based constructs and fragments are well known in the art.

[0291] Antibody fragmentation can be performed using conventional techniques. For example, an F(ab')2 fragment can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bonds, generating a Fab' fragment. Papain digestion can lead to the formation of Fab fragments. Fab, Fab', and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, microantibodies, double-chain antibodies, bispecific antibody fragments, and other fragments can also be synthesized via recombinant techniques or chemically. Techniques for generating antibody fragments are well known and documented in the art.

[0292] In all embodiments of the invention, single-domain antibodies (also known as VHH antibodies, sdAbs, DABs, dAbs, nanobodies, camel antibodies, vNAR (shark) antibodies, VH antibodies, or VL antibodies) are preferred, particularly VHH antibodies, nanobodies, camel antibodies, and vNAR (shark) antibodies. Such antibodies comprise a single monomeric variable antibody domain, typically a VH domain, which can bind an antigen (although a single VL domain with antigen-binding capability has been described and can be used). Therefore, in some such preferred embodiments, the antibody (or antigen-binding domain) of the invention comprises one (or a single) heavy chain variable region (VH or VHH), although in some embodiments, many of these individual heavy chain variable regions having the same or different sequences may coexist in the same construct or molecule.

[0293] These antibodies can be obtained or prepared using standard techniques known and documented in the art. For example, these antibodies can be obtained by immunizing a suitable animal, such as a camel or shark, with the desired antigen, and then cloning the VH domain of the resulting antibody into a suitable expression vector and selecting the conjugate. Libraries of VH domains (e.g., phage display libraries of human VH domains) can also be obtained or generated and then screened.

[0294] Due to their small size, single-domain antibodies can have relatively short half-lives, such as relatively short plasma half-lives. Therefore, these antibodies are sometimes modified to extend or prolong their half-lives. Techniques for doing so are known and described in the art, and any of these techniques can be used. Examples include attaching, conjugating, or fusing antibodies to albumin (or another protein or entity having a long (or longer) half-life), attaching, conjugating, or fusing antibodies to another protein or entity that can interact with a protein or entity having a long (or longer) half-life, attaching or conjugating antibodies to PEG (or other polymers), or attaching, conjugating, or fusing antibodies to antibodies or other proteins or entities that bind FcRn.

[0295] In some embodiments, the antibodies or antibody fragments of the present invention comprise all or part of the heavy chain constant region, such as the constant regions of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM, or IgD. Preferably, the heavy chain constant region is the IgG heavy chain constant region, such as the IgG2 heavy chain constant region, or a portion thereof. Furthermore, the antibody or antibody fragment may comprise all or part of the kappa light chain constant region or the lambda light chain constant region, or a portion thereof. All or part of these constant regions may be naturally occurring or may be wholly or partially synthetic. Suitable sequences of such constant regions are known and described in the art. When the antibodies of the present invention comprise complete complementarity of constant regions from the heavy and light chains, such antibodies are generally referred to herein as “full-length” antibodies or “all” antibodies. In some embodiments, IgG2 antibodies are preferred.

[0296] In other embodiments, it is preferable that there are no constant regions, such as no heavy chain or light chain constant regions, such as variable domains or heavy chain variable domains (VH) being the only part of the antibody present.

[0297] Antibodies or antibody fragments can be produced naturally or synthesized in whole or in part.

[0298] Many antibodies or antibody fragments contain a variable region (V) of the antibody light chain, which includes three CDR domains. L ) and antibody heavy chain variable region (V) containing three CDR domains H The VL and VH typically form antigen-binding sites.

[0299] However, it has been well demonstrated in the art that the presence of the three CDRs of the variable domain of the antibody light chain and the three CDRs of the variable domain of the heavy chain is not always necessary for antigen binding. Therefore, it is known that constructs smaller than the aforementioned classic antibody fragments are effective.

[0300] For example, camel antibodies have a broad antigen-binding library but lack a light chain. Furthermore, results from single-domain antibodies containing only the VH domain or only the VL domain show that these domains can bind antigens with acceptable high affinity and offer other advantages such as their small size and ease of production. Therefore, three CDRs can effectively bind antigens, and such single-domain antibodies (e.g., VHH antibodies, sdAbs, DABs, dAbs, nanobodies, camel antibodies, vNAR (shark) antibodies, VH antibodies, or VL antibodies, particularly VHH antibodies, nanobodies, camel antibodies, and vNAR (shark) antibodies) are preferred (e.g., VHH antibodies).

[0301] The antibodies, binding proteins, and nucleic acid molecules of this invention are generally “isolated” or “purified” molecules, provided they are distinct from any such components that may be present in situ in a human or animal body (e.g., a camel) or in tissue samples derived from a human or animal body (e.g., a camel). However, the sequences may correspond to or be substantially homologous to sequences found in a human or animal body (e.g., a camel). Therefore, as used herein, the terms “isolated” or “purified” with respect to nucleic acid molecules or sequences and proteins or peptides (e.g., antibodies) refer to such molecules when they are isolated, purified, or substantially without their natural environment (e.g., isolated or purified from a human or animal body (if they do indeed exist naturally)), or when they are produced by technical methods, including molecules produced by recombinant and synthetic methods.

[0302] It should be noted that the antibodies, etc., of the present invention are not naturally occurring, and in this respect are artificial constructs, as they do not correspond to naturally occurring molecules. For example, preferred antibodies are single-domain antibodies that can be engineered or recombined, and even in species that naturally produce such antibodies, such as camels, these species will not produce antibodies against CD163, particularly porcine CD163, unless they are experimentally induced, for example, through immunization. In other words, the antibodies, etc., of the present invention are non-natural.

[0303] As used herein, the term "fragment" refers to a biologically relevant fragment, such as a fragment that contributes to antigen binding (e.g., forms part of an antigen-binding site) and / or contributes to the functional properties of the CD163 antibody. Certain preferred fragments comprise or are derived from the heavy chain variable region (V) of the antibody of the present invention. H It consists of a structural domain or three VH CDRs.

[0304] Those skilled in the art will understand that the proteins and peptides of the present invention, such as heavy chain CDRs and light chain CDRs, heavy chain variable regions and light chain variable regions, antibodies and antibody fragments, can be prepared using any of the methods known and described in the art, but recombinant methods are most preferred.

[0305] Where appropriate, the nucleic acid fragments encoding the heavy chain variable region and the light chain variable region of the antibody of the present invention can be obtained or generated by any suitable method, such as by cloning or synthesis.

[0306] Once nucleic acid fragments encoding the heavy chain variable region and / or light chain variable region of the antibody of the present invention are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting the variable region fragments into full-length antibody molecules with appropriate constant region domains, or into antibody fragments of specific forms 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 typically integrated into one or more suitable expression vectors to facilitate the production of the antibody of the present invention, or, for example, to facilitate selection or screening, such as by integration into phage display vectors.

[0307] Possible expression vectors include, but are not limited to, entrapment, plasmid, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), provided that the vector is compatible with the host cell used. The expression vector is “suitable for transformation of the host cell”, meaning that the expression vector contains the nucleic acid molecule of the present invention and a regulatory sequence operatively linked to the nucleic acid molecule based on the host cell to be used for expression. Operable linkage means that the nucleic acid is linked to the regulatory sequence in a manner that allows nucleic acid expression.

[0308] Therefore, the present invention relates to expression vectors, such as recombinant expression vectors containing or comprising the nucleic acid molecules or fragments thereof of the present invention, as well as regulatory sequences necessary for transcription and translation of protein sequences encoded by the nucleic acid molecules of the present invention.

[0309] Expression vectors can be introduced into host cells to produce transformed host cells. The terms “transformation with…”, “transfection with…”, “transformation”, and “transfection” are intended to encompass the introduction of nucleic acids (e.g., vectors) into cells through one of the many possible techniques known in the art. Appropriate methods for transforming and transfecting host cells can be found in Sambrook et al., 1989 (Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd ed., Cold Spring Harbor Press, New York, 1989) and other laboratory textbooks.

[0310] Suitable host cells include a variety of eukaryotic and prokaryotic host cells. For example, the proteins of this invention can be expressed in yeast cells or mammalian cells. Furthermore, the proteins of this invention can be expressed in prokaryotic cells such as *Escherichia coli*.

[0311] The protein of the present invention can also be prepared by chemical synthesis using techniques known in protein chemistry, such as solid-phase synthesis.

[0312] On the other hand, expression constructs, vectors, or systems (e.g., viral or bacterial or other expression constructs, vectors, or systems) comprising one or more nucleic acid fragments or segments or molecules of the present invention are provided. Preferably, the expression construct, vector, or system is recombinant. Preferably, the construct, vector, or system further comprises regulatory sequences necessary for transcription and translation of protein sequences encoded by the nucleic acid molecules of the present invention. Preferred constructs, etc., are those that allow the antibody (or binding protein) of the present invention to be prolonged or sustained in a host target species, such as in pigs. Such expression can be transient, e.g., free, or more persistent, e.g., through genome integration, provided that sufficient levels and lengths of expression are obtained to observe therapeutic or biological effects.

[0313] On the other hand, host cells (e.g., mammalian, bacterial, or yeast host cells) or viruses comprising one or more expression constructs or expression vectors of the present invention are provided. Host cells or viruses comprising one or more nucleic acid molecules of the present invention are also provided. Host cells (e.g., mammalian host cells, bacterial host cells, or yeast host cells) or viruses expressing antibodies (or binding proteins) of the present invention form yet another aspect.

[0314] Such expression constructs, vectors, or systems, or host cells or viruses, or other nucleic acid products or fragments encoding the antibodies (or binding proteins) of the present invention, can be administered to subjects as therapeutic agents to allow the antibodies (or binding proteins) of the present invention to be generated in situ in the subjects, thereby exerting their therapeutic effects.

[0315] Another aspect of the invention provides a method for producing (or manufacturing) the antibodies of the invention, comprising the step of culturing host cells of the invention. A preferred method comprises the steps of: (i) culturing host cells containing one or more recombinant expression vectors or one or more nucleic acid sequences of the invention under conditions suitable for expressing an antibody or protein encoded thereon; and optionally (ii) isolating or obtaining the antibody or protein from the host cells or from the growth medium / supernatant. Such a production (or manufacturing) method may also include the steps 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.

[0316] In embodiments, when the antibody or protein of the present invention consists of more than one polypeptide chain (e.g., certain fragments, such as Fab fragments or whole antibodies), all polypeptides are preferably expressed in host cells from the same or different expression vectors, such that the complete protein, such as the antibody protein of the present invention, can be assembled in the host cells and isolated or purified therefrom.

[0317] On the other hand, the present invention provides a method for binding CD163, comprising contacting a composition containing CD163 with an antibody of the present invention.

[0318] In another aspect, the present invention provides a method for detecting CD163, comprising contacting a composition suspected of containing CD163 with an antibody of the present invention under conditions that effectively allow the formation of a CD163 / antibody complex, and detecting the complex thus formed.

[0319] Compositions comprising at least a first antibody (or binding protein) of the present invention constitute another aspect of the present invention. Formulations (compositions) comprising one or more antibodies of the present invention mixed with suitable diluents, carriers, or excipients constitute preferred embodiments of the present invention. Such formulations can be used for pharmaceutical purposes, such as veterinary purposes, and therefore the compositions of the present invention are preferably pharmaceutically acceptable or acceptable for administration to non-human animals, such as mammals, preferably pigs. Suitable diluents, excipients, and carriers are known to those skilled in the art.

[0320] The compositions of the present invention may be available, for example, in a form suitable for oral, nasal, parenteral, intravenous, topical, or rectal administration.

[0321] The active compounds defined herein (such as the antibodies of this invention) can be present in conventional pharmacologically administered forms, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules, or sustained-release forms. Conventional pharmaceutical excipients and typical manufacturing methods can be used to prepare these forms.

[0322] Injection solutions can be produced, for example, by conventional methods, such as by adding preservatives (e.g., parabens) or stabilizers (e.g., EDTA). The solution is then filled into injection vials or ampoules.

[0323] The appropriate dosage unit can be determined by those skilled in the art.

[0324] In the case of co-administration or combination regimens, the pharmaceutical composition may additionally contain additional active ingredients (e.g., as described elsewhere herein).

