Patent Publication Number: US-11384141-B2

Title: Serum albumin binding antibodies for tuneable half-life extension of biologics

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a U.S. National Phase application under 35 U.S.C. 371 of PCT/CA2019/050514, filed Apr. 24, 2019, which claims priority from and the benefit of U.S. Provisional Patent Application No. 62/661,871 filed on Apr. 24, 2018, the specifications of which are hereby incorporated by reference in their entireties. 
    
    
     REFERENCE TO AN ELECTRONIC SEQUENCE LISTING 
     The contents of the electronic sequence listing (SEQ.txt; Size: 75,250 bytes; and Date of Creation: Apr. 23, 2021) is herein incorporated by reference in its entirety. 
     BACKGROUND 
     (a) Field 
     The subject matter disclosed generally relates to antibodies or antigen-binding fragments that bind to serum albumin. More specifically, the subject matter relates to antibodies or antigen-binding fragments that bind to serum albumin for half-life extension of biologics, as well as compounds, pharmaceutical compositions, nucleic acid vectors, cells comprising the nucleic acid vectors, and methods of removing molecules from serum. 
     (b) Related Prior Art 
     Biologics of less than 40-50 kDa in size possess short serum half-lives due to rapid renal clearance. Strategies to prolong the serum half-life of various biologics (antibody fragments, single-domain antibodies, enzymes, growth factors, peptides) are critically important for efficacy. The half-life of biologics can be extended through various techniques, including, but not limited to PEGylation, PASylation, conjugation to carbohydrates, fusion to an IgG Fc domain, fusion to serum albumin, and fusion to an albumin binding domain or antibody binding domain that recognizes serum albumin. In the latter case, single-domain antibodies (referred to as sdAbs, V H Hs, or nanobodies), which are naturally occurring autonomous binding domains found in Camelid species, are ideal agents for which to target serum albumin for half-life extension. The flexibility V H Hs offer in terms of modularity and functionality allow for fusion to many biologics, in both N- and C-terminal orientations, without compensating target binding affinities or specificity. 
     The requirements for V H H-based half-life extension of biologics are as follows: (i) high affinity binding and species cross-reactivity of the V H H to the relevant serum albumins (human, monkey, rat, mouse) at pH 7.4, (ii) high affinity binding and species cross-reactivity of the V H H to the relevant serum albumins (human, monkey, rat, mouse) at pH 5.5, (iii) the anti-serum albumin V H H cannot compete with FcRn for albumin binding, and (iv) the anti-serum albumin V H H must retain functionality when fused to biologics through linkers. 
     On the other hand, many harmful molecules (e.g., protein-based bacterial toxin or venoms) need to be removed as quickly as possible from the body. Increasing their rate of removal will have therapeutic effects and prevent disease. To remove harmful molecules from circulation, a direct neutralizing agent (e.g., antibody) can be used to neutralize the harmful effects of the toxic molecules. Presently, direct neutralization of many toxins is not efficacious enough (the toxic substance is not removed quickly enough from serum) leaving significant room for improvement of therapeutic antibody efficacy. 
     Therefore, there is a need for additional V H Hs which target multiple serum albumin species, for the purpose of extending the serum half-life of biologics or removal of harmful molecules. 
     The following application describes the isolation, characterization, and in vivo testing of several llama-derived V H Hs which target multiple serum albumin species, for the purpose of extending the serum half-life of biologics or removal of harmful molecules. 
     SUMMARY 
     According to an embodiment, there is provided an antibody or an antigen-binding fragment that binds to serum albumin comprising three complementarity determining regions (CDR1, CDR2 and CDR3), wherein the CDR1, CDR2 and CDR3 comprise an amino acid sequence comprising: 
     
       
         
           
               
               
            
               
                   
                 1) 
               
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 GFLLRSNTM, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 IRPSGLT, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 HTRPPFQRDS 
               
               
                   
                 or 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 4) 
               
               
                   
                 ATRPPFQRDS, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 2) 
               
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 GRTFIAYAM, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
                 ITNFAGGTT, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 7) 
               
               
                   
                 AADRSAQTMRQVRPVLPY, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 3) 
               
               
                   
                 (SEQ ID NO: 8) 
               
               
                   
                 GRTFDNYVM, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 9) 
               
               
                   
                 ISGSGSIT, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 10) 
               
               
                   
                 AAGSRRTYYREPKFYPS, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 4) 
               
               
                   
                 (SEQ ID NO: 11) 
               
               
                   
                 GSTFSSSSV, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 12) 
               
               
                   
                 ITSGGST, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 NVAGRNWVPISRYSPGPY 
               
               
                   
                 or 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 14) 
               
               
                   
                 AVAGRNWVPISRYSPGPY, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 5) 
               
               
                   
                 (SEQ ID NO: 15) 
               
               
                   
                 GSIESINRM, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 16) 
               
               
                   
                 ISKGGST, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 17) 
               
               
                   
                 AAGPVWEQF, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 6) 
               
               
                   
                 (SEQ ID NO: 18) 
               
               
                   
                 GRTISLYAV, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 19) 
               
               
                   
                 ISWTDSST, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 20) 
               
               
                   
                 AADVSIRGLQKYEYDY, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 7) 
               
               
                   
                 (SEQ ID NO: 21) 
               
               
                   
                 TRTFSSYIM, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 22) 
               
               
                   
                 ISWSGRMT, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 23) 
               
               
                   
                 AADRTTAWGAPRSQYDS, 
               
               
                   
                 respectively. 
               
            
           
         
       