[0325] Another aspect of the invention provides anti-CD163 antibodies (or binding proteins) as defined herein for therapeutic purposes, particularly for the treatment or prevention of any disease or condition associated with or in which CD163 plays a role, such as pathogenicity (e.g., complete or partial pathogenicity) or a necessary role. For example, the anti-CD163 antibody of the invention can be used to treat or prevent any infection caused by a virus or other pathogen, wherein the infection is associated with CD163, or in which CD163 plays a role, such as pathogenicity (e.g., complete or partial pathogenicity) or a necessary role. In other words, according to the invention, the anti-CD163 antibody (or binding protein) can target and inhibit or reduce the function of CD163, particularly CD163 expressed on or in cells of PAMs or other CD163-positive cells. Therefore, the anti-CD163 antibody (or binding protein) as defined herein can be used to treat or prevent any disease or condition in which inhibiting CD163 or blocking or reducing CD163 function is beneficial.

[0326] Preferred embodiments provide the anti-CD163 antibody (or binding protein) of the present invention for treating or preventing infection in pigs, preferably swine virus infection. Particularly preferred is the treatment or prevention of PRRSV infection. In embodiments treating pigs, the anti-CD163 antibody (or binding protein) of the present invention is typically an anti-swine CD163 antibody (or binding protein).

[0327] CD163 is considered a possible receptor for all PRRS viruses. However, as described elsewhere herein, there are two serotypes of PRRSV: serotype 1 and serotype 2. Although serotypes 1 and 2 are phenotypically similar at several levels, their viral genotypes differ. The antibodies (or binding proteins) of the present invention can be used to treat or prevent PRRS viruses serotype 1 and / or serotype 2, such as PRRS viruses 1 and 2. In other embodiments, the present invention provides antibodies (or binding proteins) having the ability to inhibit PRRS virus serotype 2 infection, preferably specifically inhibiting PRRS virus serotype 2 infection, and thus can be used to treat or prevent PRRS virus serotype 2.

[0328] In the treatment methods and uses of the present invention, the binding protein or antibody is administered to a subject (animal or mammal, such as a pig) in need of treatment in a pharmaceutically, therapeutically, or physiologically effective amount. Therefore, the methods and uses may include the additional step of identifying the subject in need of treatment.

[0329] Treatment of a disease or symptom according to the present invention (e.g., treatment of a pre-existing disease) includes curing the disease or symptom, or any relief or alleviation of the disease (e.g., reducing the severity of the disease) or symptoms of the disease.

[0330] The methods and uses of this invention are applicable to the prevention of disease as well as the active treatment of disease (e.g., treating existing disease). Therefore, preventative and metaphylactic treatments (treatment in the face of a disease outbreak, such as treating a group of subjects after an infection and / or clinical disease has been diagnosed in a subset of the subjects, with the aim of preventing the spread of infectious diseases to close contacts and / or animals at significant risk) are also included in this invention. Therefore, in the methods and uses of this invention, treatment also includes, where appropriate, prevention, metaphylaxis, or avoidance.

[0331] Such preventative (or protective) aspects can be conveniently conducted in healthy or normal or at-risk subjects and can include complete prevention and significant prevention. Similarly, significant prevention can include a reduction (e.g., measurable or significant reduction) in the severity of the disease or symptoms compared to the expected severity or symptoms without treatment.

[0332] For example, clinical signs of PRRS infection include fetal reabsorption, stillbirth, and late abortion in pregnant sows or gilts, as well as respiratory diseases and syndromes, such as respiratory distress, in all pigs, especially piglets and piglets. Other symptoms include loss of appetite (which usually leads to reduced growth rate), fever, lethargy, respiratory distress, reproductive failure, and diarrhea (particularly in young piglets), as well as central nervous system (CNS) symptoms. Subjects with PRRSV infection also have increased susceptibility to endemic diseases such as meningitis, Glasser's disease, exudative dermatitis, mange, and bacterial bronchopneumonia (Diseases of Swine, 11th ed., edited by Jeffrey J. Zimmerman, Locke A. Karriker, Alejandro Ramirez, Kent J. Schwartz, Gregory W. Stevenson, Jianqiang Zhang, 1st ed.: March 29, 2019), which are treated with antimicrobial products such as antibiotics. Therefore, this invention plays a role in reducing the use of antimicrobial products on farms.

[0333] Therefore, the antibodies of the present invention can be used to treat or prevent clinical diseases or symptoms, such as clinical diseases or symptoms associated with PRRSV infection or downstream endemic diseases, such as those outlined above, or to reduce viral circulation, such as PRRSV circulation (e.g., the number or titer of circulating viral particles) or to prevent infection (e.g., primary infection) or new infection (e.g., secondary or subsequent infection), such as PRRSV infection (e.g., primary PRRSV infection) or new PRRSV infection (e.g., secondary or subsequent PRRSV infection).

[0334] Therefore, preferred subjects for treatment according to the present invention include all types of pigs (sometimes also referred to as swine), such as any pig (pig / swine) or pig species, including pigs of all ages and species, provided that they are susceptible to or capable of being infected by the pathogen defined herein, particularly PRRSV. Piglets, especially young piglets or live-born piglets from infected sows (up to 80% of piglets will die), are particularly preferred subjects, such as nursery pigs (e.g., weaned pigs reaching 12 weeks of age) and growing or finishing pigs (e.g., pigs reaching slaughter age), especially growing pigs. Pre-weaned piglets, such as piglets reaching 4 weeks of age (especially piglets from infected sows, where the infection can be transmitted through the mammary gland secretions of infected sows), are also preferred subjects for treatment, as are sows and pregnant sows.

[0335] In some embodiments, such as when prevention is involved, the subject is a subject at risk of being affected by the disease or condition in question, such as a subject at risk of being infected with and developing a disease by a pathogen or virus (e.g., PRRSV) as described above. Such a subject can be a healthy subject, a subject not exhibiting any symptoms of the disease, or any other suitable "at-risk" subject. In another embodiment, the subject is a subject who has, is suspected of having (or has developed), or potentially has (or has developed) the disease or condition in question as described above.

[0336] Alternatively, the present invention provides a method for treating or preventing a disease or condition associated with or in which CD163 plays a role, such as a pathogenic role (e.g., complete or partial pathogenicity) or a necessary role, comprising administering to a subject in need 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 herein.

[0337] Treatment or prevention of infection in pigs is preferred, particularly viral infection in pigs. Treatment or prevention of PRRSV infection is especially preferred, such as treatment or prevention of PRRS virus type 1 and / or PRRS virus type 2 infection, or treatment or prevention (e.g., specific treatment or prevention) of PRRS virus type 2 infection.

[0338] Therefore, another approach is provided for treating or preventing porcine PRRSV infection, such as treating or preventing porcine PRRS virus type 1 and / or 2 infection, comprising administering a therapeutically effective amount of a monoclonal antibody binding to porcine CD163 to a subject in need. Suitable CD163 antibodies (or binding proteins) for such approaches are described herein.

[0339] The embodiments of the therapeutic use of the invention described herein are applicable in comparison to this aspect of the invention.

[0340] The effective therapeutic dose will be determined based on clinical assessment and can be easily monitored.

[0341] As a further alternative consideration, the present invention provides the use of the anti-CD163 antibody (or binding protein) of the present invention, as defined herein, such as the monoclonal antibody of the present invention, in the preparation of medicaments for therapeutic purposes. Preferred therapeutic uses are described elsewhere herein, particularly for the treatment or prevention of diseases or conditions associated with or in which CD163 plays a role, such as a pathogenic role (e.g., complete or partial pathogenicity) or a necessary role. For example, the anti-CD163 antibody (or binding protein) of the present invention can be used to treat or prevent any infection caused by a virus or other pathogen, wherein the infection is associated with CD163, or wherein CD163 plays a role, such as a pathogenic role (e.g., complete or partial pathogenicity) or a necessary role. In other words, according to the present invention, the anti-CD163 antibody (or binding protein) can target and inhibit or reduce the function of CD163, particularly CD163 expressed on or in cells of PAMs or other CD163-positive cells. Therefore, the anti-CD163 antibody (or binding protein) as defined herein can be used to treat or prevent any disease or condition in which inhibiting CD163 or blocking or reducing CD163 function is beneficial.

[0342] Preferred embodiments provide the use of the anti-CD163 antibody (or binding protein) of the present invention in the preparation of a medicament for treating or preventing porcine infections, preferably porcine viral infections. Particularly preferred is the treatment or prevention of PRRSV infection, such as the treatment or prevention of PRRS virus type 1 and / or PRRS virus type 2 infection, for example, PRRS virus type 1 and PRRS virus infection, or the treatment or prevention (e.g., specific treatment or prevention) of PRRS virus type 2 infection.

[0343] Therefore, another aspect is provided regarding the use of monoclonal antibodies binding to porcine CD163 in the preparation of medicaments for the treatment or prevention of PRRS virus infection in pigs, preferably type 1 and / or type 2 PRRS virus infection. Suitable CD163 antibodies (or binding proteins) for such uses are described herein.

[0344] The embodiments of the therapeutic use of the invention described herein are applicable in comparison to this aspect of the invention.

[0345] In some embodiments, the antibodies of the present invention can be used in combination.

[0346] Any combination of VHH antibodies 49(#18), 47(#19), 48(#20), 76(#2), 77(#16), 78(#8), 150(#15), 70(#23), and 144(#1) as shown in Tables A, B, C, D, E, F, G, H, and I can be used. Therefore, 2, 3, 4, 5, 6, 7, 8, or all 9 of these can be used in combination, preferably 2 or 3, and more preferably 2.

[0347] Preferred combinations include:

[0348] One or more of 70(#23), 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0349] One or more of 144(#1) and 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0350] One or more of 150 (#15) and 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19) or 48 (#20), preferably one of them, such as 150 (#15) and 47 (#19);

[0351] One or more of 76(#2) and 70(#23) or 144(#1) or 150(#15), preferably one;

[0352] One or more of 77 (#16) and 70 (#23) or 144 (#1) or 150 (#15), preferably one;

[0353] One or more of 49 (#18) and 70 (#23) or 144 (#1) or 150 (#15), preferably one;

[0354] One or more of 47 (#19) and 70 (#23) or 144 (#1) or 150 (#15), preferably one;

[0355] One or more of 48 (#20) and 70 (#23) or 144 (#1) or 150 (#15), preferably one; or

[0356] One or more of 78 (#8) and 70 (#23) or 144 (#1) or 150 (#15), preferably one.

[0357] Preferred combinations include:

[0358] 76 (#2) and 150 (#15);

[0359] 76 (#2) and 77 (#16);

[0360] 76 (#2) and 48 (#20);

[0361] 150 (#15) and 77 (#16);

[0362] 150 (#15) and 48 (#20);

[0363] 77 (#16) and 48 (#20); or

[0364] 150(#15) and 77(#16) and 48(#20).

[0365] Any combination of VHH antibodies 57 (#11), 41 (#12), 171 (#14), and 29 (#17), as shown in Tables 1, 2, 3, and 4 respectively, can be used in this invention. Therefore, two, three, or all four can be used in combination, preferably two or three, and more preferably two.

[0366] Preferred combinations include:

[0367] One or more of 57(#11) and 41(#12) or 29(#17), preferably one, such as 57(#11) and 29(#17);

[0368] One or more of 171 (#14) and 41 (#12) or 29 (#17), preferably one;

[0369] One or more of 41(#12) and 57(#11) or 171(#14), preferably one; or

[0370] One or more of 29 (#17) and 57 (#11) or 171 (#14), preferably one.

[0371] Other combinations include:

[0372] One or more of the VHH antibodies 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, preferably one of them, and any combination of one or more of the VHH antibodies 57(#11), 41(#12), 171(#14) or 29(#17) as shown in Tables 1, 2, 3 and 4 respectively, preferably one of them.

[0373] Preferred combinations include:

[0374] 150 (#15) and 29 (#17);

[0375] 47 (#19) and 29 (#17); or

[0376] 144 (#1) and 29 (#17).

[0377] Other preferred combinations include:

[0378] One or more of 57 (#11) and 41 (#12) or 29 (#17), preferably one;

[0379] One or more of 57(#11) and 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0380] One or more of 171 (#14) and 41 (#12) or 29 (#17), preferably one;

[0381] One or more of 171(#14) and 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0382] One or more of 70 (#23) and 41 (#12) or 29 (#17), preferably one;

[0383] One or more of 70(#23), 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0384] One or more of 144(#1) and 41(#12) or 29(#17), preferably one;

[0385] One or more of 144(#1) and 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0386] One or more of 150 (#15) and 41 (#12) or 29 (#17), preferably one; or

[0387] One or more of 150 (#15) and 76 (#2), 78 (#8), 77 (#16), 49 (#18), 47 (#19) or 48 (#20), preferably one.

[0388] The preferred combinations available include:

[0389] One or more of 41 (#12), 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one;

[0390] One or more of 41(#12) and 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0391] 29(#17) and one or more of 57(#11), 171(#14), 70(#23), 144(#1) or 150(#15), preferably one; or

[0392] One or more of 29(#17) and 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one.

[0393] The preferred combinations available include:

[0394] One or more of 76(#2), 57(#11), 171(#14), 70(#23), 144(#1), or 150(#15), preferably one;

[0395] One or more of 76(#2) and 41(#12) or 29(#17), preferably one;

[0396] One or more of 77 (#16), 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one;

[0397] One or more of 77 (#16) and 41 (#12) or 29 (#17), preferably one;

[0398] One or more of 49 (#18), 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one;

[0399] One or more of 49(#18) and 41(#12) or 29(#17), preferably one;

[0400] One or more of 47(#19), 57(#11), 171(#14), 70(#23), 144(#1), or 150(#15), preferably one;

[0401] One or more of 47 (#19) and 41 (#12) or 29 (#17), preferably one;

[0402] One or more of 48 (#20), 57 (#11), 171 (#14), 70 (#23), 144 (#1), or 150 (#15), preferably one;

[0403] One or more of 48 (#20) and 41 (#12) or 29 (#17), preferably one;

[0404] One or more of 78(#8), 57(#11), 171(#14), 70(#23), 144(#1), or 150(#15), preferably one; or

[0405] One or more of 78 (#8) and 41 (#12) or 29 (#17), preferably one.

[0406] Preferred combinations include:

[0407] One or more of 57(#11) and 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0408] One or more of 171(#14) and 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0409] One or more of 70 (#23) and 41 (#12) or 29 (#17), preferably one;

[0410] One or more of 144(#1) and 41(#12) or 29(#17), preferably one;

[0411] One or more of 150 (#15) and 41 (#12) or 29 (#17), preferably one;

[0412] One or more of 41 (#12) and 70 (#23), 144 (#1) or 150 (#15), preferably one;

[0413] One or more of 41(#12) and 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0414] One or more of 29 (#17) and 70 (#23), 144 (#1) or 150 (#15), preferably one;

[0415] 29(#17) and one or more of 76(#2), 78(#8), 77(#16), 49(#18), 47(#19) or 48(#20), preferably one;

[0416] One or more of 76(#2) and 57(#11) or 171(#14), preferably one;

[0417] One or more of 76(#2) and 41(#12) or 29(#17), preferably one;

[0418] One or more of 77 (#16) and 57 (#11) or 171 (#14), preferably one;

[0419] One or more of 77 (#16) and 41 (#12) or 29 (#17), preferably one;

[0420] One or more of 49 (#18) and 57 (#11) or 171 (#14), preferably one;

[0421] One or more of 49(#18) and 41(#12) or 29(#17), preferably one;

[0422] One or more of 47(#19) and 57(#11) or 171(#14), preferably one;

[0423] One or more of 47 (#19) and 41 (#12) or 29 (#17), preferably one;

[0424] One or more of 48 (#20) and 57 (#11) or 171 (#14), preferably one;

[0425] One or more of 48 (#20) and 41 (#12) or 29 (#17), preferably one;

[0426] One or more of 78 (#8) and 57 (#11) or 171 (#14), preferably one; or

[0427] One or more of 78 (#8) and 41 (#12) or 29 (#17), preferably one.

[0428] In all of the above combinations, antibodies with three CDRs as shown in Tables A to I and Tables 1 to 4 may also be used.

[0429] Preferred combinations are those that result in improved or increased therapeutic efficacy compared to any antibody of the present invention (e.g., VHH) administered as a single active agent (monotherapy), a single antibody, or a single anti-CD163 agent, preferably those combinations that show a significant improvement or increase. Other preferred combinations are those in which the various anti-CD163 antibodies in the combination bind to different epitopes on the CD163 molecule.

[0430] For such combination therapy using two or more antibodies (or binding proteins) of the present invention, the second (or subsequent) anti-CD163 antibody of the present invention may be administered to the subject substantially simultaneously with the first anti-CD163 antibody, for example from a single pharmaceutical composition or from two pharmaceutical compositions administered closely together (at the same or similar times). Alternatively, the second (or subsequent) anti-CD163 antibody of the present invention may be administered to the subject before or after the administration of the first anti-CD163 antibody of the present invention. As used herein, “before or after administration” means “interleaved,” such that the administration of the second antibody is given to the subject at a different time than the administration of the first anti-CD163 antibody component. Generally, the two (or more) components can be effectively spaced out or administered together so that each component can exert its respective therapeutic effect, i.e., they are administered at “bioeffective amounts” at “bioeffective time intervals” and as part of the same treatment regimen.

[0431] If appropriate, combinations of the anti-CD163 antibodies (or binding proteins) of the present invention can be conveniently administered as part of the same molecule or construct, for example, by conjugation or linkage, e.g., using artificial linkers. This route of administration is particularly suitable for VHH antibodies (or other types of antibody molecules consisting of a single polypeptide chain), whose individual antibodies can be conveniently linked to a single polypeptide chain containing multiple VHH (or other) antibodies of the present invention, or combined with other VHH or other antibodies, via suitable peptide (or other) linkers (e.g., non-natural peptides or artificial linkers). In such embodiments, suitable techniques, such as spacers, are typically used to link the agents together so that each component can perform its respective function, e.g., binding to CD163. For example, in embodiments where the anti-CD163 antibodies of the present invention bind to different epitopes on CD163, such combinations of antibodies are preferred, and the construct is appropriately designed so that each individual antibody can bind to CD163, e.g., to its CD163 epitope.

[0432] Therefore, in some embodiments, the anti-CD163 antibody (or binding protein) of the present invention may be used as the sole active agent in a treatment regimen (monotherapy), or more than one anti-CD163 antibody of the present invention may be used in combination, for example as described above. In some embodiments, the anti-CD163 antibody (or binding protein) (or suitable combination) of the present invention may be used as the sole active anti-CD163 agent or the sole active anti-CD163 antibody in a treatment regimen, or they may be the sole active anti-PRRSV agent in a treatment regimen. However, in some embodiments, additional anti-CD163 agents or anti-PRRSV agents may be used.

[0433] Therefore, the anti-CD163 binding protein or antibody (or suitable combination thereof) of the present invention can be combined with one or more other (other CD163-targeting or non-CD163-targeting) active agents, such as with at least a second therapeutic agent or biological agent, wherein the anti-CD163 binding protein or antibody (or combination of such binding proteins or antibodies) of the present invention is the first.

[0434] The anti-CD163 antibody (or binding protein) (or a combination thereof) of the present invention can be combined, for example, with any other therapeutic agent, such as PRRSV, for treating the diseases discussed elsewhere herein.

[0435] For such combination therapy, generally, the second agent (non-inventory anti-CD163 antibody) can be administered to the subject substantially simultaneously with the inventory anti-CD163 antibody (or a combination of such antibodies), for example from a single pharmaceutical composition or from two pharmaceutical compositions administered closely together (at the same or similar times). Alternatively, the second agent (non-inventory anti-CD163 antibody) can be administered to the subject before or after the administration of the inventory anti-CD163 antibody component. As used herein, "before or after" means "interleaved," such that the second agent (non-inventory anti-CD163 antibody) is administered to the subject at a different time than the administration of the anti-CD163 antibody component. Typically, the two (or more) components can be effectively spaced apart or administered together to allow both components to exert their respective therapeutic effects, i.e., they are administered at "bioeffective amounts" at "bioeffective time intervals" and as part of the same treatment regimen.

[0436] The present invention also includes a kit comprising one or more antibodies or compositions of the present invention, or one or more nucleic acid molecules encoding antibodies of the present invention, or a recombinant expression vector comprising one or more nucleic acid sequences of the present invention, or a host cell or virus comprising one or more recombinant expression vectors or nucleic acid sequences of the present invention. Preferably, the kit is used for the methods and uses described herein, such as the treatment methods described herein. Preferably, the kit includes instructions for use of the kit components. Preferably, the kit is used to treat diseases or conditions as described elsewhere herein, and optionally includes instructions for using the kit components to treat such diseases or conditions. Examples equivalent to the binding proteins of the present invention are also provided.

[0437] The antibodies (or binding proteins) of the present invention, as defined herein, can also be used as molecular tools for in vitro or in vivo application and assay. Because antibodies (and some binding proteins) have antigen-binding sites, these antibodies can act as members of specific binding pairs, and these molecules can be used in any assay that requires a specific binding pair member.

[0438] Therefore, other aspects of the invention provide reagents comprising antibodies (or binding proteins) of the invention as defined herein, and the use of such antibodies (or binding proteins) as molecular tools, such as in in vitro or in vivo assays.

[0439] This article discloses a table of amino acid sequences and their sequence identifiers (SEQ ID NO).

[0440] All amino acid sequences are listed in this document from the N-terminus to the C-terminus, in accordance with the conventions of this technical field.

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453] Table J - Common Sequence

[0454]

[0455]

[0456]

[0457]

[0458]

[0459] Attached Figure Description

[0460] The invention will now be further described with reference to the following figures and in the following non-limiting examples:

[0461] Figure 1 Blocking PRRS virus type 1 BOR57 in the presence of high levels of porcine serum

[0462] Data in Figures A through I demonstrate the ability of the candidate VHH antibody to inhibit productive viral infection of BOR57 PRRS virus type 1 when incubated with porcine alveolar macrophages for 17 hours in the presence of 80% porcine serum. The candidate VHH antibody was evaluated over a dose-response range of 50 to 400 μg / mL. Data are presented as mean + / - SEM (n>3) relative to infection in the absence of the test sample, compared to a simulant control.

[0463] Figure 2 Blocking PRRS virus type 2 MN184 in a culture medium containing 10% FBS.

[0464] Data demonstrate the ability of the candidate VHH antibody to inhibit productive viral infection of MN184 PRRS virus type 2 when incubated with porcine alveolar macrophages for 17 hours in the presence of 10% FBS. The candidate antibody was evaluated over a dose response range of 50 to 300 μg / mL. Data are presented as mean + / - SEM (n>3) relative to infection in the absence of the test sample, compared to a simulant control.

[0465] Figure 3 PRRS virus type 2 specific VHH inhibitory antibody blocks type 1 virus infection.

[0466] Data demonstrated that the candidate VHH antibody inhibited productive viral infection of BOR57 PRRS virus type 1 when incubated with porcine alveolar macrophages in the presence of 80% porcine serum for 17 hours. The candidate antibody was evaluated over a dose-response range of 1 to 100 μg / mL. Data are presented as mean + / - SEM (n>3) relative to infection in the absence of the test sample, compared to a simulant control.

[0467] Figure 4 Blocking type 1 PRRS virus BOR57 using the VHH combination containing VHH15, VHH16, and VHH20.

[0468] Data show that individual VHHs can be used in combination to block infection with PRRS virus type 1. Combinations of VHH15 and VHH16, VHH15 and VHH20, and VHH16 and VHH20 showed effective blocking of productive virus infection. Each VHH in the tested VHH pairs was tested at a single concentration of 100 μg / mL. Additionally, a triple combination of VHH15 + VHH16 + VHH20 was evaluated, with each VHH present at a concentration of 50 μg / mL. Data demonstrate the potential of VHH combinations for blocking PRRS virus infection. Data are presented as a comparison with a simulant control relative to infection in the absence of the test sample.

[0469] Figure 5 Use the VHH combination containing VHH02, VHH15, VHH16, and VHH20 to block type 1 PRRS virus BOR57.

[0470] Data show that individual VHHs can be used in combination to block infection with PRRS virus type 1. Combinations of VHH02 and VHH15, VHH02 and VHH16, and VHH02 and VHH20 showed effective blocking of productive viral infection for each VHH in each VHH pair within a concentration range of 3 μg / mL to 100 μg / mL. These data demonstrate the potential of VHH combinations for blocking PRRS virus infection. Data are presented as a comparison with a simulant control relative to infection in the absence of the test sample.

[0471] Figure 6 X-ray crystallographic structure of the interaction between VHH14(02D01) and the SRCR5 domain of porcine CD163

[0472] The left image shows the fine structure of the VHH14:SRCR5 complex. On the left is the CD163:SRCR5 domain, consisting of one long β-sheet and two short β-sheets. The two shorter β-sheets are curved antiparallel towards the N-terminus, followed by a single α-helix. On the right is the core structure of VHH14(02D01), consisting of seven antiparallel β-sheets and three short α-helices positioned around the core. Each protein is labeled with its C-terminus and N-terminus.