     
     The antigen-binding fragment may be a single-domain antibody (sdAb). 
     The antibody may be an IgA, IgD, IgE, IgG, or IgM. 
     The CDR1, CDR2 and CDR3 may comprise an amino acid sequence comprising GFLLRSNTM (SEQ ID NO:1), IRPSGLT (SEQ ID NO:2), and HTRPPFQRDS (SEQ ID NO:3) or ATRPPFQRDS (SEQ ID NO:4), respectively. 
     The CDR1, CDR2 and CDR3 may comprise an amino acid sequence comprising GRTFIAYAM (SEQ ID NO:5), ITNFAGGTT (SEQ ID NO:6), and AADRSAQTMRQVRPVLPY (SEQ ID NO:7), respectively. 
     The CDR1, CDR2 and CDR3 may comprise an amino acid sequence comprising GRTFDNYVM (SEQ ID NO:8), ISGSGSIT (SEQ ID NO:9), and AAGSRRTYYREPKFYPS (SEQ ID NO:10), respectively. 
     The CDR1, CDR2 and CDR3 may comprise an amino acid sequence comprising GSTFSSSSV (SEQ ID NO:11), ITSGGST (SEQ ID NO:12), and NVAGRNWVPISRYSPGPY (SEQ ID NO:13) or AVAGRNWVPISRYSPGPY (SEQ ID NO:14), respectively. 
     The CDR1, CDR2 and CDR3 may comprise an amino acid sequence comprising GSIESINRM (SEQ ID NO:15), ISKGGST (SEQ ID NO:16), and AAGPVWEQF (SEQ ID NO:17), respectively. 
     The CDR1, CDR2 and CDR3 may comprise an amino acid sequence comprising GRTISLYAV (SEQ ID NO:18), ISWTDSST (SEQ ID NO:19), and AADVSIRGLQKYEYDY (SEQ ID NO:20), respectively. 
     The CDR1, CDR2 and CDR3 may comprise an amino acid sequence comprising TRTFSSYIM (SEQ ID NO:21), ISWSGRMT (SEQ ID NO:22), and AADRTTAWGAPRSQYDS (SEQ ID NO:23), respectively. 
     The antibody or an antigen-binding fragment may be humanized or partially humanized. 
     According to another embodiment, there is provided a compound comprising an antibody or an antigen-binding fragment according to the present invention. 
     The antibody or an antigen-binding fragment may be linked to the compound via a linker. 
     The linker may be an amino acid sequence that allows for the functional linking of the compound to the antibody or an antigen-binding fragment. 
     The amino acid sequence may comprise about 3 to about 40 amino acids. 
     The linker sequence may be (GGGGS) n  (SEQ ID NO:81) wherein n ≥1, or any suitable linker. 
     The antibody or an antigen-binding fragment may be fused to an antibody or an antigen-binding fragment, operable to bind a target epitope. 
     The antibody or an antigen-binding fragment may be linked to a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, or combinations thereof, wherein each of the antibody or an antigen-binding fragment and the linked peptide, polypeptide, protein, enzyme, antibody, antibody fragment, or combinations thereof are functional. 
     According to another embodiment, there is provided a composition comprising the compound of the present invention, and a pharmaceutically acceptable diluent, carrier or excipient. 
     According to another embodiment, there is provided a nucleic acid vector comprising a nucleotide sequence encoding a compound of the present invention. 
     According to another embodiment, there is provided a cell comprising the nucleic acid vector of the present invention for expressing the compound of the present invention. 
     According to another embodiment, there is provided a cell for expressing the compound of the present invention. 
     According to another embodiment, there is provided a method of removing a molecule from serum, comprising administering a compound according to the present invention specific to the molecule, wherein the antibody or an antigen-binding fragment comprises CDR1, CDR2 and CDR3 comprising an amino acid sequence comprising GRTFDNYVM (SEQ ID NO:8), ISGSGSIT (SEQ ID NO:9), and AAGSRRTYYREPKFYPS (SEQ ID NO:10), respectively. 
     According to another embodiment, there is provided a use of a compound according to the present invention specific to a molecule for removing the molecule from serum, wherein the sdAb comprises CDR1, CDR2 and CDR3 comprising an amino acid sequence comprising GRTFDNYVM (SEQ ID NO:8), ISGSGSIT (SEQ ID NO:9), and AAGSRRTYYREPKFYPS (SEQ ID NO:10), respectively. 
     According to another embodiment, there is provided a solid support for purification of albumin, derivatives thereof, or fragments thereof comprising a solid or semi-solid medium linked to an antibody or an antigen-binding fragment according to the present invention or a compound according to the present invention. 
     According to another embodiment, there is provided a method of purifying albumin comprising contacting an albumin containing sample with a solid support according to the present invention. 
     Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which: 
         FIG. 1  illustrates the results from immunization of a llama in order to generate serum albumin binding single domain antibodies (V H Hs). An ELISA shows the llama serum response to various serum albumins day 42 post immunization with human serum albumin (HSA). Pre-immune serum drawn before the first immunization is shown as a control. RhSA: rhesus serum albumin; MSA: mouse serum albumin; RSA: rat serum albumin. 
         FIG. 2A  illustrates the sequences of wild-type serum albumin binding V H Hs and their humanized variants. The sequences of isolated (llama, wild-type) and humanized V H Hs are shown. The international ImMunoGeneTics information (IMGT) numbering system is used to distinguish framework regions (FRs) and complementarity determining regions (CDRs). The sequences of (A) R11 (SEQ ID NO:24), R11-H0 (SEQ ID NO:25), R11-H1 (SEQ ID NO:26), R11-H2 (SEQ ID NO:27), R11-H3 (SEQ ID NO:28), R11-H4 (SEQ ID NO:29), R11-H5 (SEQ ID NO:30), R11-H6 (SEQ ID NO:31) V H Hs, (B) R28 (SEQ ID NO:32), R28-H0 (SEQ ID NO:33), R28-H1 (SEQ ID NO:34), R28-H2 (SEQ ID NO:35), R28-H3 (SEQ ID NO:36), R28-H4 (SEQ ID NO:37), R28-H5 (SEQ ID NO:38) V H Hs, (C) M75 (SEQ ID NO:39), M75-H0 (SEQ ID NO:40), M75-H1 (SEQ ID NO:41), M75-H2 (SEQ ID NO:42), M75-H3 (SEQ ID NO:43), M75-H4 (SEQ ID NO:44), M75-H5 (SEQ ID NO:45) V H Hs, (D) M79 (SEQ ID NO:46), M79-H0 (SEQ ID NO:47), m79-H1 (SEQ ID NO:48), M79-H2 (SEQ ID NO:49), M79-H3 (SEQ ID NO:50), M79-H4 (SEQ ID NO:51), M79-H5 (SEQ ID NO:52) V H Hs are provided. 
         FIG. 2B  provides sequences of wild-type serum albumin binding V H Hs and their humanized variants. The sequences of isolated (llama, wild-type) and humanized V H Hs are shown. The international ImMunoGeneTics information (IMGT) numbering system is used to distinguish framework regions (FRs) and complementarity determining regions (CDRs). The sequences of (E) H18 (SEQ ID NO:53), H18-H0 (SEQ ID NO:54), H18-H1 (SEQ ID NO:55), H18-H2 (SEQ ID NO:56), H18-H3 (SEQ ID NO:57), H18-H4 (SEQ ID NO:58), H18-H5 (SEQ ID NO:59) V H Hs, (F) Rh34 (SEQ ID NO:60),Rh34-H0 (SEQ ID NO:61), Rh34-H1 (SEQ ID NO:62), Rh34-H2 (SEQ ID NO:63), Rh34-H3 (SEQ ID NO:64), Rh34-H4 (SEQ ID NO:65), Rh34-H5 (SEQ ID NO:66), V H Hs, and (G) Rh46 (SEQ ID NO:67), Rh46-H0 (SEQ ID NO:68), Rh46-H1 (SEQ ID NO:69), Rh46-H2 (SEQ ID NO:70), Rh46-H3 (SEQ ID NO:71), Rh46-H4 (SEQ ID NO:72), Rh46-H5 (SEQ ID NO:73) V H Hs are provided. 
         FIG. 3  illustrates the biophysical characterization of serum albumin binding V H Hs. In this example, various assays are shown to characterize the V H Hs. (A) Representative size exclusion chromatography (SEC) profiles illustrating the V H Hs are strictly monomeric and non-aggregating. (B) Representative surface plasmon resonance (SPR) sensorgrams demonstrating the cross-reactive binding of V H Hs to various serum albumins at pH 7.4. HSA: human serum albumin; RhSA: rhesus serum albumin; MSA: mouse serum albumin; RSA: rat serum albumin. 
         FIG. 4  illustrates the SPR binding analyses of serum albumin binding V H Hs at pH 5.5. The representative SPR sensorgrams illustrate pH-sensitive binding to various serum albumins. (A) M79 V H H binding to HSA, RSA and RhSA at pH 5.5. (B) M75 V H H (at a 100 nM injection) very weakly binds HSA and does not recognize RSA at pH 5.5. HSA: human serum albumin; RhSA: rhesus serum albumin; RSA: rat serum albumin. 
         FIG. 5  illustrates SPR-based epitope binning experiments. The SPR-based epitope binning experiments identified the different epitope bins targeted by the pool of serum albumin binding V H Hs. (A) Representative SPR sensorgrams of V H H co-injection experiments of various V H H+V H H combinations. (B) Graphical representation of the three epitopes targeted by the serum albumin V H Hs. R11 and M79 bind the same or a completely overlapping epitope, R28 binds an epitope that is partially overlapping with the R11/M79 epitope and M75 binds an epitope distinct from R11, M79 and R28. 
         FIG. 6  illustrates SPR-based human FcRn (h-FcRn) binding assays. The assays are used to show that the serum albumin binding V H Hs do not block the interaction of serum albumin with h-FcRn. (A) Graphical representation of the assay design. (B) Top panels showing that h-FcRn does not bind to immobilized HSA at pH 7.4, but does bind at pH 5.5. Lower panels showing R11, R28 and M79 binding to immobilized to HSA at pH 5.5, then injection of h-FcRn illustrates h-FcRn is free to bind HSA, demonstrating the V H Hs do not compete with h-FcRn binding to HSA. 
         FIG. 7  illustrates that serum albumin V H Hs can be formatted as N-terminal or C-terminal fusions and remain functional. A representative V H H (B39, SEQ ID NO:74; Murase et al, 2014) was used to demonstrate that the serum albumin binding V H Hs can be placed at either the N-terminus or C-terminus of the representative V H H (B39) and retain their ability to bind human serum albumin. (A) Schematic representation of construct designs. (B) SPR assays demonstrating functional binding of constructs to  C. difficile  toxin B (via B39 V H H). (C) SPR assays demonstrating functional binding of constructs to human serum albumin (via R11, R28, M75 or M79 V H Hs). 
         FIG. 8  illustrates the half-life extension of a monovalent single domain antibody in rats. An anti-toxin B V H H B39 is fused to serum albumin binding V H Hs and evaluated in vivo. (A) Design of V H H-V H H dimers that are either control or test articles. B39 is a  C. difficile  toxin B binding antibody. (B) Size exclusion chromatography profiles. (C) ELISA standard curves using toxin B coated on ELISA wells, V H H-V H H constructs added and detected with anti-HA-IgG HRP. (D) Rat in vivo half-life profiles of V H H-V H H fusions injected at 1 mg/kg. The data show that three V H Hs (R11, R28 and M79) significantly extend B39 half-life (31.1-46.1 h) and that the pH sensitive V H H (M75) increases B39 half-life (4.3 h) but to a lesser extent than the aforementioned V H Hs. The half-life of unfused B39 and B39-A20.1 control constructs are ˜0.5 h and 1.4 h, respectively. 
         FIG. 9  illustrates the half-life extension of a dimeric single domain antibody in rats. The anti-toxin A V H H-V H H dimer, consisting of anti- C. difficile  toxin A V H Hs A20 (SEQ ID NO:75) and A26 (SEQ ID NO:76) (Hussack et al 2011a), are fused to serum albumin binding V H Hs and evaluated in vivo. (A) Design of V H H-V H H dimers that are either control or test articles. A20 and A26 bind unique  C. difficile  toxin A epitopes. (B) ELISA standard curves using toxin A coated on ELISA wells, V H H-V H H-V H H constructs added and detected with anti-His6-IgG HRP. This demonstrates the constructs retain binding to toxin A. (C) SPR assays demonstrating binding of the fusion proteins to human and rat serum albumin surfaces. (D) SPR co-injection assay demonstrating the A20-A26-M75 and A20-A26-M79 fusion proteins can simultaneously bind to toxin A on the SPR surface and to human serum albumin in solution. The control A20-A26 can only bind to toxin A and does not bind human serum albumin in solution, as expected. (E) Rat in vivo half-life profiles of V H H-V H H-V H H fusions injected at 1 mg/kg. The data show a half-life of 1.8 h for the A20-A26 control construct compared to half-lives of 6.8 h and 45.0 h for A20-A26 fused to M75 and M79, respectively. 
         FIG. 10  illustrates the half-life extension of a growth factor binding protein. A growth factor CIBP2 (SEQ ID NO:77)(WO 2008019491 A1; UniProtKB ref #P18065) is fused to serum albumin binding V H Hs and evaluated in vivo. (A) Design of CIBP2-V H Hs that are either control or test articles. (B) Rat in vivo half-life profiles of CIBP2-V H H fusions injected at 1 mg/kg, as determined by MRM mass spectrometry analysis. The data show rapid clearance of the CIBP2 control construct (half-life could not be calculated) compared to half-lives of 4.9 h and 40.3 h for CIBP2 fused to M75 and M79, respectively. 
         FIG. 11  illustrates half-life extension of a blood brain barrier penetrating antibody FC5 (Muruganandam et al, 2002) fused to an amyloid-β binding peptide (ABP (SEQ ID NO:79); Chakravarthy et al, 2014) construct in rats. FC5-ABP is fused to serum albumin binding V H Hs and evaluated in vivo. (A) Design of FC5-ABP-V H Hs that are either control or test articles. (B) Rat in vivo half-life profiles of FC5-ABP-V H H fusions injected at 1 mg/kg, as determined by MRM mass spectrometry analysis. The data show a half-life of 1.1 h for the FC5-ABP control compared to a half-life of 25.4 h for the FC5-ABP-M79 construct. 
         FIG. 12  illustrates the half-life extension of an enzyme. In this example, an enzyme (IDS, SEQ ID NO:80) important in lysosomal storage disease is fused to serum albumin binding V H Hs R28 and M79 and evaluated in vivo. (A) Design of IDS-V H Hs. (B) Size exclusion chromatography profiles. (C) SPR binding profiles of IDS-V H Hs to rat serum albumin with Kos of 55.7 nM and 339 nM for IDS-R28 and IDS-M79, respectively. (D) Rat in vivo half-life profiles of IDS-V H H fusions. The data show a half-life of 0.9 h for the control IDS-C1 enzyme compared to half-lives ranging from 2.8 h to 4.4 h for various IDS-R28 and IDS-M79 constructs tested at different concentrations. 
         FIG. 13  illustrates the half-life extension of a monovalent single domain antibody in rats using humanized serum albumin binding V H Hs. In this example an anti-toxin B V H H B39 is fused to three representative humanized serum albumin binding V H Hs (M75-H1, SEQ ID NO: 41; R28-H5, SEQ ID NO: 38; R11-H6, SEQ ID NO: 31) and evaluated in vivo. (A) Schematic representation of constructs designed for testing. (B) ELISA standard curves using toxin B coated on ELISA wells, V H H-V H H constructs added and detected with anti-HA-IgG HRP. (C) Rat in vivo half-life profiles of B39-humanized V H H fusions injected at 1 mg/kg (or 0.5 mg/kg for B39-R11-H6). Serum antibody concentrations were determined by ELISA and obtained from standard curves. The data show a half-life of 3.8 h for B39-M75-H1, 51.2 h for B39-R28-H5 and 41.4 h for B39-R11-H6. The unfused B39 half-life was determined to be 0.5 h ( FIG. 8 ). The data show that all three humanized V H Hs (M75-H1, R28-H5 and R11-H6) extend B39 half-life and that the pH sensitive V H H (M75-H1) increases B39 half-life to a lesser extent. This trend is consistent with the half-life extension of the wild-type versions of these V H Hs. Importantly, V H H humanization did not negatively impact V H H function as in vivo half-life in rats was essentially identical to the durations observed for wild-type V H Hs (Table 2). 
         FIG. 14  illustrates crude domain mapping on HSA. Serum albumin is comprised of three major domains (domain 1, DI; domain 2, DII; domain 3, DIII). These domains were expressed and purified from mammalian cells (HEK293 6E) either alone or as fusions (DI, DII, DIII, DI-DII, DII-DIII) to determine the location of V H H binding. (A) Schematic of HSA domains synthesized and cloned into the pTT5 expression vector. Numbers refer to amino acid positions of the mature human serum albumin after signal and pro-peptide cleavage. (B) SDS-PAGE of the constructs expressed and purified. DI and DIII domains could not be expressed. (C) SPR sensorgrams demonstrating the response from injection of 100 nM of each anti-serum albumin V H H (M75, M79, R11 or R28) over amine-coupled surfaces of DII, DI-DII or DII-DIII. (D) Summary of V H H reactivity for various serum albumin domains. From the domain mapping binding studies: M75 binds DI, and M79, R11 and R28 all bind DII of human serum albumin. This is consistent with previous FcRn competition experiments that showed V H Hs did not interfere with FcRn binding to human serum albumin (in DIII). This is also consistent with epitope binning experiments that showed M75 bound an HSA epitope that was distinct from the M79/R11/R28 binding site. 
         FIG. 15  illustrates the impact of pH on the binding affinity of V H Hs for human and rat serum albumin. (A) Affinity of V H Hs for HSA at various pHs. (B) Affinity of M75 for HSA, HSA DI-DII and RSA at various pHs. 
     