[0473] The figure on the right is an example of the outlined electron density (2m|Fo|-D|Fc|) at the 1σ level. Protein chains and individual amino acids are plotted as lines. The data show a clear interaction between tyrosine (Y59) and aspartic (D62) residues in the CDR2 domain of VHH14 and a pair of leucine residues (Leu 526, Leu 527) in the porcine SRCR5 domain. Further interactions were also observed between leucine 104 of VHH14 (CDR3) and a pair of amino acids in serine 507 (S507) and glutamic acid 509 (E509) of SRCR5. The crystal structure was refined to 2.0 to 2.3 Å. Leu(L)52 in this figure corresponds to Leu(L)527 in the full-length CD163 molecule shown in SEQ ID NO: 116. In this figure, Leu(L)51 corresponds to Leu(L)526 in the full-length CD163 molecule shown in SEQ ID NO:116. In this figure, Ser(S)32 corresponds to Leu(L)507 in the full-length CD163 molecule shown in SEQ ID NO:116. In this figure, Glu(E)34 corresponds to Leu(L)509 in the full-length CD163 molecule shown in SEQ ID NO:116. Asp(D)62, Tyr(Y)59, and Leu(L)104 in VHH 02D01 are shown in SEQ ID NO:17' or 99 (Table 3). Detailed Implementation

[0474] Example

[0475] Example 1: Immunization, library generation, screening, and clonal selection

[0476] Materials and methods:

[0477] immunity

[0478] Single-domain antibodies were obtained from llamas immunized with recombinant proteins. Llamas were injected with porcine CD163 Fc fusion antigen formulations (pCD163-SRCR1-9-huFc and pCD163-SRCR4-7-huFc) prepared in incomplete Freund's adjuvant. Animals were immunized with six subcutaneous injections at weekly intervals (two 100 μg injections followed by four 50 μg injections). One week after the final booster immunization, serum was collected, and antibody titers against pCD163-SRCR1-9-huFc and pCD163-SRCR4-7-huFc were determined by ELISA.

[0479] In this ELISA, 96-well plates (Maxisorp; Nunc) were coated with recombinant protein. After blocking and adding diluted serum samples, the presence of anti-pCD163 antibody was demonstrated using mouse anti-camel IgG2 / 3 (EMD microwells; product number MAC131), followed by anti-mouse immunoglobulin peroxidase conjugate (JIR, product number 715-035-150).

[0480] Library Construction

[0481] RNA was extracted from the PBMCs of three immunized llamas (400 ml each). 40 μg of RNA was used for cDNA synthesis using random primers. The cDNA was amplified by a single PCR using unlabeled primers annealed at the leader sequence and the CH1 region of the hinge, followed by a second PCR amplification to introduce restriction endonuclease sites for cloning the VHH gene in the pDCL1 phagemid vector. The library was electroporated into TG1 *E. coli* cells, and the bacterial glycerol stock solution of the immunoglobulin was stored at -80°C (FL1158 and FL1159).

[0482] choose

[0483] Phage production from the llama VHH library pool was used in two consecutive rounds of phage display selection using pCD163 recombinant protein or porcine alveolar macrophages (pPAM). Selection rounds on the recombinant protein were performed using 10 μg / ml pCD163-SRCR1-9-huFc or pCD163-SRCR4-7-huFc at pH 7.4 (PBS buffer), followed by washing of non-specific phages and then elution of specific phages with trypsin (total elution). Selection rounds on pPAM were performed using 5E106 cells at pH 7.4 (PBS buffer), followed by washing of non-specific phages and then elution of specific phages with trypsin (total elution). The eluted phages were serially diluted and used for TG1 infection exponential growth. Infected TG1 was plated on LBCarb100Glu 2% plates, and enrichment values ​​were calculated against the background (no antigen used for selection).

[0484] ELISA screening

[0485] Single clones from the second round of selection criteria were picked into 96-well plates and used as periplasmic extracts (PEs) to test their binding to pCD163-SRCR4-7-huFc or pCD163-SRCR5-6-huFc proteins via a binding ELISA at pH 7.0. For the PE binding ELISA, MaxiSorp... TM High protein-binding capacity 96-well ELISA plates were coated with 1 μg / ml pCD163-SRCR4-7-huFc protein, diluted in PBS, and incubated overnight at 4°C. The next day, the plates were washed three times with 0.05% PBS Tween (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 0.05% PBS Tween (pH 7.4), and each well was incubated with 20 μl of P.E. and 80 μl of 1% Marvel / PBS (pH 7.4) at room temperature with shaking for 1 hour. The plate was washed three times with 0.05% PBS Tween (pH 7.4) and incubated with 100 μl of anti-c-Myc antibody (Roche; product number 11667203001) followed by a second antibody, DAM-HRP (JIR; product number 715-035-150), in 1% Marvel / PBS (pH 7.4) at room temperature with shaking for 1 hour. The plate was washed three times with 0.05% PBS Tween (pH 7.4) and the substrate solution (TMB solution) was added. The reaction was terminated with H2SO4, and the plate was read at 450 nm using a plate reader.

[0486] Cell screening (FACS):

[0487] Periplasmic extract (PE) from the selected clone was incubated with an anti-c-myc antibody (Roche; product number 11667203001) specific to the c-myc tag present in soluble VHH at room temperature (RT) with stirring for 30 minutes. The mixture (P.E + anti-c-myc antibody) was added to the pPAM or pPAMΔ5 domain (for cells lacking SRCR domain 5) and incubated at 4°C with gentle shaking for 60 minutes.

[0488] The cells were washed three times with 150 μl / well of FACS buffer and incubated with 50 μl / well of the secondary antibody GAM-APC at 4°C for 30 minutes with shaking in the dark.

[0489] Cells were washed three times with 150 μl / well of FACS buffer and resuspended in 75 μl / well of FACS buffer for testing on an FACS instrument (Attune). TM Measurements were performed in the RL-1 channel (APC channel) of NxT, and a total of 10,000 cells were obtained for each sample.

[0490] sequencing

[0491] Positive conjugates were sent for sequencing. Clones were classified into families based on their different HCDR3 sequences.

[0492] Results and Discussion:

[0493] Table 5: Summary of ELISA Data

[0494]

[0495] Table 6: Evaluation of candidate binding to primary alveolar macrophages by flow cytometry

[0496]

[0497] After immunizing llamas with recombinant CD163, phage clones were screened through two rounds of phage panning. Phage candidates were confirmed by ELISA using recombinant CD163 expression constructs (pCD163SRCR1-9-Fc or pCD163-SRCR4-7-Fc) compared to control human IgG. As shown in Table 5, several candidates exhibited selective binding to porcine CD163.

[0498] Further screening was conducted to assess the ability of clones to bind to native membrane-bound CD163 on isolated primary porcine alveolar macrophages, which were prepared from cells recovered from bronchoalveolar lavage of donor animals (Burkard C. et al., PLOS Pathogens 2017). Candidate phages were demonstrated to bind to native membrane-bound CD163 at 4°C and room temperature when the CD163 receptor was more likely to be internalized via endocytosis.

[0499] The selective binding of all candidates to the SRCR5 domain of CD163 was further evaluated by selecting candidates that could not bind to porcine alveolar macrophages isolated from pigs, which have a deletion of this domain in the CD163 gene (Burkard C. et al., PLOS Pathogens 2017). All selected candidates failed to bind to PAM isolated from these animals, thus demonstrating preferential binding to the SRCR5 domain of porcine CD163.

[0500] Therefore, immunization of llamas with a recombinant CD163 protein construct resulted in the successful isolation of a candidate phage antibody that can bind to CD163 expressed on primary porcine alveolar macrophages, specifically targeting the SRCR5 domain, which is known to be essential for PRRS virus infection in these cells.

[0501] References:

[0502] Burkard C, Lillico SG, Reid E, Jackson B, Mileham AJ, Ait-Ali T et al., (2017), Precise engineering of porcine PRRSV resistance: macrophages from genome-edited pigs lacking the CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLOS Pathogens 13(2):e1006206.doi:10.1371 / journal.ppat.1006206

[0503] Example 2: Determination of the affinity of anti-CD163 VHH antibody for porcine CD163

[0504] Materials and methods:

[0505] The binding affinity of each identified VHH antibody candidate to porcine CD163 was determined by surface plasmon resonance.

[0506] Expression and purification of VHH candidate antibodies

[0507] Synthetic genes encoding VHH variable domains with FLAG and His tags 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. ExpiCHO-S cells were transfected with 23 leader region sequences using 40 μg of total DNA plasmid constructs. 25 mL of cells were used for protein production for 8 days (32°C, 5% CO2). The generated VHH antibody was captured from the clear supernatant using a HisTrap HP 5mL IMAC column (GE Healthcare, product number 17-5248-02) on a Pure 25FPLC system. The eluted antibody peak fractionation buffer was replaced with 1x PBS pH 7.4 and concentrated using a 3kDa MCO rotary concentrator (Amicon, product number UFC900324). The purified protein was analyzed for the presence of the correct strand by size exclusion chromatography (aSEC) and SDS-PAGE.

[0508] Affinity Measurement

[0509] To evaluate the affinity of the selected purified clones for pCD163, pCD163-SRCR1-9-huFc or pCD163-SRCR4-7-huFc proteins were amine-conjugated onto a CM5 sensing vector (GE Healthcare). Surface cytoplasmic resonance (SPR) (Biacore 3000, GE Healthcare) was used to determine the binding kinetics of the selected single-domain antibody at pH 7.4. Approximately 2000 RU of pCD163-SRCR1-9-huFc or pCD163-SRCR4-7-huFc protein was immobilized onto the CM5 chip at a concentration of 20 or 30 μg / ml in acetate buffer at pH 5.0 or pH 5.5 using a standard amine conjugation procedure. QC of immobilization was performed using a commercially available anti-huFc antibody (JIR, product number 109-005-098) at a concentration of 30.0 μg / ml.

[0510] During binding kinetic measurements, 1x HBS-EP pH 7.4 was used as the run buffer. Purified VHH was injected at 3-fold dilutions (300 nM; 100 nM; 33 nM; 11 nM; 3.7 nM; 1.2 nM; 0.4 nM; 0 nM) for 120 s at a flow rate of 30 μl / min. RU levels were restored to baseline 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 and mass transfer fit was applied to the set sample curves using the simultaneous fitting option of BIAevaluation software to calculate the kinetic constants of antibody-antigen interactions, including association rate (ka), dissociation rate (kd), and affinity (KD). Curves were removed from the fit after visual inspection of residues and consideration of Chi2 values; a minimum of four curves were considered in the simultaneous fit.

[0511] Results and Discussion:

[0512] Table 7: Determination of VHH's affinity for porcine CD163

[0513]

[0514] As described above, phage candidates were expressed and purified as VHHs. As mentioned above, the binding of each VHH to the full-length or truncated form of CD163 was characterized by surface plasmon resonance. The binding and dissociation rates of each antibody were determined, and affinity assays were calculated. As shown in Table 7, the KD values ​​of the antibody candidates ranged from 1 to 75 nM. The relative affinities to the full-length or truncated pCD163-SRCR4-7 constructs were generally similar.

[0515] Example 3: Candidate CD163-specific antibody inhibits porcine respiratory and reproductive (PRRS) virus infection in primary porcine alveolar macrophages.

[0516] Materials and methods:

[0517] PRRS Virus Infection Protocol

[0518] reagents

[0519] VHH candidate antibodies were aliquoted at a concentration of 1 mg / mL.

[0520] Control antibody:

[0521] Primary antibody: Anti-PRRS 1AC7, Ingenasa

[0522] Secondary antibody: Goat anti-mouse IgG (H+L) Alexa Fluor Plus 488, ThermoFisher, A32723

[0523] Culture medium:

[0524] Complete RPMI (10% FBS or 80% (high) porcine serum, Ultraglutamine, Pen / Strep)

[0525] PAM isolation: as described by Burkard et al., PLOS Pathogens 2017, involves the isolation of porcine alveolar macrophages.

[0526] Virus isolates:

[0527] Type 1 virus: BOR57 isolate (Roslin Institute, Edinburgh, UK)

[0528] Type 2 virus: MN184 American strain (Han et al., 2006)

[0529] Infection protocol

[0530] Day 1 - Seed Cells

[0531] Porcine alveolar macrophages were seeded at 20 million cells / plate in 48-well plates of complete RPMI and incubated overnight in a CO2 incubator.