    
    
     DETAILED DESCRIPTION 
     The present invention is directed to a technology for extending the serum half-life of biologics, or increasing the rate of removal and neutralization of harmful molecules. In embodiments there is disclosed an antibody or an antigen-binding fragment that binds to serum albumin comprising four framework regions (FR1 to FR4) and three complementarity determining regions (CDR1, CDR2 and CDR3). According to an embodiment, the antibody or an antigen binding fragment may be a single domain antibody (sdAb) that binds to serum albumin comprising four framework regions (FR1 to FR4) and three complementarity determining regions (CDR1, CDR2 and CDR3). 
     The CDR1, CDR2 and CDR3 of the invention may comprise any one of the following amino acid sequence: 
     
       
         
           
               
               
            
               
                   
                 1) 
               
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 GFLLRSNTM, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                 IRPSGLT, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 HTRPPFQRDS 
               
               
                   
                 or 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 4) 
               
               
                   
                 ATRPPFQRDS, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 2) 
               
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 GRTFIAYAM, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
                 ITNFAGGTT, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 7) 
               
               
                   
                 AADRSAQTMRQVRPVLPY, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 3) 
               
               
                   
                 (SEQ ID NO: 8) 
               
               
                   
                 GRTFDNYVM, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 9) 
               
               
                   
                 ISGSGSIT, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 10) 
               
               
                   
                 AAGSRRTYYREPKFYPS, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 4) 
               
               
                   
                 (SEQ ID NO: 11) 
               
               
                   
                 GSTFSSSSV, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 12) 
               
               
                   
                 ITSGGST, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 13) 
               
               
                   
                 NVAGRNWVPISRYSPGPY 
               
               
                   
                 or 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 14) 
               
               
                   
                 AVAGRNWVPISRYSPGPY, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 5) 
               
               
                   
                 (SEQ ID NO: 15) 
               
               
                   
                 GSIESINRM, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 16) 
               
               
                   
                 ISKGGST, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 17) 
               
               
                   
                 AAGPVWEQF, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 6) 
               
               
                   
                 (SEQ ID NO: 18) 
               
               
                   
                 GRTISLYAV, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 19) 
               
               
                   
                 ISWTDSST, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 20) 
               
               
                   
                 AADVSIRGLQKYEYDY, 
               
               
                   
                 respectively; or 
               
               
                   
                   
               
               
                   
                 7) 
               
               
                   
                 (SEQ ID NO: 21) 
               
               
                   
                 TRTFSSYIM, 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 22) 
               
               
                   
                 ISWSGRMT, 
               
               
                   
                 and 
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 23) 
               
               
                   
                 AADRTTAWGAPRSQYDS, 
               
               
                   
                 respectively. 
               
            
           
         
       
     
     According to embodiments, the sdAb of the present invention may be the R11 sdAb (SEQ ID NO:24), and humanized versions thereof (H0 to H6) SEQ ID NOS:25-31); the R28 sdAb (SEQ ID NO:32), and humanized versions thereof (H0 to H5) SEQ ID NOS:33-38); the M75 sdAb (SEQ ID NO:39), and humanized versions thereof (H0 to H5) SEQ ID NOS:40-45); the M79 sdAb (SEQ ID NO:46), and humanized versions thereof (H0 to H5) SEQ ID NOS:47-52); the H18 sdAb (SEQ ID NO:53), and humanized versions thereof (H0 to H5) SEQ ID NOS:54-59); the Rh34 sdAb (SEQ ID NO:60), and humanized versions thereof (H0 to H5) SEQ ID NOS:61-66); and the Rh46 sdAb (SEQ ID NO:67), and humanized versions thereof (H0 to H5) SEQ ID NOS:68-73). See Tables 1 to Tables 3 below, and  FIGS. 2A and 2B . 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Wild-type V H H affinities for serum albumins from various species. 
               
            
           
           
               
               
               
               
            
               
                   
                 K D  (nM) at pH 7.4 
                 K D  (nM) at pH 5.5 
                 h-FcRn 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 V H H 
                 HSA 
                 RhSA 
                 RSA 
                 MSA 
                 HSA 
                 RhSA 
                 RSA 
                 MSA 
                 blocking? 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 R11 
                 93 
                 159 
                 22 
                  14 #   
                 29 
                 116 
                 30 
                 — 
                 No 
               
               
                 R28 
                 12 
                 38 
                 0.4 
                 160 
                 1.8 
                 6 
                 0.3 
                 — 
                 No 
               
               
                 M75 
                 1.2 
                 2.9 
                 315 
                 510 
                 735 
                 — 
                 n.b. 
                 — 
                 — 
               
               
                 M79 
                 122 
                 85 
                 13 
                 406 #   
                 7 
                 2 
                 7 
                 — 
                 No 
               
               
                 H18 
                 34 
                 48 
                 n.b. 
                 — 
                 46 
                 42 
                 n.b. 
                 — 
                 No 
               
               
                 Rh34 
                 286 
                 333 
                 n.b. 
                 — 
                 129 
                 167 
                 n.b. 
                 — 
                 No 
               
               
                 Rh46 
                 80 
                 81 
                 n.b. 
                 — 
                 41 
                 30 
                 n.b. 
                 — 
                 No 
               
               
                   
               
               
                 Unless noted, all values were determined by single cycle kinetic (SCK) SPR measurements on a Biacore T200 
               
               
                   # determined by ITC 
               
               
                 “—”: not determined 
               
               
                 n.b.: no binding 
               
               
                 HSA: human serum albumin 
               
               
                 RhSA: rhesus serum albumin 
               
               
                 RSA: rat serum albumin 
               
               
                 MSA: mouse serum albumin 
               
               
                 h-FcRn: human neonatal Fc receptor 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 In vivo serum half-lives of various anti-serum albumin V H H-fusions in rat 
               
            
           
           
               
               
               
            
               
                   
                   
                 Serum  
               
               
                 Fusion protein description 
                 Name 
                 T 1/2  (h)* 
               
               
                   
               
               
                 Monovalent V H H + 
                 B39 control 
                 ~0.5 α   
               
               
                 anti-serum albumin V H H 
                 B39-A20.1 control 
                  1.4 
               
               
                   
                 B39-R11 
                 46.1 
               
               
                   
                 B39-R28 
                 31.1 
               
               
                   
                 B39-M75 
                  4.3 
               
               
                   
                 B39-M79 
                 31.9 
               
               
                 Biparatopic V H H-V H H + 
                 A20-A26 control 
                  1.8 
               
               
                 anti-serum albumin V H H 
                 A20-A26-M75 
                  6.8 
               
               
                   
                 A20-A26-M79 
                 45.0 
               
               
                 Growth factor binding protein + 
                 CIBP2 control 
                 n.d. #   
               
               
                 anti-serum albumin V H H 
                 CIBP2-M75 
                  4.9 #   
               
               
                   
                 CIBP2-M79 
                 40.3 #   
               
               
                 BBB carrier V H H + peptide + 
                 FC5-ABP control 
                  1.1 α,#   
               
               
                 anti-serum albumin V H H 
                 FC5-ABP-M75 
                  0.5 α,#   
               
               
                   
                 FC5-ABP-M79 
                 25.4 #   
               
               
                 Enzyme +  
                 IDS-C1 control (140 
                  0.9 α,#   
               
               
                 anti-serum albumin V H H 
                 nmol/kg) 
                   
               
               
                   
                 IDS-R28 (80 nmol/kg) 
                  2.8 #   
               
               
                   
                 IDS-M79 (80 nmol/kg) 
                  3.1 #   
               
               
                   
                 IDS-R28 (160 nmol/kg) 
                  4.4 #   
               
               
                   
                 IDS-M79 (160 nmol/kg) 
                  3.6 #   
               
               
                 Monovalent V H H + humanized 
                 B39-M75-H1 
                  3.8 
               
               
                 anti-serum albumin V H H 
                 B39-R28-H5 
                 51.2 
               
               
                   
                 B39-R11-H6 
                 41.4 
               
               
                   
               
               
                 *Mean serum T 1/2  (terminal half-life, in h, unless otherwise noted), from n = 3 rats per group, determined by non-compartmental analysis using PK Solver v2.0 (Zhang et al, 2010). 
               
               
                   α alpha half-life, β half-life could not be determined 
               
               
                   # determined by MRM analysis 
               
               
                 BBB: blood brain barrier 
               
               
                 n.d.: could not be determined 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Biophysical characteristics of wild-type and humanized anti-serum albumin V H H S . 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Stability 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 # positions 
                 Yield 1   
                 SEC (% ) 
                 (SEC, % 
                 T m (° C.) 
                 K D HSA 
                 K D RhSA 
                 K D RSA 
                 K D HSA 
                 K D RhSA 
                 K D RSA 
               
               
                 V H H 
                 humanized 
                 (mg) 
                 main peak 2   
                 main peak) 3   
                 ±SEM 4   
                 (nM) 5   
                 (nM) 5   
                 (nM) 5   
                 (nM) 6   
                 (nM) 6   
                 (nM) 6   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 R11 
                 0 
                 5.25 
                 94.3 
                 98.2 
                 69.64 ± 0.09 
                 93.1 
                 159 
                 61.7 
                 28.9 
                 116 
                 29.9 
               
               
                 R11-H0 
                 19 
                 0.38 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 R11-H1 
                 17 
                 0.46 
                 85.8 
                 93.8 
                 — 
                 n.b. 
                 n.b. 
                 n.b. 
                 — 
                 — 
                 — 
               
               
                 R11-H2 
                 16 
                 0.85 
                 50.6 
                 — 
                 — 
                 n.b. 
                 n.b. 
                 n.b. 
                 — 
                 — 
                 — 
               
               
                 R11-H3 
                 14 
                 1.87 
                 96.0 
                 99.0 
                 61.64 ± 0.09 
                 c.b. 
                 c.b. 
                 c.b. 
                 — 
                 — 
                 — 
               
               
                 R11-H4 
                 12 
                 6.37 
                 96.5 
                 98.5 
                 68.58 ± 0.07 
                 c.b. 
                 c.b. 
                 c.b. 
                 — 
                 — 
                   
               
               
                 R11-H5 
                 10 
                 3.89 
                 96.3 
                 93.2 
                 65.16 ± 0.11 
                 307 
                 643 
                 196 
                 98.2 
                 525 
                 160 
               
               
                 R11-H6 
                 8 
                 7.06 
                 96.3 
                 99.1 
                 67.47 ± 0.09 
                 252 
                 561 
                 151 
                 67.5 
                 511 
                 123 
               
               
                 R28 
                 0 
                 5.50 
                 95.4 
                 90.2 
                 71.56 ± 0.20 
                 12.1 
                 38.2 
                 0.4 
                 1.8 
                 5.9 
                 0.3 
               
               
                 R28-H0 
                 15 
                 0.09 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 R28-H1 
                 13 
                 1.77 
                 85.3 
                 96.6 
                 66.13 ± 0.12 
                 162 
                 2710 
                 5.3 
                 15.2 
                 28.7 
                 6.5 
               
               
                 R28-H2 
                 11 
                 2.97 
                 93.7 
                 98.8 
                 66.55 ± 0.14 
                 147 
                 2630 
                 4.1 
                 6.6 
                 668 
                 1 
               
               
                 R28-H3 
                 9 
                 7.43 
                 95.5 
                 96.6 
                 70.88 ± 0.08 
                 51.3 
                 625 
                 2.4 
                 47.2 
                 1270 
                 1.8 
               
               
                 R28-H4 
                 7 
                 1.98 
                 89.5 
                 80.9 
                 66.84 ± 0.23 
                 11.5 
                 1740 
                 1.1 
                 51.6 
                 341 
                 5.3 
               
               
                 R28-H5 
                 6 
                 4.49 
                 95.8 
                 93.5 
                 68.12 ± 0.26 
                 11.1 
                 145 
                 0.4 
                 2 
                 7.3 
                 3.5 
               
               
                 M75 
                 0 
                 5.88 
                 96.9 
                 93.5 
                 79.77 ± 0.07 
                 1.2 
                 2.9 
                 315 
                 735 
                 n.b 
                 n.b. 
               
               
                 M75-H0 
                 15 
                 0.43 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 M75-H1 
                 13 
                 5.82 
                 97.9 
                 97.0 
                 78.03 ± 0.08 
                 3.2 
                 6.1 
                 659 
                 — 
                 — 
                 — 
               
               
                 M75-H2 
                 11 
                 6.23 
                 94.4 
                 96.9 
                 73.16 ± 0.13 
                 7.8 
                 63.6 
                 2490 
                 — 
                 — 
                 — 
               
               
                 M75-H3 
                 9 
                 5.53 
                 93.5 
                 99.1 
                 78.61 ± 0.08 
                 4.8 
                 17.7 
                 1260 
                 — 
                 — 
                 — 
               
               
                 M75-H4 
                 7 
                 5.46 
                 95.9 
                 97.7 
                 75.98 ± 0.09 
                 2.8 
                 6.7 
                 377 
                 — 
                 — 
                 — 
               
               
                 M75-H5 
                 5 
                 5.78 
                 94.2 
                 99.0 
                 80.88 ± 0.07 
                 2.1 
                 4.6 
                 62.4 
                 — 
                 — 
                 — 
               
               
                 M79 
                 0 
                 9.83 
                 98.1 
                 98.2 
                 67.16 ± 0.09 
                 122 
                 84.6 
                 12.6 
                 7.1 
                 2.0 
                 7.1 
               
               
                 M79-H0 
                 19 
                 0.27 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 M79-H1 
                 17 
                 0.27 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 M79-H2 
                 16 
                 8.20 
                 96.7 
                 98.5 
                 63.26 ± 0.09 
                 76.2 
                 62.1 
                 10.4 
                 6.5 
                 2.6 
                 5.9 
               
               
                 M79-H3 
                 14 
                 4.99 
                 98.7 
                 99.2 
                 60.94 ± 0.09 
                 53.3 
                 36.7 
                 8.7 
                 9.6 
                 2.9 
                 12.2 
               
               
                 M79-H4 
                 11 
                 7.63 
                 98.3 
                 95.7 
                 64.66 ± 0.08 
                 58.9 
                 38.7 
                 10 
                 8.4 
                 2.9 
                 8.8 
               
               
                 M79-H5 
                 9 
                 8.20 
                 98.1 
                 98.6 
                 70.44 ± 0.12 
                 103 
                 86.5 
                 20.2 
                 8.1 
                 4.2 
                 6.6 
               
               
                 H18 
                 0 
                 8.38 
                 98.0 
                 — 
                 72.76 ± 0.11 
                 33.9 
                 47.6 
                 n.b. 
                 46.2 
                 42.2 
                 n.b. 
               
               
                 H18-H0 
                 14 
                 0.11 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 H18-H1 
                 12 
                 0.82 
                 67.6 
                 — 
                 61.65 ± 0.14 
                 52.8 
                 91.5 
                 n.b. 
                 56.3 
                 92.2 
                 n.b. 
               
               
                 H18-H2 
                 10 
                 0.12 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 H18-H3 
                 8 
                 10.54 
                 93.5 
                 — 
                 69.52 ± 0.11 
                 19.1 
                 38.2 
                 n.b. 
                 22.3 
                 57.6 
                 n.b. 
               
               
                 H18-H4 
                 7 
                 10.84 
                 92.3 
                 — 
                 68.92 ± 0.14 
                 58.6 
                 34.4 
                 n.b. 
                 57.1 
                 72.7 
                 n.b. 
               
               
                 H18-H5 
                 5 
                 12.03 
                 92.3 
                 — 
                 69.60 ± 0.09 
                 59.4 
                 91.7 
                 n.b. 
                 46.6 
                 70.7 
                 n.b. 
               
               
                 Rh34 
                 0 
                 0.65 
                 91.6 
                 — 
                 agg 
                 286 
                 333 
                 n.b. 
                 129 
                 167 
                 n.b. 
               
               
                 Rh34-H0 
                 16 
                 0.04 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 Rh34-H1 
                 14 
                 0.53 
                 63.8 
                 — 
                 — 
                 824 
                 1350 
                 n.b. 
                 1560 
                 1920 
                 n.b. 
               
               
                 Rh34-H2 
                 12 
                 2.56 
                 76.0 
                 — 
                 — 
                 1330 
                 1470 
                 n.b. 
                 2320 
                 3080 
                 n.b. 
               
               
                 Rh34-H3 
                 10 
                 3.71 
                 86.3 
                 — 
                 — 
                 1370 
                 1260 
                 n.b. 
                 2310 
                 1810 
                 n.b. 
               
               
                 Rh34-H4 
                 9 
                 1.61 
                 92.1 
                 — 
                 — 
                 647 
                 739 
                 n.b. 
                 974 
                 2250 
                 n.b. 
               
               
                 Rh34-H5 
                 7 
                 2.53 
                 94.1 
                 — 
                 — 
                 410 
                 589 
                 n.b. 
                 524 
                 1420 
                 n.b. 
               
               
                 Rh46 
                 0 
                 4.25 
                 97.5 
                 — 
                 65.38 ± 0.04 
                 80.1 
                 80.8 
                 n.b. 
                 41.3 
                 29.6 
                 n.b. 
               
               
                 Rh46-H0 
                 19 
                 0.08 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
                 — 
               
               
                 Rh46-H1 
                 17 
                 5.15 
                 95.2 
                 — 
                 66.29 ± 0.16 
                 583 
                 707 
                 n.b. 
                 97.7 
                 40.8 
                 n.b. 
               
               
                 Rh46-H2 
                 15 
                 9.83 
                 96.6 
                 — 
                 67.44 ± 0.09 
                 426 
                 456 
                 n.b. 
                 87.3 
                 44.1 
                 n.b. 
               
               
                 Rh46-H3 
                 13 
                 10.13 
                 94.1 
                 — 
                 72.44 ± 0.09 
                 289 
                 415 
                 n.b. 
                 70.3 
                 34.0 
                 n.b. 
               
               
                 Rh46-H4 
                 11 
                 6.95 
                 94.8 
                 — 
                 67.55 ± 0.12 
                 237 
                 405 
                 n.b. 
                 51.5 
                 25.2 
                 n.b. 
               
               
                 Rh46-H5 
                 9 
                 14.26 
                 94.6 
                 — 
                 69.31 ± 0.10 
                 233 
                 343 
                 n.b. 
                 54.9 
                 24.1 
                 n.b. 
               
               
                   
               
               
                   1 Purified mg of V H H from 250 mL  E. coli  cultures (periplasmic extraction, IMAC purification) 
               
               
                   2 SEC monomer peak area after IMAC purification of V H H S  (Superdex 75 Increase column, 0.5 mL/min, HBS-EP buffer) 
               
               
                   3 SEC monomer peak area after 4 week storage of peak fractions from 2  at 4° C. (Superdex 75 Increase column, 0.5 mL/min, HBS-EP buffer) 
               
               
                   4 Determined by circular dichroism spectroscopy unfolding at 210 nm (25-106° C., 1° C./min, 100 mM NaPi buffer) 
               
               
                   5 SPR performed at pH 7.4, using single-cycle kinetic measurements on a Biacore T200 
               
               
                   6 SPR performed at pH 5.5, using single-cycle kinetic measurements on a Biacore T200 
               
               
                 HSA: human serum albumin 
               
               
                 RhSA: rhesus serum albumin 
               
               
                 RSA: rat serum albumin 
               
               
                 “—”: not determined 
               
               
                 n.b.: no binding by SPR 
               
               
                 c.b.: complex binding by SPR, data not analyzable 
               
               
                 agg: aggregation upon heating, T m , not determined 
               
            
           
         
       