[0532] Day 2 - VHH Treatment and Infection Attack

[0533] 1. Pretreatment (30 minutes before infection)

[0534] a. Aspirating culture medium from cells

[0535] b. Add 100 μL of culture medium to both the untreated uninfected control and the untreated infected control.

[0536] c. Add 20 μL of PBS from 100 μL of culture medium to the infected control treated with the simulant.

[0537] d. Add an appropriate amount of VHH stock solution to 100 μL of culture medium to the treated infected sample.

[0538] e. Place the plate back into the CO2 incubator for 30 minutes.

[0539] 2. Thaw the original virus solution and sonicate it for 15 seconds before use.

[0540] 3. Infection attack (2 hours)

[0541] a. Remove the culture medium from the cells and preserve the medium containing VHH for overnight incubation.

[0542] b. Add 100 μL of culture medium to the untreated, uninfected control.

[0543] c. Add 10 μL of virus from 100 μL of culture medium to the untreated infected control.

[0544] d. Add 10 μL of virus from 100 μL of culture medium to 20 μL of PBS and then add the virus to the infected control treated with the simulant.

[0545] e. Add an appropriate amount of VHH stock solution and 10 μL of virus from 100 μL of culture medium to the treated infected sample.

[0546] f. Gently stir the plate and return it to the CO2 incubator.

[0547] g. Gently stir the plate every 15 minutes for 2 hours.

[0548] 4. Incubate overnight (15 hours)

[0549] a. Aspirating culture medium from cells

[0550] b. Add 100 μL of culture medium to both the untreated uninfected control and the untreated infected control.

[0551] c. Add 20 μL of PBS from 100 μL of culture medium to the infected control treated with the simulant.

[0552] d. Add the VHH-containing culture medium retained from the pretreatment step to the appropriate sample.

[0553] e. Return the plate to the CO2 incubator for 15 hours.

[0554] Day 3 - In-hole fixation and staining protocol

[0555] 5. Aspirate culture medium from cells

[0556] 6. Fix cells in 4% formaldehyde / PBS++ (containing calcium and magnesium) solution for 30 minutes at room temperature.

[0557] 7. Wash once with PBS++

[0558] 8. Communicate with Triton-X (1%, in PBS++) for 5 minutes at room temperature.

[0559] 9. Wash once with PPBS++ or blocking solution (PBS++ / 5% FBS).

[0560] 10. Block with blocking solution (PBS++ / 5% FBS) for 20 minutes at room temperature.

[0561] 11. Add the primary antibody anti-PRRS 1AC7 at a dilution of 1:5000 to all wells except for the unstained control and the control containing only the secondary antibody.

[0562] 12. Incubate at room temperature for 1 hour.

[0563] 13. Wash three times with PBS++

[0564] 14. Add the second antibody, goat anti-mouse IgG (H+L) Alexa Fluor Plus 488, at a ratio of 1:5000 to all wells except the unstained control.

[0565] 15. Incubate at room temperature for 45 minutes.

[0566] 16. Wash three times with PBS++

[0567] 17. Add 300 μL PBS++

[0568] 18. Scrape the cells with the tip of a wide-bore P200 pipette, then scrape around the edge of the well with a normal P200 tip, then wash the surface of the well three times with P1000, collect the cells and transfer them to FACs tubes.

[0569] 19. Measurements were taken on a Fortessa x20 (voltage: FSC = 418, SSC = 308 & B530-30 = 386).

[0570] Results and discussion:

[0571] The following describes the ability of a single VHH to block the infection of PAM host cells by members of the PRRS virus family. The assays described above were used to measure the extent of viral infection, quantified as the ability to spread the virus by FACS measurement after a 17-hour infection cycle. The data provided are shown in… Figure 1 and Figure 2 The results are summarized in Table 8 below.

[0572] Table 8: Blocking PRRS Virus Infection

[0573]

[0574] nd-Undetermined

[0575] The data clearly show that VHH can inhibit type 1 (see also...) Figure 1 ) and Type 2 (see also) Figure 2 Productive infection of porcine alveolar macrophages by PRRS isotypes. VHHs showing activity in infection assays were divided into VHHs effective against both type 1 and type 2 PRRS infection (VHH 001, 002, 008, 015, 016, 018, 019, 020, and 023), and VHHs showing no inhibitory activity against type 1 PRRS infection but selective inhibitory activity against type 2 PRRS infection (VHH 011, 012, 014, and 017). The data clearly demonstrate that VHHs can inhibit productive infection of porcine alveolar macrophages by both type 1 and type 2 PRRS isotypes.

[0576] At concentrations up to 300 μg / mL, some VHHs showed inhibitory activity only in type 2 virus infections (VHH011, 012, 014, 017). Figure 2 These candidate VHHs showed specificity against type 2 PRRS virus and no inhibitory activity in type 1 virus infection assays at similar dose-response concentrations up to 100 μg / mL. Figure 3 ).

[0577] References:

[0578] Burkard C. et al., Precise engineering of porcine PRRSV resistance: Macrophages from genome-edited pigs lacking the CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function. PLOS Pathogens, 13(2)2017:e1006206

[0579] Han J, Y. Wang, KSFaaberg. Complete genome analysis of RFLP 184 isolate of porcine reproductive and respiratory syndrome virus, Virus Research, 122 (2006), p. 175.

[0580] Example 4: A combination of candidate CD163-specific antibodies inhibits porcine respiratory and reproductive (PRRS) virus infection in primary porcine alveolar macrophages.

[0581] Materials and methods:

[0582] PRRS Virus Infection Protocol

[0583] reagents

[0584] VHH candidate antibodies were aliquoted at a concentration of 1 mg / mL.

[0585] Control antibody:

[0586] Primary antibody: Anti-PRRS 1AC7, Ingenasa

[0587] Secondary antibody: Goat anti-mouse IgG (H+L) Alexa Fluor Plus 488, ThermoFisher, A32723

[0588] Culture medium:

[0589] Complete RPMI (10% FBS or 80% high porcine serum, Ultraglutamine, Pen / Strep)

[0590] PAM isolation: as described by Burkard et al., PLOS Pathogens 2017, involves the isolation of porcine alveolar macrophages.

[0591] Virus isolates:

[0592] Type 1 virus: BOR57 isolate (Roslin Institute, Edinburgh, UK)

[0593] Type 2 virus: MN184 American strain (Han et al., 2006)

[0594] Infection protocol

[0595] Day 1 - Seed Cells

[0596] Porcine alveolar macrophages were seeded at 20 million cells / plate in 48-well plates of complete RPMI and incubated overnight in a CO2 incubator.

[0597] Day 2 - VHH Treatment and Infection Attack

[0598] 1. Pretreatment (30 minutes before infection)

[0599] a. Aspirating culture medium from cells

[0600] b. Add 100 μL of culture medium to both the untreated uninfected control and the untreated infected control.

[0601] c. Add 20 μL of PBS from 100 μL of culture medium to the infected control treated with the simulant.

[0602] d. Add an appropriate amount of VHH stock solution to 100 μL of culture medium to the treated infected sample.

[0603] e. Place the plate back into the CO2 incubator for 30 minutes.

[0604] 2. Thaw the original virus solution and sonicate it for 15 seconds before use.

[0605] 3. Infection attack (2 hours)

[0606] a. Remove the culture medium from the cells and preserve the medium containing VHH for overnight incubation.

[0607] b. Add 100 μL of culture medium to the untreated, uninfected control.

[0608] c. Add 10 μL of virus from 100 μL of culture medium to the untreated infected control.

[0609] d. Add 10 μL of virus from 100 μL of culture medium to 20 μL of PBS and then add the virus to the infected control treated with the simulant.

[0610] e. Add an appropriate amount of VHH stock solution (or VHH combination) and 10 μL of virus from 100 μL of culture medium to the treated infected sample.

[0611] f. Gently stir the plate and return it to the CO2 incubator.

[0612] g. Gently stir the plate every 15 minutes for 2 hours.

[0613] 4. Incubate overnight (15 hours)

[0614] a. Aspirating culture medium from cells

[0615] b. Add 100 μL of culture medium to both the untreated uninfected control and the untreated infected control.

[0616] c. Add 20 μL of PBS from 100 μL of culture medium to the infected control treated with the simulant.

[0617] d. Add the VHH-containing culture medium retained from the pretreatment step to the appropriate sample.

[0618] e. Return the plate to the CO2 incubator for 15 hours.

[0619] Day 3 - In-hole fixation and staining protocol

[0620] 5. Aspirate culture medium from cells

[0621] 6. Fix cells in 4% formaldehyde / PBS++ (containing calcium and magnesium) solution for 30 minutes at room temperature.

[0622] 7. Wash once with PBS++

[0623] 8. Communicate with Triton-X (1%, in PBS++) for 5 minutes at room temperature.

[0624] 9. Wash once with PPBS++ or blocking solution (PBS++ / 5% FBS).

[0625] 10. Block with blocking solution (PBS++ / 5% FBS) for 20 minutes at room temperature.

[0626] 11. Add the primary antibody anti-PRRS 1AC7 at a dilution of 1:5000 to all wells except for the unstained control and the control containing only the secondary antibody.

[0627] 12. Incubate at room temperature for 1 hour.

[0628] 13. Wash three times with PBS++

[0629] 14. Add the second antibody, goat anti-mouse IgG (H+L) Alexa Fluor Plus 488, at a ratio of 1:5000 to all wells except the unstained control.

[0630] 15. Incubate at room temperature for 45 minutes.

[0631] 16. Wash three times with PBS++

[0632] 17. Add 300 μL PBS++

[0633] 18. Scrape the cells with the tip of a wide-well P200 pipette, then scrape around the edge of the well with a normal P200 tip, and then wash the surface of the well three times with P1000. Collect the cells and transfer them to FACs tubes.

[0634] 19. Measurements were taken on a Fortessa x20 (voltage: FSC = 418, SSC = 308 & B530-30 = 386).

[0635] Results and discussion:

[0636] The following describes the ability of a single VHH to block the infection of PAM host cells by members of the PRRS virus family. The assays described above were used to measure the extent of viral infection, quantified as the ability to spread the virus by FACS measurement after a 17-hour infection cycle. The data presented are in Figure 4and Figure 5 As shown in the image.

[0637] Combinations of VHH 15, 16, and 20 were used for infection assays with BOR57 type 1 PRRS virus in the presence of 10% FBS. Specific combinations of 100 μg each of VHH 15 and VHH 16, VHH 15 and VHH 20, VHH16 and VHH20, and a triplet combination of 50 μg each of VHH 15 + VHH 16 + VHH 20 showed infectivity that reduced the infection potential of type 1 PRRS virus to extremely low levels (see [link to relevant documentation]). Figure 4 ).

[0638] The potential of different VHH pair combinations was investigated in an infection assay using BOR57 type 1 PRRS virus in the presence of 10% FBS. VHH02 (01B04) was used as a partner in combination with VHH15 (02G01), VHH16 (02H11), and VHH20 (03H11) to assess the potential for blocking infection. Combinations were tested with increasing VHH concentrations in each partner. Data suggest that combinations of VHHs can be used to block type 1 PRRS virus infection (see [link]). Figure 5 ).

[0639] Data show that the combination of VHHs can enhance the inhibitory effect of type 1 PRRS isotypes on productive infection of porcine alveolar macrophages, possibly to a greater extent than VHH candidates alone. The data suggest that individual VHHs can be combined to potentially enhance the blocking effect against infection of PRRS virus family members.

[0640] References:

[0641] Burkard C. et al., ibid.; Han J, Y. Wang, KSFaaberg, ibid.

[0642] Example 5: Determination of the binding of anti-CD163 VHH antibody to porcine CD163 SRCR5

[0643] Materials and methods:

[0644] X-ray crystallography was performed on the porcine CD163 SRCR5 domain bound to VHH antibody candidate 2D01 (VHH14).

[0645] Expression and purification of VHH14 antibody

[0646] Synthetic genes encoding VHH variable domains with FLAG and His tags 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. ExpiCHO-S cells were transfected with 23 leader region sequences using 40 μg of total DNA plasmid constructs. 25 mL of cells were used for protein production for 8 days (32°C, 5% CO2). The generated VHH antibody was captured from the clear supernatant using a HisTrap HP 5mL IMAC column (GE Healthcare, product number 17-5248-02) on a Pure 25FPLC system. The eluted antibody peak fractionation buffer was replaced with 1x PBS pH 7.4 and concentrated using a 3kDa MCO rotary concentrator (Amicon, product number UFC900324). The purified protein was analyzed for the presence of the correct strand by size exclusion chromatography (aSEC) and SDS-PAGE.