     
     According to other embodiments, the antibody or an antigen-binding fragment of the present invention may be sdAb having sequences substantially identical to sdAb R11, R28, M75, M79, H18, Rh34 and Rh46, operable to bind to serum albumin from multiple species, including, but not limited to, human, monkey, rat, and mouse. A substantially identical sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutation to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, physico-chemical or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered “substantially identical” polypeptides. A conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity). According to one embodiment, these conservative amino acid mutations may be made to the framework regions of the sdAb while maintaining the CDR sequences listed above and the overall structure of the CDR of the antibody or fragment; thus the specificity and binding of the antibody are maintained. According to another embodiment, these conservative amino acid mutations may be made to the framework regions of the sdAb and the CDR sequence listed above while maintaining the antigen-binding function of the overall structure of the CDR of the antibody or fragment; thus the specificity and binding of the antibody are maintained. 
     In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term “basic amino acid” it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term “neutral amino acid” (also “polar amino acid”), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term “hydrophobic amino acid” (also “non-polar amino acid”) is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). “Acidic amino acid” refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D). 
     Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and as found at ca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any other appropriate software that is known in the art. 
     The substantially identical sequences of the present invention may be at least 90% identical; in another example, the substantially identical sequences may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical, or any percentage therebetween, at the amino acid level to sequences described herein. Importantly, the substantially identical sequences retain the activity and specificity of the reference sequence. In a non-limiting embodiment, the difference in sequence identity may be due to conservative amino acid mutation(s). In a non-limiting example, the present invention may be directed to an antibody or antigen-binding fragment comprising a sequence at least 95%, 98%, or 99% identical to that of the antibodies described herein. 
     The antibody or an antigen-binding fragment of the present invention may be used for example to improve the half-life of the compounds in serum, by targeting an albumin moiety. As used herein, the expression “targeting an albumin moiety” is intended to mean that the antibody or an antigen-binding fragment of the present invention are enabled to bind to serum albumin and particularly to human, rhesus, mouse and rat serum albumin. 
     The term “antibody”, also referred to in the art as “immunoglobulin” (Ig), as used herein refers to a protein constructed from paired heavy and light polypeptide chains; various Ig isotypes exist, including IgA, IgD, IgE, IgG, and IgM. When an antibody is correctly folded, each chain folds into a number of distinct globular domains joined by more linear polypeptide sequences. For example, the immunoglobulin light chain folds into a variable (V L ) and a constant (C L ) domain, while the heavy chain folds into a variable (V H ) and three constant (C H 1, C H 2, C H 3) domains. Interaction of the heavy and light chain variable domains (V H  and V L ) results in the formation of an antigen binding region (Fv). Each domain has a well-established structure familiar to those of skill in the art. 
     The light and heavy chain variable regions are responsible for binding the target antigen and can therefore show significant sequence diversity between antibodies. The constant regions show less sequence diversity, and are responsible for binding a number of natural proteins to elicit important biochemical events. The variable region of an antibody contains the antigen-binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The majority of sequence variability occurs in six hypervariable regions, three each per variable heavy (V H ) and light (V L ) chain; the hypervariable regions combine to form the antigen-binding site, and contribute to binding and recognition of an antigenic determinant. The specificity and affinity of an antibody for its antigen is determined by the structure of the hypervariable regions, as well as their size, shape, and chemistry of the surface they present to the antigen. Various schemes exist for identification of the regions of hypervariability, the two most common being those of Kabat and of Chothia and Lesk. Kabat and Wu (1991) define the “complementarity-determining regions” (CDR) based on sequence variability at the antigen-binding regions of the V H  and V L  domains. Chothia and Lesk (1987) define the “hypervariable loops” (H or L) based on the location of the structural loop regions in the V H  and V L  domains. These individual schemes define CDR and hypervariable loop regions that are adjacent or overlapping, those of skill in the antibody art often utilize the terms “CDR” and “hypervariable loop” interchangeably, and they may be so used herein. The CDR/loops are identified herein according to the IMGT nomenclature scheme (i.e., CDR1, 2 and 3, for each variable region). 
     An “antibody fragment” or “antigen-binding fragment” as referred to herein may include any suitable antigen-binding antibody fragment known in the art. The antibody fragment may be a naturally-occurring antibody fragment, or may be obtained by manipulation of a naturally-occurring antibody or by using recombinant methods. For example, an antibody fragment may include, but is not limited to a Fv, single-chain Fv (scFv; a molecule consisting of V L  and V H  connected with a peptide linker), Fab, F(ab) 2 , single-domain antibody (sdAb; a fragment composed of a single V L  or V H  or a V H H), and multivalent presentations of any of these. Antibody fragments such as those just described may require linker sequences, disulfide bonds, or other type of covalent bond to link different portions of the fragments; those of skill in the art will be familiar with the requirements of the different types of fragments and various approaches and various approaches for their construction. 
     In a non-limiting example, the antigen-binding fragment of the present invention may be an sdAb derived from naturally-occurring sources (i.e. in effect, an additional sdAb as the albumin binding sdAb of the present invention). Heavy chain antibodies of camelid origin (Hamers-Casterman et al, 1993) lack light chains and thus their antigen binding sites consist of one domain, termed V H H. SdAbs have also been observed in shark and are termed V NAR  (Nuttall et al, 2003). Other sdAbs may be engineered based on human Ig heavy and light chain sequences (Jespers et al, 2004; To et al, 2005). As used herein, the term “sdAb” includes those sdAb directly isolated from V H , V H H, V L , or V NAR  reservoir of any origin through phage display or other technologies, sdAb derived from the aforementioned sdAb, recombinantly produced sdAb, as well as those sdAb generated through further modification of such sdAb by humanization, affinity maturation, stabilization, solubilization, camelization, or other methods of antibody engineering. Also encompassed by the present invention are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the sdAb. 
     SdAbs possess desirable properties for antibody molecules, such as high thermostability, high detergent resistance, relatively high resistance to proteases (Dumoulin et al, 2002) and high production yield (Arbabi-Ghahroudi et al, 1997); they can also be engineered to have very high affinity by isolation from an immune library (Li et al, 2009) or by in vitro affinity maturation (Davies &amp; Riechmann, 1996). Further modifications to increase stability, such as the introduction of non-canonical disulfide bonds (Hussack et al, 2011a,b; Kim et al, 2012), may also be brought to the sdAb. 
     A person of skill in the art would be well-acquainted with the structure of a single-domain antibody (see, for example, 3DWT, 2P42 in Protein Data Bank). An sdAb comprises a single immunoglobulin domain that retains the immunoglobulin fold; most notably, only three CDR/hypervariable loops form the antigen-binding site. However, and as would be understood by those of skill in the art, not all CDR may be required for binding the antigen. For example, and without wishing to be limiting, one, two, or three of the CDR may contribute to binding and recognition of the antigen by the sdAb of the present invention. The CDR of the sdAb or variable domain are referred to herein as CDR1, CDR2, and CDR3. 
     The present invention further encompasses an antibody or an antigen-binding fragment that is “humanized” using any suitable method known in the art, for example, but not limited to CDR grafting and veneering. Humanization of an antibody or an antigen-binding fragment comprises replacing an amino acid in the sequence with its human counterpart, as found in the human consensus sequence, without loss of antigen-binding ability or specificity; this approach reduces immunogenicity of the antibody or antigen-binding fragment when introduced into human subjects. In the process of CDR grafting, one or more than one of the CDR defined herein may be fused or grafted to a human variable region (V H , or V L ), to other human antibody (IgA, IgD, IgE, IgG, and IgM), to other human antibody fragment framework regions (Fv, scFv, Fab) or to other proteins of similar size and nature onto which CDR can be grafted (Nicaise et al, 2004). In such a case, the conformation of the one or more than one hypervariable loop is likely preserved, and the affinity and specificity of the antibody or an antigen-binding fragment for its target (i.e., human/rhesus/rat/mouse serum albumin, collectively referred to as serum albumin) is likely minimally affected. CDR grafting is known in the art and is described in at least the following: U.S. Pat. Nos. 6,180,370, 5,693,761, 6,054,297, 5,859,205, and European Patent No. 626390. Veneering, also referred to in the art as “variable region resurfacing”, involves humanizing solvent-exposed positions of the antibody or fragment; thus, buried nonhumanized residues, which may be important for CDR conformation, are preserved while the potential for immunological reaction against solvent-exposed regions is minimized. Veneering is known in the art and is described in at least the following: U.S. Pat. Nos. 5,869,619, 5,766,886, 5,821,123, and European Patent No. 519596. Persons of skill in the art would also be amply familiar with methods of preparing such humanized antibody fragments and humanizing amino acid positions. 
     The antibody or an antigen-binding fragment used with the present invention may also comprise additional sequences to aid in expression, detection or purification of a recombinant antibody or an antigen-binding fragment. Any such sequences or tags known to those of skill in the art may be used. For example, and without wishing to be limiting, the antibody or antigen-binding fragment may comprise a targeting or signal sequence (for example, but not limited to ompA or pelB), a detection/purification tag (for example, but not limited to c-Myc, HA, HisS, or His6), or a combination thereof. In another example, the additional sequence may be a biotin recognition site such as that described by Cronan et al in WO 95/04069 or Voges et al in WO/2004/076670. As is also known to those of skill in the art, linker sequences may be used in conjunction with the additional sequences or tags, or may serve as a detection/purification tag. 
     In another embodiment, there is disclosed a compound comprising antibody or an antigen-binding fragment according to the present invention. In embodiments, the antibody or an antigen-binding fragment of the compound may be linked to the remainder of the compound via a linker (also known as a linker sequence. As known to those of skill in the art, linker sequences may be used in conjunction with the antibody or antigen-binding fragment of the present invention of the compound of the present invention. As used herein, the term “linker sequences” is intended to mean short peptide sequences that occur between protein domains. Linker sequences are often composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. The linker sequence can be any linker sequence known in the art that would allow for the antibody and polypeptide of a compound, of the present invention to be operably linked for the desired function. The linker may be any sequence in the art (either a natural or synthetic linker) that allows for an operable fusion comprising an antibody or fragment linked to a polypeptide. For example, the linker sequence may be a linker sequence L such as (GGGGS) n , wherein n equal to or greater than 1, or from about 1 to about 5, or from about 1 to 15, or n may be any number of linker that would allow for the operability of the compound of the present invention. In another example, the linker may be an amino acid sequence, for example, an amino acid sequence that comprises about 3 to about 40 amino acids, or about 5 to about 40 amino acids, or about 10 to about 40 amino acids, or about 15 to about 40 amino acids, or about 20 to about 40 amino acids, or about 25 to about 40 amino acids, or about 30 to about 40 amino acids, or about 35 to about 40 amino acids, or about 3 to about 35 amino acids, or about 5 to about 35 amino acids, or about 10 to about 35 amino acids, or about 15 to about 35 amino acids, or about 20 to about 35 amino acids, or about 25 to about 35 amino acids, or about 30 to about 35 amino acids, or about 3 to about 30 amino acids, or about 5 to about 30 amino acids, or about 10 to about 30 amino acids, or about 15 to about 30 amino acids, or about 20 to about 30 amino acids, or about 25 to about 30 amino acids, or about 3 to about 25 amino acids, or about 5 to about 25 amino acids, or about 10 to about 25 amino acids, or about 15 to about 25 amino acids, or about 20 to about 25 amino acids, or about 3 to about 20 amino acids, or about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or about 15 to about 20 amino acids, or about 3 to about 15 amino acids, or about 5 to about 15 amino acids, or about 10 to about 15 amino acids, or about 15 to about 20 amino acids, or about 3 to about 10 amino acids, or about 5 to about 10 amino acids, or about 3 to about 5 amino acids, or about 3, 5, 10, 15, 20, 25, 30, 35, or 40 amino acids. 
     According to an embodiment, the antibody or an antigen-binding fragment of the compound may be fused to any one of a peptide, polypeptide (e.g. growth factor CIBP2, antimicrobial cyclic peptides), a protein, an enzyme or polypeptide [such as for example iduronate-2-sulfatase (IDS), acid beta-glucosidase (GCase), serine proteases, growth factors], an antibody or a fragment operable to bind a target epitope (e.g. anti-microbial antibodies, anti-inflammatory antibodies, intrabodies, BBB-crossing antibodies, neurodegeneration targets antibodies, ion channel targeting antibodies for pain, imaging, diagnostic, affinity purification reagents, anti-cancer targets, checkpoint inhibitors, GPCR targeting antibodies), or combinations thereof, in which both the antibody or an antigen-binding fragment and the rest of the compound remain functional for their intended purpose. In a preferred embodiment, the compound may be fused to an antibody or an antigen-binding fragment, operable to bind a target epitope. 
     The antibody or antigen-binding fragment of the present invention may also be in a multivalent display format, also referred to herein as multivalent presentation. Multimerization may be achieved by any suitable method of known in the art. For example, and without wishing to be limiting in any manner, multimerization may be achieved using self-assembly molecules such as those described in Zhang et al (2004a; 2004b) and W02003/046560, where pentabodies are produced by expressing a fusion protein comprising the antibody or antigen-binding fragment of the present invention and the pentamerization domain of the B-subunit of an AB5 toxin family (Merritt &amp; Hol, 1995). A multimer may also be formed using the multimerization domains described by Zhu et al. (2010); this form, referred to herein as a “combody” form, is a fusion of the antibody or fragment of the present invention with a coiled-coil peptide resulting in a multimeric molecule (Zhu et al., 2010). Other forms of multivalent display are also encompassed by the present invention. For example, and without wishing to be limiting, the antibody or antigen-binding fragment may be presented as a dimer, a trimer, or any other suitable oligomer. This may be achieved by methods known in the art (Spiess et al, 2015), for example direct linking connection (Nielsen et al, 2000), c-jun/Fos interaction (de Kruif &amp; Logtenberg, 1996), “Knob into holes” interaction (Ridgway et al, 1996). 
     Another method known in the art for multimerization is to dimerize the antibody or antigen-binding fragment using an Fc domain, for example, but not limited to human Fc domains. The Fc domains may be selected from various classes including, but not limited to, IgG, IgM, or various subclasses including, but not limited to IgG1, IgG2, etc. In this approach, the Fc gene in inserted into a vector along with the sdAb gene to generate a sdAb-Fc fusion protein (Bell et al, 2010; Iqbal et al, 2010); the fusion protein is recombinantly expressed then purified. For example, and without wishing to be limiting in any manner, multivalent display formats may encompass chimeric or humanized formats of antibodies V H H of the present invention linked to an Fc domain, or bi or tri-specific antibody fusions with two or three antibodies V H H recognizing unique epitopes. Such antibodies are easy to engineer and to produce, can greatly extend the serum half-life of sdAb, and may be excellent tumor imaging reagents (Bell et al., 2010). 
     The Fc domain in the multimeric complex as just described may be any suitable Fc fragment known in the art. The Fc fragment may be from any suitable source; for example, the Fc may be of mouse or human origin. In a specific, non-limiting example, the Fc may be the mouse Fc2b fragment or human Fc1 fragment (Bell et al, 2010; Iqbal et al, 2010). The Fc fragment may be fused to the N-terminal or C-terminal end of the V H H or humanized versions of the present invention. 
     Each subunit of the multimers described above may comprise the same or different antibodies or antigen-binding fragments of the present invention, which may have the same or different specificity. Additionally, the multimerization domains may be linked to the antibody or antigen-binding fragment using a linker, as required; such a linker should be of sufficient length and appropriate composition to provide flexible attachment of the two molecules, but should not hamper the antigen-binding properties of the antibody. As defined above, the linker sequence can be any linker known in the art that would allow for the compound of the present invention to be prepared and be operable for the desired function. For example, such a linker sequence should be of sufficient length and appropriate composition to provide flexible attachment of the two molecules, but should not hamper the antigen-binding properties of the antibody. 
     According to another embodiment, the present invention also encompasses a composition comprising one or more than one of the compound as described herein. The composition may comprise a single sdAb and/or compound as described above, or may be a mixture of sdAb or compounds. Furthermore, in a composition comprising a mixture of sdAb or compounds of the present invention, the sdAb or compound may have the same specificity, or may differ in their specificities; for example, and without wishing to be limiting in any manner, the composition may comprise sdAb or compounds specific to albumin (same or different epitope). 
     The composition may also comprise a pharmaceutically acceptable diluent, excipient, or carrier. The diluent, excipient, or carrier may be any suitable diluent, excipient, or carrier known in the art, and must be compatible with other ingredients in the composition, with the method of delivery of the composition, and is not deleterious to the recipient of the composition. The composition may be in any suitable form; for example, the composition may be provided in suspension form, powder form (for example, but limited to lyophilised or encapsulated), capsule or tablet form. For example, and without wishing to be limiting, when the composition is provided in suspension form, the carrier may comprise water, saline, a suitable buffer, or additives to improve solubility and/or stability; reconstitution to produce the suspension is effected in a buffer at a suitable pH to ensure the viability of the antibody or antigen-binding fragment. Dry powders may also include additives to improve stability and/or carriers to increase bulk/volume; for example, and without wishing to be limiting, the dry powder composition may comprise sucrose or trehalose. In a specific, non-limiting example, the composition may be so formulated as to deliver the antibody or antigen-binding fragment to the gastrointestinal tract of the subject. Thus, the composition may comprise encapsulation, time release, or other suitable technologies for delivery of the sdAb or compounds of the present invention. It would be within the competency of a person of skill in the art to prepare suitable compositions comprising the present sdAb or compounds. 
     The invention also encompasses nucleic acid vector comprising a nucleotide sequence encoding a sdAb or a compound of the present invention, as well as cells comprising the nucleic acid vector, for expressing the sdAb or compound of the present invention, and cells for expressing the sdAb or compound of the present invention. 
     According to another embodiment, there is provided a method of removing a molecule from serum, comprising administering a compound according to the present invention, specific to the molecule, wherein the sdAb comprises CDR1, CDR2 and CDR3 comprising an amino acid sequence comprising GRTFDNYVM (SEQ ID NO:8), ISGSGSIT (SEQ ID NO:9), and AAGSRRTYYREPKFYPS (SEQ ID NO:10), respectively. 
     According to another embodiment, there is provided a use of a compound according to the present invention which is specific to a molecule, for removing the molecule from serum, wherein the sdAb comprises CDR1, CDR2 and CDR3 comprising an amino acid sequence comprising GRTFDNYVM (SEQ ID NO:8), ISGSGSIT (SEQ ID NO:9), and AAGSRRTYYREPKFYPS (SEQ ID NO:10), respectively. 
     According to another embodiment, there is provided a solid support for purification of albumin, derivatives thereof, or fragments thereof comprising a solid or semi-solid medium linked to an antibody or an antigen-binding fragment according to the present invention or a compound according to any one of claims  12  to  15 . 
     According to another embodiment, there is provided a method of purifying albumin comprising contacting an albumin containing sample with a solid support according to the present invention. 
     The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope. 
     Serum Albumin V H H Isolation 
     Example 1 
     Llama Immunization 
     One male llama ( Lama glama ) was immunized by Cedarlane (Burlington, ON, Canada) four times with 100 μg of human serum albumin (HSA; Sigma, Oakville, ON, Canada) in 1 mL of phosphate-buffered saline (PBS), pH 7.4, emulsified in an equal volume of Freund&#39;s complete adjuvant for the priming immunization (day 0) or Freund&#39;s incomplete adjuvant for the boosting immunizations (days 21, 28 and 35). Pre-immune blood was drawn before the first injection on day 1 and served as a negative control. One week after the final immunization, serum and peripheral blood mononuclear cells (PBMCs) were obtained from the animal (day 42). 
     Example 2 
     Fractionation of Serum 
     Pre-immune and day 42 llama sera were fractionated by protein G and protein A chromatography (Hi Trap, GE Healthcare, Mississauga, ON, Canada) and eluted by acidic elution. Serum fractions A1 (HCAb), A2 (HCAb), G1 (HCAb), and G2 (clgG) were neutralized with Tris pH 8.8 and dialyzed against PBS pH 7.4 for storage at 4° C. IgG serum fractions were measured using 1.3 AbS 280nm =1 mg/mL. 
     Example 3 
     ELISA of Whole and Fractionated Serum 
     Total serum (pre-immune and day 42), as well as the resulting fractionated sera, A1 (HCAb), A2 (HCAb), G1 (HCAb), and G2 (conventional IgG), were analyzed for specific binding to serum albumins from human, rhesus, rat and mouse (HSA, RhSA, MSA and RSA, respectively) by ELISA. Wells of NUNC MaxiSorp™ microtiter plates were coated overnight at 4° C. with 1.5 μg of each serum albumin in 100 μL PBS. The next day, wells were blocked with 300 μL of PBS containing 5% (w/v) skim milk and 0.05% (v/v) Tween-20 for 1.5 h at 37° C., then sera were diluted in PBS, added to wells and incubated for 1 h. Wells were washed 3× with PBS containing 0.1% Tween-20 (PBS-T), incubated with HRP-conjugated goat anti-llama IgG (Cedarlane) diluted to 1:10000 in PBS, then washed again 3× with PBS-T. Wells were developed with 100 μL of tetramethylbenzidine substrate (Mandel Scientific, Guelph, ON, Canada) then after 5 min, the reaction was stopped with 100 μL of 1 M H2504 and the absorbance at 450 nm was measured using a Multiskan™ FC photometer (Thermo-Fisher, Ottawa, ON, Canada)( FIG. 1 ). 
     Example 4 
     Library Construction 
     A phage-displayed V H H library was constructed from the heavy-chain-only antibody repertoire of the immunized llama as described previously (Hussack et al, 2011a; Baral et al, 2013). Briefly, total cellular RNA was extracted from approximately 5×10 7  peripheral blood mononuclear cells (PBMCs) using a PureLink® RNA Mini Kit (Life Technologies, Carlsbad, Calif.), pooled, then reverse transcribed using SuperScript® VILO™ MasterMix (Life Technologies) as per the manufacturer&#39;s instructions. Rearranged V H H genes were amplified using two rounds of semi-nested PCR and cloned into the pMED1 phagemid vector, and then phage were rescued from library-bearing  Escherichia coli  TG1 cells by superinfection with M13KO7 helper phage (Life Technologies) and purified by polyethylene glycol precipitation, essentially as previously described (Hussack et al, 2011a). 
     Example 5 
     Panning 
     The phage-displayed V H H library was panned, essentially as described (Hussack et al, 2011a; Baral et al, 2013), for a single round simultaneously against HSA, RhSA, MSA and RSA immobilized in separate wells. Briefly, wells of NUNC MaxiSorp™ microtiter plates (Thermo-Fisher) were coated overnight at 4° C. with 5 μg of each serum albumin in 100 μL of PBS. The next day, wells were blocked for 1.5 h at 37° C. with 300 μL of PBS containing 5% (w/v) skim milk and 0.05% (v/v) Tween-20, then ˜10 12  phage particles (diluted in 100 μL PBS containing 20% (v/v) SuperBlock™ (Life Technologies)) were applied to each well and incubated at room temperature for 2 h. The wells were washed five times with PBS containing 0.05% (v/v) Tween-20 (PBS-T), five times with PBS and then bound phage were eluted sequentially with 100 μL of 100 mM triethylamine followed by 100 μL of 100 mM glycine, pH 2.0. Both high and low pH phage elutions were neutralized with 50 μL of 1 M Tris.HCl, pH 8.0, pooled and titered. As a control, the library was panned against an antigen-free well containing only blocking solution. 
     Example 6 
     Next Generation DNA Sequencing 
     The original library phage and the phage eluted from each panning (HSA, RhSA, MSA and RSA) were used directly as templates for next generation sequencing (NGS). Approximately 10 6  phage particles were used as template in 25 μL PCR reactions containing 1×ABI Buffer II, 1.5 mM MgCl 2 , 200 μM each dNTP (Thermo-Fisher), 5 pmol each of primers NGS-MJ7 (5′CGCTCTTCCGATCTCTGNNNNNGCCCAGCCGGCCATGGCC) and NGS-MJ8 (5′TGCTCTTCCGATCTGACNNNNNTGAGGAGACGGTGACCTGG) and 1 U of AmpliTaq® Gold DNA polymerase (Life Technologies) and cycled as follows on an GeneAmp® PCR System 9700 thermal cycler (Applied BioSystems, Foster City, Calif.): 95° C. for 7 min; 35 cycles of (94° C. for 30 s, 55° C. for 45 s, and 72° C. for 2 min); 72° C. for 10 min. The resulting amplicons were purified using PureLink® PCR purification kits (Life Technologies) with a ≥300 bp size cutoff according to the manufacturer&#39;s instructions. Each sample was individually barcoded in a second “tagging” 50 μL PCR reaction containing 1× Phusion HF Buffer, 1.5 mM MgCl 2 , 200 μM each dNTP, 10 pmol of each primer pair P5-seqF (5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT TCCGATCTCTG) and P7-index1-seqR (5′ CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGT GCTCTTCCGATCTGAC) sequences. 0.25 U Phusion High-Fidelity DNA polymerase (Thermo-Fisher) and 5 μL first-round PCR as template, then cycled as follows: 98° C. for 30 s; 20 cycles of (98° C. for 10 s, 65° C. for 30 s, and 72° C. for 30 s); 72° C. for 5 min. The final five amplicons (derived from library phage, HSA output phage, RhSA output phage, MSA output phage and RSA output phage) were pooled and purified from 1% (w/v) agarose gels using a QIAquick® gel extraction kit (QIAGEN, Toronto, ON, Canada), desalted using Agencourt AMPure XP beads (Beckman-Coulter, Pasadena, Calif.), then sequenced on a MiSeq Sequencing System (Illumina, San Diego, Calif.) using a 500-cycle MiSeq Reagent Kit V2 and a 5% PhiX genomic DNA spike. From each sample, 1.8-2.4 million reads were generated, of which 0.4-1.2 million were used for analysis after assembly using FLASH (default parameters; (Magoc and Salzberg, 2011) and quality filtering using the FAST-X toolkit with a stringency of Q30 over 95% of each read (Schmieder and Edwards, 2011). The DNA sequence of each V H H was then translated in silico, and the CDR3 sequence (IMGT positions 105-117) parsed using conserved N-terminal amino acid consensus sequences (YYC). For each panning, the set of CDR3 sequences derived from the output phage was compared to the set from the V H H library; for each shared CDR3 sequence, an enrichment score was calculated as the frequency in the output phage divided by the frequency in the library. This frequency score was used as a first-pass approximation of the binding behaviour of V H Hs in the library and used for identification of putative serum albumin-binding V H Hs with a range of predicted cross-species reactivity. A fold-enrichment of 10 was used as a cut-off for putative serum albumin binding. 
     Example 7 
     Subcloning Expression and Purification 
     The DNA sequences of seven V H  Hs were synthesized commercially in the pSJF2 expression vector (GenScript, Piscataway, N.J.) and each construct was produced in  E. coli . Briefly, 1 L 2×YT cultures containing 100 μg/mL ampicillin, 0.1% (w/v) glucose and 0.5 mM IPTG were inoculated with single plasmid-bearing  E. coli  TG1 colonies and grown overnight at 37° C. with 220 rpm shaking. The next morning, periplasmic proteins were extracted by osmotic shock. The resulting supernatant was dialyzed overnight into immobilized metal affinity chromatography buffer A (10 mM HEPES buffer pH7.0, 500 mM NaCl) and sterile filtered. Protein was purified by IMAC using 5 mL HiTrap™ Chelating HP IMAC columns (GE Healthcare), under the control of an ÄKTA™ Express (GE Healthcare). A step-wise gradient of 500 mM imidazole in the above buffer A was used for protein elution. Proteins were stored at 4° C. 
     Example 8 
     Phage ELISA and Soluble ELISA 
     Wells of NUNC MaxiSorp™ microtiter plates were coated overnight at 4° C. with 1.5 μg of each serum albumin in 100 μL PBS. The next day, wells were blocked with 300 μl of PBS containing 5% (w/v) skim milk and 0.05% (v/v) Tween-20 for 1.5 h at 37° C., then serially diluted V H Hs or V H H-bearing phage were added to wells and incubated for 1 h. Wells were washed 3× with PBS containing 0.1% Tween-20 (PBS-T), then incubated in either horseradish peroxidase (HRP)-conjugated rabbit anti-His6 (Cedarlane) or anti-M13 (GE Healthcare) secondary antibody, respectively, both at a dilution of 1:5,000, then washed again 3× with PBS-T and developed with 100 μL of tetramethylbenzidine substrate (Mandel Scientific, Guelph, ON, Canada). After 5 min, the reaction was stopped with 100 μL of 1 M H2504 and the absorbance at 450 nm was measured using a Multiskan™ FC photometer (Thermo-Fisher). 
     Example 9 
     Size Exclusion Chromatography 
     V H Hs were purified by size exclusion chromatography (SEC) using a Superdex™ 75 10/300 GL column (GE Healthcare) under the control of an ÄKTA™-FPLC (GE Healthcare). Briefly 250-500 μg of sample were applied at a flow rate of 0.5 mL/min in a mobile phase that consisted of phosphate buffered saline (PBS pH 7.0). Fractions of 0.5 mL of monomeric V H H were collected. The results are shown in  FIG. 3A . 