[0647] Expression and purification of porcine SRCR5

[0648] A synthetic gene encoding the porcine CD163 SRCR5 region with a ^x HIS tag was subcloned into pTXBac1 (a proprietary vector) and then transformed into *E. coli* DH10Bac to generate a recombinant baculovirus plasmid. The recombinant baculovirus plasmid was used to transform *Spodoptera frugiperda* (Sf) cells to generate the P1 virus clone. Routine examination of protein expression in cells and culture medium samples from the P1 clone was performed to identify high-expression clones. High-expression clones were amplified by infecting Sf cells with the P1 virus stock solution to generate the P2 virus stock solution, which was subsequently used to express the recombinant SRCR5 protein in Sf1 cells over a 72-hour period at a multiplicity of infection (MOI) ~1.

[0649] Production was scaled up in 10L cultures of Sf cells. Optimal expression conditions were used as described above. Culture supernatant was equilibrated with phosphate-buffered saline (PBS) pH 7.5 before purification using the HIS tag on an IMAC according to standard protocols. Samples were washed with PBS pH 7.5 and 0.1% Triton X-114 buffer. Proteins were eluted with imidazole according to the manufacturer's instructions. The eluted samples were exchanged with PBS buffer at pH 7.5 and then further purified on cobalt-resin. Samples were eluted with imidazole after washing (as described above). The resulting elution buffer was replaced with 20 mM Tris-HCl, pH 7.4, 150 mM NaCl.

[0650] Table 9:

[0651]

[0652] Crystallization & X-ray Structure Determination Study

[0653] CD163 SRCR5: A VHH14 complex at a concentration of 11.84 mg / mL was prepared in PBS pH 7.4 buffer. Growth was carried out in 0.2 M NaCl, 0.1 M phosphate / citrate buffer pH 4.5, and 20% w / v PEG 8000. Various crystals were rapidly frozen in liquid nitrogen after the addition of a freezing solution containing: 0.14 M NaCl, 0.07 M phosphate / citrate buffer pH 4.5, 13.9% PEG 8000, and 46% ethylene glycol.

[0654] Data was collected at 100K on the BioMAX beamline of the Swedish MAX IV. The beamline is equipped with an Eiger 16M hybrid pixel detector.

[0655] The structures were determined using Phaser software and two homologous proteins (PDB codes: 5DA4 and 5JFB), and their compositions were measured. and It can simulate 103 amino acids of the CD163 SRCR5 domain (bold Table 9) and 122 amino acids of VHH014 (02D01), with residue 4 marked in bold forward (Table 9).

[0656] Results and Discussion:

[0657] The structure of CD163 SRCR5: The VHH014 complex reveals a mono-monomer SRCR5 domain (left) that interacts with the mono-monomer VHH014 antibody (right), as shown. Figure 6 As shown, the electron density map reveals specific interactions between residues in VHH014 and amino acids identified in the CD163:SRCR5 domain. Specifically, based on the XYAD / E / N motif in CDR2 of VHH14, leucine 526 and leucine 527 within the porcine CD163 SRCR5 domain appear to interact with specific conserved residues, a motif shared among all identified VHH candidates. VHH14 (02D01) is of interest because it does not inhibit type 1 PRRS virus infection but is able to reduce type 2 PRRS family virus infection. The dileucine motif identified in porcine CD163SRCR5 can represent a feature very important for the interaction between type 2 PRRS virus and SRCR5 and for productive infection of porcine alveolar macrophages. sequence list <110> Eco Animal Health Ltd. <120> CD163 antibody or binding protein <130> P22113986WP <160> 118 <170> PatentIn version 3.5 <210> 1 <211> 123 <212> PRT <213> llamas <400> 1 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Val Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ala Pro Ser Arg Tyr 20 25 30 Val Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Ala Trp Ser Gly Arg Ala Pro Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala 85 90 95 Gly Gly Glu Gly Ala Ile Arg Trp Thr Thr Leu Asp Ala Tyr Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 2 <211> 5 <212> PRT <213> llamas <400> 2 Arg Tyr Val Met Gly 1 5 <210> 3 <211> 16 <212> PRT <213> llamas <400> 3 Gly Ile Ala Trp Ser Gly Arg Ala Pro Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> llamas <400> 4 Gly Glu Gly Ala Ile Arg Trp Thr Thr Leu Asp Ala Tyr Asp Tyr 1 5 10 15 <210> 5 <211> 30 <212> PRT <213> llamas <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Val Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ala Pro Ser 20 25 30 <210> 6 <211> 14 <212> PRT <213> llamas <400> 6 Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Phe Val Ala 1 5 10 <210> 7 <211> 32 <212> PRT <213> Llama <400> 7 Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala Gly 20 25 30 <210> 8 <211> 11 <212> PRT <213> Llama <400> 8 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 9 <211> 123 <212> PRT <213> Llama <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Val Gly Ser 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Thr Ser Gly Arg Thr Pro Ser Arg Tyr 20 25 30 Val Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Trp Ser Gly Arg Ala Pro Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala 85 90 95 Gly Gly Glu Gly Ala Ile Lys Trp Thr Thr Leu Asp Ala Tyr Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 10 <211> 5 <212> PRT <213> Llama <400> 10 Arg Tyr Val Met Gly 1 5 <210> 11 <211> 16 <212> PRT <213> Llama <400> 11 Ala Ile Ser Trp Ser Gly Arg Ala Pro Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 12 <211> 15 <212> PRT <213> Llama <400> 12 Gly Glu Gly Ala Ile Lys Trp Thr Thr Leu Asp Ala Tyr Asp Tyr 1 5 10 15 <210> 13 <211> 30 <212> PRT <213> llamas <400> 13 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Val Gly Ser 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Thr Ser Gly Arg Thr Pro Ser 20 25 30 <210> 14 <211> 14 <212> PRT <213> llamas <400> 14 Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Phe Val Ala 1 5 10 <210> 15 <211> 32 <212> PRT <213> llamas <400> 15 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala Gly 20 25 30 <210> 16 <211> 11 <212> PRT <213> llamas <400> 16 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 17 <211> 123 <212> PRT <213> Llama <400> 17 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Val Gly Gly<着 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Pro Ser Arg Tyr 20 25 30 Val Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Ala Trp Ser Gly Arg Ala Pro Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Val Ile Ser Arg Asp Ser Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Val Tyr Tyr Cys Ala 85 90 95 Gly Gly Glu Gly Ala Ile Leu Trp Thr Thr Pro Gly Ala Tyr Asn Tyr 100 105 110<00着01598>Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 18 <211> 5 <212> PRT <213> Llama <400> 18 It should be noted that there seems to be a "着" character in the original text at line 12 which might be an error. I've translated it as best as possible while keeping all the tags intact.Arg Tyr Val Met Gly 1 5 <210> 19 <211> 16 <212> PRT <213> llamas <400> 19 Gly Ile Ala Trp Ser Gly Arg Ala Pro Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 20 <211> 15 <212> PRT <213> llamas <400> 20 Gly Glu Gly Ala Ile Leu Trp Thr Thr Pro Gly Ala Tyr Asn Tyr 1 5 10 15 <210> twenty one <211> 30 <212> PRT <213> llamas <400> twenty one Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Val Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Pro Ser 20 25 30 <210> twenty two <211> 14 <212> PRT <213> llamas <400> twenty two Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Phe Val Ala 1 5 10 <210> twenty three <211> 32 <212> PRT <213> Llama <400> 23 Arg Phe Val Ile Ser Arg Asp Ser Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Val Tyr Tyr Cys Ala Gly 20 25 30 <210> 24 <211> 11 <212> PRT <213> Llama <400> 24 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 25 <211> 121 <212> PRT <213> Llama <400> 25 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Val Thr Tyr 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala His Arg Trp Ser Gly Ser Ala Tyr Tyr Ala Glu His Ala Asp 50 55 60 Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Tyr Ala Lys Asn Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys His Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala Gly Val Gly Ser Ala Ala Gln Tyr Arg Tyr Trp Gly 100 105 110 Arg Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 26 <211> 5 <212> PRT <213> llamas <400> 26 Thr Tyr Ser Met Gly 1 5 <210> 27 <211> 19 <212> PRT <213> llamas <400> 27 Ala His Arg Trp Ser Gly Ser Ala Tyr Tyr Ala Glu His Ala Asp Ser 1 5 10 15 Val Glu Gly <210> 28 <211> 10 <212> PRT <213> llamas <400> 28 Gly Val Gly Ser Ala Ala Gln Tyr Arg Tyr 1 5 10 <210> 29 <211> 30 <212> PRT <213> llamas <400> 29 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Val 20 25 30 <210> 30 <211> 14 <212> PRT <213> llamas <400> 30 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 31 <211> 32 <212> PRT <213> llamas <400> 31 Arg Phe Thr Ile Ser Arg Asp Tyr Ala Lys Asn Met Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys His Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 32 <211> 11 <212> PRT <213> llamas <400> 32 Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 33 <211> 121 <212> PRT <213> llamas <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ala Pro Gly 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala His Arg Trp Ser Gly Ser Ala Tyr Tyr Ala Asp Tyr Ala Asp 50 55 60 Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Tyr Ala Lys Asn Met 65 70 75 80 Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala Gly Val Gly Ser Ala Ala Gln Tyr Thr Tyr Trp Gly 100 105 110 Arg Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 34 <211> 5 <212> PRT <213> Llama <400> 34<00​​​​​​​​​​​<400> 35 Ala His Arg Trp Ser Gly Ser Ala Tyr Tyr Ala Asp Tyr Ala Asp Ser 1 5 10 15 Val Glu Gly <210> 36 <211> 10 <212> PRT <213> llamas <400> 36 Gly Val Gly Ser Ala Ala Gln Tyr Thr Tyr 1 5 10 <210> 37 <211> 30 <212> PRT <213> llamas <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ala 20 25 30 <210> 38 <211> 14 <212> PRT <213> llamas <400> 38 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 39 <211> 32 <212> PRT <213> llamas <400> 39 Arg Phe Thr Ile Ser Arg Asp Tyr Ala Lys Asn Met Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 40 <211> 11 <212> PRT <213> Llama <400> 40 Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 41 <211> 121 <212> PRT <213> LlamaT <400> 41 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Gly Thr Tyr 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala His Arg Trp Ser Gly Ser Ala Tyr Tyr Ala Glu His Ala Asp 50 55 60 Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Tyr Ala Lys Asn Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys His Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala Gly Val Gly Ser Glu Ala Gln Tyr Arg Tyr Trp Gly 100 105 110 Arg Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 42 <211> 5 <212> PRT <213> llamas <400> 42 Thr Tyr Ser Met Gly 1 5 <210> 43 <211> 19 <212> PRT <213> llamas <400> 43 Ala His Arg Trp Ser Gly Ser Ala Tyr Tyr Ala Glu His Ala Asp Ser 1 5 10 15 Val Glu Gly <210> 44 <211> 10 <212> PRT <213> llamas <400> 44 Gly Val Gly Ser Glu Ala Gln Tyr Arg Tyr 1 5 10 <210> 45 <211> 30 <212> PRT <213> llamas <400> 45 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Gly 20 25 30 <210> 46 <211> 14 <212> PRT <213> llamas <400> 46 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 47 <211> 32 <212> PRT <213> llamas <400> 47 Arg Phe Thr Ile Ser Arg Asp Tyr Ala Lys Asn Met Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys His Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 48 <211> 11 <212> PRT <213> llamas <400> 48 Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 49 <211> 124 <212> PRT <213> llamas <400> 49 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser Ser Tyr 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Thr Trp Asn Gly Tyr Ile Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Thr Phe Ser Thr Thr Ser Pro Ile Ser Arg Thr Tyr Asn 100 105 110 Tyr Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 50 <211> 5 <212> PRT <213> Llama <400> 50 Ser Tyr Ser Met Gly 1 5 <210> 51<​​​​​​​​​​​​​​​​​​​ <213> llamas <400> 52 Thr Thr Phe Ser Thr Thr Ser Pro Ile Ser Arg Thr Tyr Asn Tyr 1 5 10 15 <210> 53 <211> 30 <212> PRT <213> llamas <400> 53 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Thr Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser 20 25 30 <210> 54 <211> 14 <212> PRT <213> llamas <400> 54 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 55 <211> 32 <212> PRT <213> llamas <400> 55 Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Phe Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Glu Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 56 <211> 11 <212> PRT <213> Llama <400> 56 Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 57 <211> 120 <212> PRT <213> Llama <400> 57 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ser Arg Thr Ser Ser Thr Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Asp Phe Val 35 40 45 Ala Ile Ile Ser Phe Gly Gly Thr Phe Tyr Ala Asp Ser Val Lys Gly 50 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 65 70 75 80 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 85 90 95 Gly Arg Thr Leu Ser Lys Arg Ala Asp Ser Tyr Ala Ser Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 58 <211> 5 <212> PRT <213> llamas <400> 58 Thr Tyr Ala Met Gly 1 5 <210> 59 <211> 15 <212> PRT <213> llamas <400> 59 Ile Ile Ser Phe Gly Gly Thr Phe Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 60 <211> 13 <212> PRT <213> llamas <400> 60 Gly Arg Thr Leu Ser Lys Arg Ala Asp Ser Tyr Ala Ser 1 5 10 <210> 61 <211> 30 <212> PRT <213> llamas <400> 61 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ser Arg Thr Ser Ser 20 25 30 <210> 62 <211> 14 <212> PRT <213> llamas <400> 62 Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Asp Phe Val Ala 1 5 10 <210> 63 <211> 32 <212> PRT <213> Llama <400> 63 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 64 <211> 11 <212> PRT <213> Llama <400> 64 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 65 <211> 123 <212> PRT <213> Llama <400> 65 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Gly Thr Leu Ala Met Tyr 20 25 30 Ala Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Lys Phe Val 35 40 45 Ala Ala Ile Asn Thr Ser Gly Arg Tyr Ser Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Thr 65 70 75 80 Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Asp Lys Gly Asn Trp Ala Leu Ala Met Ser Tyr Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 66 <211> 5 <212> PRT <213> Llama <400> 66 Met Tyr Ala Met Ser 1 5 <210> 67 <211> 17 <212> PRT <213> Llama <400> 67 Ala Ile Asn Thr Ser Gly Arg Tyr Ser Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 68 <211> 14 <212> PRT <213> Llama <400> 68 Thr Asp Lys Gly Asn Trp Ala Leu Ala Met Ser Tyr Asp Tyr 1 5 10 <210> 69 <211> 30 <212> PRT <213> llamas <400> 69 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Ser Leu Ser Cys Ala Ala Ser Gly Gly Thr Leu Ala 20 25 30 <210> 70 <211> 14 <212> PRT <213> llamas <400> 70 Trp Phe Arg Gln Ala Pro Gly Lys Asp Arg Lys Phe Val Ala 1 5 10 <210> 71 <211> 32 <212> PRT <213> llamas <400> 71 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Thr Leu Gln 1 5 10 15 Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 72 <211> 11 <212> PRT <213> llamas <400> 72 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 73 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 <220> <221> MISC_FEATURE <222> (1)..(1) <223> Any amino acid <220> <221> MISC_FEATURE <222> (3)..(3) <223> Any amino acid <400> 73 Xaa Ile Xaa Trp Ser Gly Arg Ala Pro Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 74 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 <220> <221> MISC_FEATURE <222> (1)..(1) <223> Gly or Ala <220> <221> MISC_FEATURE <222> (3)..(3) <223> Ala or Ser <400> 74 Xaa Ile Xaa Trp Ser Gly Arg Ala Pro Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 75 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 <220> <221> MISC_FEATURE <222> (6)..(6) <223> Any amino acid <220> <221> MISC_FEATURE <222> (10)..(10) <223> Any amino acid <220> <221> MISC_FEATURE <222> (11)..(11) <223> Any amino acid <220> <221> MISC_FEATURE <222> (14)..(14) <223> Any amino acid <400> 75 Gly Glu Gly Ala Ile Xaa Trp Thr Thr Xaa Xaa Ala Tyr Xaa Tyr 1 5 10 15 <210> 76 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 <220> <221> MISC_FEATURE <222> (6)..(6) <223> Arg or Lys or Leu <220> <221> MISC_FEATURE <222> (10)..(10) <223> Leu or Pro <220> <221> MISC_FEATURE <222> (11)..(11) <223> Asp or Gly <220> <221> MISC_FEATURE <222> (14)..(14) <223> Asp or Asn <400> 76 Gly Glu Gly Ala Ile Xaa Trp Thr Thr Xaa Xaa Ala Tyr Xaa Tyr 1 5 10 15 <210> 77 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 <220> <221> MISC_FEATURE <222> (1)..(1) <223> Any amino acid <220> <221> MISC_FEATURE <222> (2)..(2) <223> Any amino acid <400> 77 Xaa Xaa Ser Met Gly 1 5 <210> 78 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR1 <220> <221> MISC_FEATURE <222> (1)..(1) <223> Thr or Pro <220> <221> MISC_FEATURE <222> (2)..(2) <223> Tyr or Gly <400> 78 Xaa Xaa Ser Met Gly 1 5 <210> 79 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 <220> <221> MISC_FEATURE <222> (12)..(12) <223> Any amino acid <220> <221> MISC_FEATURE <222> (13)..(13) <223> Any amino acid <400> 79 Ala His Arg Trp Ser Gly Ser Ala Tyr Tyr Ala Xaa Xaa Ala Asp Ser 1 5 10 15 Val Glu Gly <210> 80 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR2 <220> <221> MISC_FEATURE <222> (12)..(12) <223> Glu or Asp <220> <221> MISC_FEATURE <222> (13)..(13) <223> His or Tyr <400> 80 Ala His Arg Trp Ser Gly Ser Ala Tyr Tyr Ala Xaa Xaa Ala Asp Ser 1 5 10 15 Val Glu Gly <210> 81 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 <220> <221> MISC_FEATURE <222> (5)..(5) <223> Any amino acid <220> <221> MISC_FEATURE <222> (9)..(9) <223> Any amino acid <400> 81 Gly Val Gly Ser Xaa Ala Gln Tyr Xaa Tyr 1 5 10 <210> 82 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Heavy chain CDR3 <220> <221> MISC_FEATURE <222> (5)..(5) <223> Ala or Glu <220> <221> MISC_FEATURE <222> (9)..(9) <223> Arg or Thr <400> 82 Gly Val Gly Ser Xaa Ala Gln Tyr Xaa Tyr 1 5 10 <210> 83 <211> 122 <212> PRT <213> llamas <400> 83 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser Val Tyr 20 25 30 Gly Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Ser Gly Thr Thr Gly Ser Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​1 5 10 15 Gly <210> 86 <211> 13 <212> PRT <213> llamas <400> 86 Gly Gly Arg Val Tyr Ile Thr Thr Ser Ser Ser Trp Ala Tyr 1 5 10 <210> 87 <211> 30 <212> PRT <213> llamas <400> 87 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser 20 25 30 <210> 88 <211> 14 <212> PRT <213> llamas <400> 88 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 89 <211> 32 <212> PRT <213> llamas <400> 89 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Ala 20 25 30 <210> 90 <211> 11 <212> PRT <213> Llama <400> 90 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 91 <211> 127 <212> PRT[[ID=2L]] <213> Llama <400> 91 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Arg Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val ]>35 40 45 Ala Ala Ile Ala Trp Ser Thr Gly Ser Thr Tyr Tyr Ala Asn Ser Val 50 55 60 Lys Gly Arg Phe Ala Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Glu Thr Arg Tyr Cys Ser Gly Phe Gly Cys Leu Asp Pro Arg 100 105 110 It seems there might be some minor formatting issues in the original text (like an extra '>' in some lines which might be a typo). I've translated it as accurately as possible. If you have any further clarifications or corrections regarding the original text, feel free to let me know.Thr Tyr Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 92 <211> 5 <212> PRT <213> llamas <400> 92 Arg Tyr Ala Met Gly 1 5 <210> 93 <211> 17 <212> PRT <213> llamas <400> 93 Ala Ile Ala Trp Ser Thr Gly Ser Thr Tyr Tyr Ala Asn Ser Val Lys 1 5 10 15 Gly <210> 94 <211> 18 <212> PRT <213> llamas <400> 94 Glu Thr Arg Tyr Cys Ser Gly Phe Gly Cys Leu Asp Pro Arg Thr Tyr 1 5 10 15 Gly Ser <210> 95 <211> 30 <212> PRT <213> llamas <400> 95 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser 20 25 30 <210> 96 <211> 14 <212> PRT <213> llamas <400> 96 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala 1 5 10 <210> 97 <211> 32 <212> PRT <213> llamas <400> 97 Arg Phe Ala Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 98 <211> 11 <212> PRT <213> llamas <400> 98 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 99 <211> 124 <212> PRT <213> llamas <400> 99 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Asp 20 25 30 Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ile 35 40 45 Ala Gly Ile Gly Arg Ser Gly Gly Ser Ile Tyr Tyr Ala Asp Ala Val 5…​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<212> PRT <213> llamas <400> 102 Arg Gln Arg Ile Gly Leu Val Val Gly Ala Leu Gly Tyr Asp Tyr 1 5 10 15 <210> 103 <211> 30 <212> PRT <213> llamas <400> 103 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser 20 25 30 <210> 104 <211> 14 <212> PRT <213> llamas <400> 104 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ile Ala 1 5 10 <210> 105 <211> 32 <212> PRT <213> llamas <400> 105 Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 20 25 30 <210> 106 <211> 11 <212> PRT <213> Llama <400> 106 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 107 <211> 129 <212> PRT <213> Llama <400> 107 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Asn Ser Ile Thr Ser Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Ala Asp Ser Gly Leu Phe Ser Gly Ser Ser Cys Leu Lys Tyr Arg 100 105 110 Ala Met Arg Phe Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 108 <211> 5 <212> PRT <213> llamas <400> 108 Asp Tyr Thr Ile Gly 1 5 <210> 109 <211> 17 <212> PRT <213> llamas <400> 109 Cys Ile Asn Ser Ile Thr Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 110 <211> 20 <212> PRT <213> llamas <400> 110 Asp Ser Gly Leu Phe Ser Gly Ser Ser Cys Leu Lys Tyr Arg Ala Met 1 5 10 15 Arg Phe Gly Ser 20 <210> 111 <211> 30 <212> PRT <213> llamas <400> 111 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp 20 25 30 <210> 112 <211> 14 <212> PRT <213> llamas <400> 112 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ser 1 5 10 <210> 113 <211> 32 <212> PRT <213> llamas <400> 113 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Ala 20 25 30 <210> 114 <211> 11 <212> PRT <213> llamas <400> 114 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 115 <211> 101 <212> PRT <213> pig <400> 115 Pro Arg Leu Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val 1 5 10 15 Gln His Gly Asp Thr Trp Gly Thr Val Cys Asp Ser Asp Phe Ser Leu 20 25 30 Glu Ala Ala Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val 35 40 45 Ser Leu Leu Gly Gly Ala His Phe Gly Glu Gly Ser Gly Gln Ile Trp 50 55 60 Ala Glu Glu Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys 65 70 75 80 Pro Val Ala Pro Arg Pro Asp Gly Thr Cys Ser His Ser Arg Asp Val 85 90 95 Gly Val Val Cys Ser 100 <210> 116 <211> 1115 <212> PRT <213> Pig <400> 116 Met Asp Lys Leu Arg Met Val Leu His Glu Asn Ser Gly Ser Ala Asp 1 5 10 15 Phe Arg Arg Cys Ser Ala His Leu Ser Ser Phe Thr Phe Ala Val Val 20 25 30 Ala Val Leu Ser Ala Cys Leu Val Thr Ser Ser Leu Gly Gly Lys Asp 35 40 45 Lys Glu Leu Arg Leu Thr Gly Gly Glu Asn Lys Cys Ser Gly Arg Val 50 55 60 Glu Val Lys Val Gln Glu Glu Trp Gly Thr Val Cys Asn Asn Gly Trp 65 70 75 80 Asp Met Asp Val Val Ser Val Val Cys Arg Gln Leu Gly Cys Pro Thr 85 90 95 Ala Ile Lys Ala Thr Gly Trp Ala Asn Phe Ser Ala Gly Ser Gly Arg 100 105 110 Ile Trp Met Asp His Val Ser Cys Arg Gly Asn Glu Ser Ala Leu Trp 115 120 125 Asp Cys Lys His Asp Gly Trp Gly Lys His Asn Cys Thr His Gln Gln 130 135 140 Asp Ala Gly Val Thr Cys Ser Asp Gly Ser Asp Leu Glu Met Gly Leu 145 150 155 160 Val Asn Gly Gly Asn Arg Cys Leu Gly Arg Ile Glu Val Lys Phe Gln 165 170 175 Gly Arg Trp Gly Thr Val Cys Asp Asp Asn Phe Asn Ile Asn His Ala 180 185 190 Ser Val Val Cys Lys Gln Leu Glu Cys Gly Ser Ala Val Ser Phe Ser 195 200 205 Gly Ser Ala Asn Phe Gly Glu Gly Ser Gly Pro Ile Trp Phe Asp Asp 210 215 220 Leu Val Cys Asn Gly Asn Glu Ser Ala Leu Trp Asn Cys Lys His Glu 225 230 235 240 Gly Trp Gly Lys His Asn Cys Asp His Ala Glu Asp Ala Gly Val Ile 245 250 255 Cys Leu Asn Gly Ala Asp Leu Lys Leu Arg Val Val Asp Gly Val Thr 260 265 270 Glu Cys Ser Gly Arg Leu Glu Val Lys Phe Gln Gly Glu Trp Gly Thr 275 280 285 Ile Cys Asp Asp Gly Trp Asp Ser Asp Asp Ala Ala Val Ala Cys Lys 290 295 300 Gln Leu Gly Cys Pro Thr Ala Val Thr Ala Ile Gly Arg Val Asn Ala 305 310 315 320 Ser Glu Gly Thr Gly His Ile Trp Leu Asp Ser Val Ser Cys His Gly 325 330 335 His Glu Ser Ala Leu Trp Gln Cys Arg His His Glu Trp Gly Lys His 340 345 350 Tyr Cys Asn His Asp Glu Asp Ala Gly Val Thr Cys Ser Asp Gly Ser 355 360 365 Asp Leu Glu Leu Arg Leu Lys Gly Gly Gly Ser His Cys Ala Gly Thr 370 375 380 Val Glu Val Glu Ile Gln Lys Leu Val Gly Lys Val Cys Asp Arg Ser 385 390 395 400 Trp Gly Leu Lys Glu Ala Asp Val Val Cys Arg Gln Leu Gly Cys Gly 405 410 415 Ser Ala Leu Lys Thr Ser Tyr Gln Val Tyr Ser Lys Thr Lys Ala Thr 420 425 430 Asn Thr Trp Leu Phe Val Ser Ser Cys Asn Gly Asn Glu Thr Ser Leu 435 440 445 Trp Asp Cys Lys Asn Trp Gln Trp Gly Gly Leu Ser Cys Asp His Tyr 450 455 460 Asp Glu Ala Lys Ile Thr Cys Ser Ala His Arg Lys Pro Arg Leu Val 465 470 475 480 Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu Val Gln His Gly Asp 485 490 495 Thr Trp Gly Thr Val Cys Asp Ser Asp Phe Ser Leu Glu Ala Ala Ser 500 505 510 Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val Val Ser Leu Leu Gly 515 520 525 Gly Ala His Phe Gly Glu Gly Ser Gly Gln Ile Trp Ala Glu Glu Phe 530 535 540 Gln Cys Glu Gly His Glu Ser His Leu Ser Leu Cys Pro Val Ala Pro 545 550 555 560 Arg Pro Asp Gly Thr Cys Ser His Ser Arg Asp Val Gly Val Val Cys 565 570 575 Ser Arg Tyr Thr Gln Ile Arg Leu Val Asn Gly Lys Thr Pro Cys Glu 580 585 590 Gly Arg Val Glu Leu Asn Ile Leu Gly Ser Trp Gly Ser Leu Cys Asn 595 600 605 Ser His Trp Asp Met Glu Asp Ala His Val Leu Cys Gln Gln Leu Lys 610 615 620 Cys Gly Val Ala Leu Ser Ile Pro Gly Gly Ala Pro Phe Gly Lys Gly 625 630 635 640 Ser Glu Gln Val Trp Arg His Met Phe His Cys Thr Gly Thr Glu Lys 645 650 655 His Met Gly Asp Cys Ser Val Thr Ala Leu Gly Ala Ser Leu Cys Ser 660 665 670 Ser Gly Gln Val Ala Ser Val Ile Cys Ser Gly Asn Gln Ser Gln Thr 675 680 685 Leu Ser Pro Cys Asn Ser Ser Ser Ser Asp Pro Ser Ser Ser Ile Ile 690 695 700 Ser Glu Glu Asn Gly Val Ala Cys Ile Gly Ser Gly Gln Leu Arg Leu 705 710 715 720 Val Asp Gly Gly Gly Arg Cys Ala Gly Arg Val Glu Val Tyr His Glu 725 730 735 Gly Ser Trp Gly Thr Ile Cys Asp Asp Ser Trp Asp Leu Asn Asp Ala 740 745 750 His Val Val Cys Lys Gln Leu Ser Cys Gly Trp Ala Ile Asn Ala Thr 755 760 765 Gly Ser Ala His Phe Gly Glu Gly Thr Gly Pro Ile Trp Leu Asp Glu 770 775 780 Ile Asn Cys Asn Gly Lys Glu Ser His Ile Trp Gln Cys His Ser His 785 790 795 800 Gly Trp Gly Arg His Asn Cys Arg His Lys Glu Asp Ala Gly Val Ile 805 810 815 Cys Ser Glu Phe Met Ser Leu Arg Leu Ile Ser Glu Asn Ser Arg Glu 820 825 830 Thr Cys Ala Gly Arg Leu Glu Val Phe Tyr Asn Gly Ala Trp Gly Ser 835 840 845 Val Gly Arg Asn Ser Met Ser Pro Ala Thr Val Gly Val Val Cys Arg 850 855 860 Gln Leu Gly Cys Ala Asp Arg Gly Asp Ile Ser Pro Ala Ser Ser Asp 865 870 875 880 Lys Thr Val Ser Arg His Met Trp Val Asp Asn Val Gln Cys Pro Lys 885 890 895 Gly Pro Asp Thr Leu Trp Gln Cys Pro Ser Ser Pro Trp Lys Lys Arg 900 905 910 Leu Ala Ser Pro Ser Glu Glu Thr Trp Ile Thr Cys Ala Asn Lys Ile 915 920 925 Arg Leu Gln Glu Gly Asn Thr Asn Cys Ser Gly Arg Val Glu Ile Trp 930 935 940 Tyr Gly Gly Ser Trp Gly Thr Val Cys Asp Asp Ser Trp Asp Leu Glu 945 950 955 960 Asp Ala Gln Val Val Cys Arg Gln Leu Gly Cys Gly Ser Ala Leu Glu 965 970 975 Ala Gly Lys Glu Ala Ala Phe Gly Gln Gly Thr Gly Pro Ile Trp Leu 980 985 990 Asn Glu Val Lys Cys Lys Gly Asn Glu Thr Ser Leu Trp Asp Cys Pro 995 1000 1005 Ala Arg Ser Trp Gly His Ser Asp Cys Gly His Lys Glu Asp Ala 1010 1015 1020 Ala Val Thr Cys Ser Glu Ile Ala Lys Ser Arg Glu Ser Leu His 1025 1030 1035 Ala Thr Gly Arg Ser Ser Phe Val Ala Leu Ala Ile Phe Gly Val 1040 1045 1050 Ile Leu Leu Ala Cys Leu Ile Ala Phe Leu Ile Trp Thr Gln Lys 1055 1060 1065 Arg Arg Gln Arg Gln Arg Leu Ser Val Phe Ser Gly Gly Glu Asn 1070 1075 1080 Ser Val His Gln Ile Gln Tyr Arg Glu Met Asn Ser Cys Leu Lys 1085 1090 1095 Ala Asp Glu Thr Asp Met Leu Asn Pro Ser Gly Asp His Ser Glu 1100 1105 1110 Val Gln 1115 <210> 117 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> His-tagged Porcine SRCR5 <400> 117 Met Pro Arg Leu Val Gly Gly Asp Ile Pro Cys Ser Gly Arg Val Glu 1 5 10 15 Val Gln His Gly Asp Thr Trp Gly Thr Val Cys Asp Ser Asp Phe Ser 20 25 30 Leu Glu Ala Ala Ser Val Leu Cys Arg Glu Leu Gln Cys Gly Thr Val 35 40 45 Val Ser Leu Leu Gly Gly Ala His Phe Gly Glu Gly Ser Gly Gln Ile 50 55 60 Trp Ala Glu Glu Phe Gln Cys Glu Gly His Glu Ser His Leu Ser Leu 65 70 75 80 Cys Pro Val Ala Pro Arg Pro Asp Gly Thr Cys Ser His Ser Arg Asp 85 90 95 Val Gly Val Val Cys Ser Gly His His His His His His 100 105 <210> 118 <211> 143 <212> PRT <213> Synthetic Sequence <220> <223> His-tagged VHH14 (02D01) <400> 118 Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Asp 20 25 30 Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ile 35 40 45 Ala Gly Ile Gly Arg Ser Gly Gly Ser Ile Tyr Tyr Ala Asp Ala Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Gln Arg Ile Gly Leu Val Val Gly Ala Leu Gly Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Asp Tyr Lys Asp 115 120 125 Asp Asp Asp Lys Gly Gly Gly Gly Ser His His His His His His 130 135 140

Claims

1. A monoclonal antibody binding porcine CD163, said monoclonal antibody comprising an antigen-binding domain of an SRCR5 domain binding porcine CD163, said antigen-binding domain comprising at least one heavy chain variable region, said heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein said heavy chain variable region comprises: (i) The variable heavy chain VH CDR1, composed of the amino acid sequence SEQ ID NO: 10, (ii) the variable heavy chain VH CDR2 composed of the amino acid sequence SEQ ID NO: 11, and, (iii) VH CDR3, a variable heavy chain composed of the amino acid sequence SEQ ID NO:

12.

2. A monoclonal antibody binding porcine CD163, said monoclonal antibody comprising an antigen-binding domain of an SRCR5 domain binding porcine CD163, said antigen-binding domain comprising at least one heavy chain variable region, said heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein said heavy chain variable region comprises: (i) The variable heavy chain VH CDR1 composed of the amino acid sequence SEQ ID NO: 2, (ii) VHCDR2, a variable heavy chain composed of the amino acid sequence SEQ ID NO: 3, and, (iii) VHCDR3, a variable heavy chain consisting of the amino acid sequence SEQ ID NO:

4.

3. A monoclonal antibody binding porcine CD163, said monoclonal antibody comprising an antigen-binding domain of the SRCR5 domain of porcine CD163, said antigen-binding domain comprising at least one heavy chain variable region, said heavy chain variable region comprising three complementarity-determining regions (CDRs), wherein said heavy chain variable region comprises: (i) The variable heavy chain VH CDR1, composed of the amino acid sequence SEQ ID NO: 18, (ii) the variable heavy chain VH CDR2 composed of the amino acid sequence SEQ ID NO: 19, and, (iii) VHCDR3, a variable heavy chain consisting of the amino acid sequence SEQ ID NO:

20.

4. The monoclonal antibody according to any one of claims 1 to 3, wherein the monoclonal antibody is a single-domain antibody.

5. The monoclonal antibody according to any one of claims 1 to 4, wherein the monoclonal antibody can inhibit PRRSV type 1 and type 2 infection.

6. A combination of two or more monoclonal antibodies according to any one of claims 1 to 3, or a combination of a monoclonal antibody according to any one of claims 1 to 3 with one or more additional CD163-targeting active agents.

7. One or more nucleic acid molecules comprising a nucleotide sequence encoding a monoclonal antibody according to any one of claims 1 to 5.

8. One or more expression vectors comprising the nucleic acid molecule according to claim 7.

9. One or more host cells comprising the expression vector according to claim 8 or the nucleic acid molecule according to claim 7, or expressing a monoclonal antibody according to any one of claims 1 to 5.

10. A method for producing a monoclonal antibody according to any one of claims 1 to 5, the method comprising the steps of: (i) culturing a host cell containing one or more expression vectors as defined in claim 8 or one or more nucleic acid molecules as defined in claim 7 under conditions suitable for expressing an antibody; and optionally, (ii) isolating or obtaining the expressed antibody from the host cell or from a growth medium / supernatant.

11. A composition comprising a monoclonal antibody according to any one of claims 1 to 5, a combination of monoclonal antibodies according to claim 6, one or more nucleic acid molecules according to claim 7, or an expression vector according to claim 8.

12. Use of a monoclonal antibody according to any one of claims 1 to 5, or a combination of monoclonal antibodies according to claim 6, or one or more nucleic acid molecules according to claim 7, or an expression vector according to claim 8, in the preparation of a medicament or composition for the treatment or prevention of porcine PRRS virus infection.

13. The use according to claim 12, for the treatment or prevention of PRRSV type 1 and / or type 2 infection.

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