     Example 10 
     Isothermal Titration Calorimetry 
     ITC experiments were performed at 25° C. using a MicroCal Auto-ITC 200  (GE Healthcare). To avoid buffer artifacts all serum albumins and V H Hs were buffer exchanged into PBS using SEC. Settings included 18 automatically defined injections of 2 μL over 5 s and a syringe stirring at 1000 rpm. Concentrations of 50 μM were used for the V H H titrants in the syringe and concentrations of 5 μM of the various serum albumins were in the cell. Data analysis was performed with the Origin software package (GE Healthcare). 
     Example 11 
     SPR Binding Assays at pH 7.4 
     For SPR, a total of 1362-1471 resonance units of each serum albumin protein were immobilized in 10 mM acetate buffer, pH 4.5, on CM5 or CM5 series S sensor chips (GE Healthcare) using an amine coupling kit (GE Healthcare). Kinetic analyses were carried out on a Biacore 3000 or Biacore T200 instrument (GE Healthcare) at 25° C. by injecting V H Hs at various concentration ranges, in HBS-EP+ buffer (10 mM HEPES buffer, pH 7.4 containing 150 mM NaCl, 3 mM EDTA and 0.005% (v/v) surfactant P20) and at a flow rate of 20 μL/min. Data were analyzed using BIAevaluation software version 4.1 (GE Healthcare) and fitted to a 1:1 binding model. Results are shown in  FIG. 3B  and Table 1. 
     Example 12 
     SPR Binding Assays at pH 5.5 
     SPR experiments were repeated exactly as described above with the exception of the running buffer which was adjusted to pH 5.5 (HBSP-MES: 10 mM HEPES buffer, pH 5.5, 10 mM MES, 150 mM NaCl, 0.005% P20). SEC-purified fractions of monomeric V H Hs were also buffer exchanged into the same pH 5.5 buffer before running the SPR experiments. The results are shown in  FIGS. 4A, 4B  and Table 1. 
     Example 13 
     SPR-Based Epitope Binning 
     The SPR-based epitope binning experiments identified the different epitope bins targeted by the pool of V H Hs.  FIG. 5A  shows SPR sensorgrams of V H H co-injection experiments of various V H H+V H H combinations (injected at 10-20×K D  concentrations).  FIG. 5B  shows a graphical representation of the three epitope bins targeted by the serum albumin V H Hs. R11 and M79 bind the same epitope, R28 binds an epitope that is partially overlapping with the R11/M79 epitope and M75 binds an epitope distinct of R11, M79 and R28. 
     Example 14 
     SPR FcRn Competition Binding Assay 
     All FcRn binding assays were performed at pH 5.5 using HBSP-MES running buffer. Briefly, human serum albumin was immobilized on a CM5 sensor chip as described above. Human FcRn (h-FcRn, produced recombinantly by NRC) was flowed over immobilized HSA at 2 μM in control experiments to demonstrate binding. To ensure anti-serum albumin V H Hs did not compete with h-FcRn for albumin binding, a co-injection SPR assay was set up as follows. Serum albumin V H Hs were first injected over the HSA surface at concentrations that were 10× their K D  for 120 sat a flow rate of 20 μL/min. Immediately following the first 120 s injection, a second injection followed that contained the V H H and 2 μM h-FcRn. In cases where the V H H did not compete for albumin binding with h-FcRn, sensorgrams show two unique and additive binding responses. The results are shown in  FIGS. 6A and 6B . 
     Anti-Serum Albumin V H H Fusion Proteins 
     Example 15 
     Synthesis of Anti-Serum Albumin V H H Fusion Proteins 
     Anti-serum albumin V H Hs were synthesized as fusion proteins to the  C. difficile  toxin B V H H (B39; Murase et al, 2014) by Genscript using a (GGGGS) 5  linker. A control construct consisting of a  C. difficile  toxin A V H H (A20; Hussak et al, 2011a) fused to B39 was synthesized as a control, as well as the B39 V H H monomer. Sequences were subcloned into the expression vector PSJF2H with N-terminal HA and 6 His tags. Plasmid DNA (5 μg) were diluted into 50 μl of nuclease-free water to produce DNA stocks (100 ng/μL) stored at −20° C.  FIG. 7A  illustrates the different constructs prepared. 
     Example 16 
     Expression and Purification 
     Approximately 5 μL of Zymo Research Mix and Go TG1  E. coli  competent cells (Cedarlane) were aliquoted into PCR tubes placed on ice. To this, 0.5 μL of DNA plasmid stock was added to cells and incubated on ice for 10 min. The cells were plated onto pre-warmed (at 37° C.) 2YT+ampicillin plates for incubation overnight at 32° C. V H H fusions were expressed using a 5-day M9 minimal media method as previously described (Baral et al, 2013). After induction of protein expression, cell cultures were harvested at 5,000 rpm for 20 min (4° C.), the supernatant was decanted, and the cell contents were extracted from the cell pellet by whole cell lysis. Briefly, each pellet was resuspended in 100 mL of ice-cold lysis buffer (50 mM Tris-HCl buffer, pH 8.0, 25 mM NaCl, 2 mM EDTA, pH 8.0) and frozen at −80° C. for 1 h. Next, pellets were thawed at room temperature with the addition of DTT and PMSF (final of 1 mM and 2 mM, respectively). Freshly prepared lysozyme was added to each culture (150 μg/mL final concentration) and incubated for 30 min. DNAse was added (200 μL of 15 units/μL) for further 30 min incubation. The slurry was then centrifuged at 8,000 rpm for 30 min at 4° C. The resulting supernatant was dialyzed overnight into immobilized metal affinity chromatography (IMAC) buffer A (10 mM HEPES, pH 7.0, 500 mM NaCl) and sterile filtered. Protein was purified by IMAC using 5 mL HiTrap™ Chelating HP columns (GE Healthcare), under the control of an ÄKTA™ Express (GE Healthcare). A step-wise gradient of 500 mM imidazole in the above buffer was used for protein elution. Proteins were stored at 4° C. 
     Example 17 
     Size Exclusion Chromatography and SPR 
     Size exclusion chromatography was performed on all purified V H H-V H H fusions with a Superdex 75™ column under the control of an ÄKTA™-FPLC (GE Healthcare) to determine their aggregation state and to provide samples for SPR analysis. Briefly, V H Hs were applied at concentrations of 500 μg with a flow rate of 0.5 mL/min in a mobile phase that consisted of HBS-EP running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% (v/v) P20 surfactant)(GE Healthcare). Approximately 0.5 mL samples were collected and sent for SPR. A Biacore 3000 instrument was used to assess the functionality of V H H-V H H fusion proteins, essentially as described above with the exception that a single concentration injection of V H H-V H H over the human serum albumin and toxin B surfaces was performed. The results are shown in  FIGS. 7B and 7C . 
     Example 18 
     Endotoxin Removal 
     To remove endotoxins, affinity purified fusion proteins were concentrated to 5 mL volume for passage through a HiLoad 1660 S75 size exclusion column (GE Healthcare) under control of an ÄKTA™-FPLC. Briefly, the column was cleaned with 0.5 M NaOH followed by 50% isopropanol to remove endotoxins. V H H fusion protein samples (5 mL) were injected onto the column at 1 mL/min in PBS, pH 7.5, endotoxin-free buffer (Sigma). Collected samples were concentrated on Amicon spin columns (Millipore) to a 1 mL volume and filtered through 0.22 μM filter (Millipore). Samples were then processed on  Proteus  NoEndo™ Mini spin column kits (Generon, Berkshire, UK) as per the manufacturer&#39;s instructions. Samples were tested for endotoxin levels prior to rat PK studies. 
     Example 19 
     Rat PK Studies 
     Male Wistar rats (˜200 g) were injected (i.v.) with equimolar amounts of V H H fusion protein (˜0.25 mg/mL) for a total dose of −1 mg/kg equivalent, with endotoxin levels in the range of 0.14-4 EU/mg. Groups of three rats per fusion protein were tested. Serum (50 μL) was collected at nine time points for up to 168 h. Serum samples were frozen at −20° C. until analysis. The results are shown in  FIG. 8A-D . 
     Example 20 
     ELISA on PK Serum Samples 
     ELISAs were performed to determine the serum half-life of V H H fusion proteins in serum, using purified proteins and standard curves. The B39 V H H antigen, TcdB-RBD (Murase et al, 2014), was coated at 0.3 μg/well in PBS pH 7.4 overnight at 4° C. The next day, wells were blocked in 2% milk in PBS, pH 7.4, for 1 h at 37° C. Next, 100 μL of serum (diluted 1:10, 1:50 or 1:250 in PBS, depending on serum time point) were added to wells in duplicate. Standard curves were also produced on each plate. Serum samples were incubated at room temperature for 1 h. Following three washes with PBS-Tween 20 (0.05%, v/v), a secondary antibody of anti-HA-HRP (1:5000 dilution) in PBS pH 7.4 was added to each well and incubated for 1 h at room temperature. A final set of three washes preceded the addition of the HRP substrate tetramethylbenzidine (Mandel Scientific). The reaction was stopped with 1.5 M phosphoric acid, and the absorbance was measured using a plate reader at 450 nm. The results are shown in  FIG. 8C . 
     Additional Anti-Serum Albumin V H H Fusion Protein Examples 
     Example 21 
     Synthesis of Additional Anti-Serum Albumin V H H Fusion Constructs 
     DNA encoding the following nine constructs were synthesized and subcloned as described above. A20-A26, A20-A26-M75, A20-A26-M79, FC5-ABP, FC5-ABP-M75, FC5-ABP-M79, CIBP2, CIBP2-M75, and CIBP2-M79 were all subcloned into the mammalian expression vector pTT5™ (Durocher et al, 2002) with HA and His6 tags. Plasmid DNA (5 μg) were diluted into 50 μL of nuclease-free water to produce DNA stocks (100 ng/μL) stored at −20° C. Iduronate-2-sulfatase (IDS; UniProtKB ref #P22304) enzyme-V H H conjugates, IDS-R28 and IDS-M79, were designed, expressed and purified by Oxyrane (Gent, Belgium).  FIGS. 9A, 10A, 11A and 12A  illustrate the different constructs prepared. 
     Example 22 
     Transformation and Plasmid Preparation 
     Approximately 5 μL of Zymo Research Mix and Go TG1  E. coli  competent cells (Cedarlane) were aliquoted into PCR tubes placed on ice. To this, 0.5 μL of DNA plasmid stock was added to cells and incubated on ice for 10 min. The cells were plated onto pre-warmed (at 37° C.) 2YT+ampicillin plates for incubation overnight at 32° C. Starter cultures of 5 mL of 2YT+ampicillin were inoculated with a single colony and grown at 37° C. for 4 h at which point 1 mL was transferred into 200 mL of 2YT+ampicillin in 500 mL ultra-yield flasks with an air top seal for overnight incubation at 37° C. Plasmid extraction was performed using the endo-free plasmid Maxi prep kit (Thermo-Fisher, Ottawa, ON, Canada). Yields of 300-400 μg of plasmid were obtained for transfection into HEK293-6E cells. 
     Example 23 
     Expression and Purification 
     Mammalian expression was performed essentially as described previously (Durocher et al, 2002). HEK293-6E mammalian cells were cultured from frozen in enriched F17 media at 5% CO 2 , 60% humidity, 37° C. and 100 rpm shaking. Cultures (100 mL) were transfected with 100 μg DNA/100 μL PEIpro transfection reagent (Polyplus, Illkirch, France) at a cell density of 1.5×10 6 -1.7×10 6  cells/mL, 99% cell viability. Cells were fed after 24 h with 2% TNI in enriched F17 media and were then grown for 5 d before harvesting. Harvested cultures were spun at 4000 rpm for 15 min on bench top centrifuge. Supernatants were filtered through a 0.22 μM filter (Millipore), dialyzed into endo-free PBS pH 7.4 (Sigma), then loaded onto an IMAC nickel affinity column (GE Healthcare) on the AKTA system and purified as described earlier. Endotoxins were removed and measured as described above. 
     Example 24 
     Rat PK Studies 
     Rat PK studies were performed exactly as described above (1 mg/kg equivalent) for the other anti-serum albumin V H H fusion proteins. The results are shown in  FIGS. 9E, 10B, 11B, 12D and 13B . One set of rats ( FIG. 13B ; B39-R11-H6 test group) received 0.5 mg/kg equivalent. 
     Example 25 
     ELISA Analysis of A20-A26 PK Serum Samples 
     ELISA was performed to determine the serum half-life of A20-A26 fusion proteins (with or without fusion to an anti-serum albumin V H H) in serum. The A20-A26 antigen,  C. difficile  toxin A (List Biological Laboratories, Campbell, Calif.) was coated at 0.1 μg/well in PBS, pH 7.4, overnight at 4° C. The next day, wells were blocked in 2% (w/v) milk in PBS, pH 7.5, for 1 h at 37° C. Next, serum samples (diluted 1:100, 1:1,000, 1:5,000 or 1:10,000 in PBS, depending on the fusion protein and time point) were added to wells in duplicate. ELISA plates were incubated at room temperature for 1 h. Following 3 washes with PBS-Tween 20 (0.05%, v/v), secondary antibody of anti-His-HRP (1:5,000 dilution) in PBS, pH 7.4, was added to each well and incubated for 1 h at room temperature. A final set of three washes preceded the addition of the HRP substrate tetramethylbenzidine (Mandel). The reaction was stopped with 1.5 M sulfuric acid, and the absorbance was measured using a plate reader at 450 nm. Serial dilutions of purified proteins were run on the same plates to generate standard curves. The results are shown in  FIG. 9B . 
     Example 26 
     SPR Analysis 
     SPR assays were used to demonstrate the binding of the fusion proteins to human and rat serum albumin surfaces ( FIG. 9C ). A SPR co-injection assay demonstrate that the A20-A26-M75 and A20-A26-M79 fusion proteins can simultaneously bind to toxin A on the surface and to human serum albumin in solution. The control A20-A26 can only bind to toxin A and does not bind human serum albumin in solution, as expected ( FIG. 9D ). The IDS-R28 and IDS-M79 constructs ( FIG. 12A ) were SEC purified ( FIG. 12B ) before confirming that they retain the ability to bind rat serum albumin in SP ( FIG. 12C ). 
     Example 27 
     MRM Mass Spectrometry Analysis of FC5-ABP, CIBP2 and IDS Fusion Proteins in Rat Serum 
     Using purified protein constructs as controls [CIBP2, CIBP2-M75, CIBP2-M79 ( FIG. 10A ), FC5-ABP, FC5-ABP-M75, FC5-ABP-M79 ( FIG. 11A ) and, and IDS, IDS-R28, IDS-M79 ( FIG. 12A )], MRM mass spectrometry analysis was used to determine the serum concentrations of the above fusion proteins in rats, essentially as previously described (Haqqani et al, 2013;  FIGS. 10B, 11B and 12D ). 
     Example 28 
     Identification of Lead Humanized V H Hs and In Vivo Testing 
     Humanized V H Hs were designed ( FIG. 2 ), expressed in  E. coli , purified by immobilized metal affinity chromatography and assessed by SEC, thermal unfolding (T m ) and binding affinity at pH 7.4 and pH 5.5 for albumins from various species. Lead humanized V H Hs were fused to B39 V H H for in vivo half-life extension studies in rats (Table 3,  FIG. 13 ). Based on the biophysical properties described in Table 3 (expression yield, lack of aggregation, preservation of thermal stability and serum albumin binding affinities), the lead humanized version of each V H H identified are: M75-H1 (SEQ ID NO: 41), M79-H2 (SEQ ID NO: 49), R28-H5 (SEQ ID NO: 38) and R11-H6 (SEQ ID NO: 31). Of these, three examples were fused to the B39 V H H and the serum half-life evaluated in rats, confirming that humanization of the wild-type V H H sequences did not negatively impact half-life extension ( FIG. 13C ). 
     Example 29 
     Human Serum Albumin Domain Mapping 
     To identify which domain of serum albumin the V H Hs described bind, the three major domains of HSA were expressed in mammalian HEK293-6E cells as either individual domains (DI, DII and DIII) or two neighboring domains (DI-DII and DII-DIII). HSA domains were purified by affinity chromatography, subjected to SEC, and used for SPR binding experiments ( FIG. 14 ). The results demonstrated that M75 binds to HSA domain 1 (DI) and R11, R28 and M79 bind to HSA domain 2 (DII). This data is consistent with FcRn competition assays in that none of the V H Hs compete with FcRn for albumin binding that occurs in domain 3 of HSA. 
     Example 30 
     Impact of pH on V H H Affinities for Serum Albumin 
     V H H affinities (K D s) for human and rat serum albumin as a function of pH were determined ( FIG. 15 ), to illustrate the unique pH sensitivity of the M75 V H H for HSA, HSA DI-DII and for RSA. The affinity of M75 for HSA drops significantly, from K D =1.2 nM at pH 7.4 to K D =735 nM at pH 5.5. The affinity of M75 for RSA is K D =315 nM at pH 7.4 while at pH 5.5 the affinity could not be measured because there was no evidence binding. In addition, flowing 50 μM of M75 V H H over RSA surfaces at pH 6.0 did not show a trace of binding. In comparison, the other three V H Hs maintain nearly identical binding affinities for HSA at pHs 7.4 and 5.5. 
     While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure. 
     
       
         
           
               
            
               
                   
               
               
                 SEQUENCES 
               
            
           
           
               
               
               
            
               
                 SEQ ID NO: 
                 Sequence 
                 Description 
               
               
                   
               
               
                 SEQ ID NO: 1 
                 GFLLRSNTM 
                 CDR1-R11 
               
               
                   
               
               
                 SEQ ID NO: 2 
                 IRPSGLT 
                 CDR2-R11 
               
               
                   
               
               
                 SEQ ID NO: 3 
                 HTRPPFQRDS 
                 CDR3.1-R11 
               
               
                   
               
               
                 SEQ ID NO: 4 
                 ATRPPFQRDS 
                 CDR3.2-R11 
               
               
                   
               
               
                 SEQ ID NO: 5 
                 GRTFIAYAM 
                 CDR1-R28 
               
               
                   
               
               
                 SEQ ID NO: 6 
                 ITNFAGGTT 
                 CDR2-R28 
               
               
                   
               
               
                 SEQ ID NO: 7 
                 AADRSAQTMRQVRPVLPY 
                 CDR3-R28 
               
               
                   
               
               
                 SEQ ID NO: 8 
                 GRTFDNYVM 
                 CDR1-M75 
               
               
                   
               
               
                 SEQ ID NO: 9 
                 ISGSGSIT 
                 CDR2-M75 
               
               
                   
               
               
                 SEQ ID NO: 10 
                 AAGSRRTYYREPKFYPS 
                 CDR3-M75 
               
               
                   
               
               
                 SEQ ID NO: 11 
                 GSTFSSSSV 
                 CDR1-M79 
               
               
                   
               
               
                 SEQ ID NO: 12 
                 ITSGGST 
                 CDR2-M79 
               
               
                   
               
               
                 SEQ ID NO: 13 
                 NVAGRNWVPISRYSPGPY 
                 CDR3.1-M79 
               
               
                   
               
               
                 SEQ ID NO: 14 
                 AVAGRNWVPISRYSPGPY 
                 CDR3.2-M79 
               
               
                   
               
               
                 SEQ ID NO: 15 
                 GSIESINRM 
                 CDR1-H18 
               
               
                   
               
               
                 SEQ ID NO: 16 
                 ISKGGST 
                 CDR2-H18 
               
               
                   
               
               
                 SEQ ID NO: 17 
                 AAGPVWEQF 
                 CDR3-H18 
               
               
                   
               
               
                 SEQ ID NO: 18 
                 GRTISLYAV 
                 CDR1-Rh34 
               
               
                   
               
               
                 SEQ ID NO: 19 
                 ISWTDSST 
                 CDR2-Rh34 
               
               
                   
               
               
                 SEQ ID NO: 20 
                 AADVSIRGLQKYEYDY 
                 CDR3-Rh34 
               
               
                   
               
               
                 SEQ ID NO: 21 
                 TRTFSSYIM 
                 CDR1-Rh46 
               
               
                   
               
               
                 SEQ ID NO: 22 
                 ISWSGRMT 
                 CDR2-Rh46 
               
               
                   
               
               
                 SEQ ID NO: 23 
                 AADRTTAWGAPRSQYDS 
                 CDR3-Rh46 
               
               
                   
               
               
                 SEQ ID NO: 24 
                 QVKLEESGGGLVQAGGSLRLSCVGPGFLLRSNTMGWY 
                 R11 sdAb 
               
               
                   
                 RQAPGKERELVAFIRPSGLTNYNDAVQGRFTISRDNAK 
                   
               
               
                   
                 NTVYLQMNALKPEDTAVYYCHTRPPFQRDSWGQGTQV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 25 
                 EVQLVESGGGLVQPGGSLRLSCAASGFLLRSNTMGWV 
                 R11-H0 
               
               
                   
                 RQAPGKGLEWVSFIRPSGLTNYNDAVQGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCATRPPFQRDSWGQGTLV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 26 
                 EVQLVESGGGLVQPGGSLRLSCAASGFLLRSNTMGWY 
                 R11-H1 
               
               
                   
                 RQAPGKGLELVSFIRPSGLTNYNDAVQGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCATRPPFQRDSWGQGTLV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 27 
                 EVQLVESGGGLVQPGGSLRLSCAASGFLLRSNTMGWY 
                 R11-H2 
               
               
                   
                 RQAPGKGLELVSFIRPSGLTNYNDAVQGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCHTRPPFQRDSWGQGTLV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 28 
                 EVQLVESGGGLVQPGGSLRLSCAASGFLLRSNTMGWY 
                 R11-H3 
               
               
                   
                 RQAPGKERELVSFIRPSGLTNYNDAVQGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCHTRPPFQRDSWGQGTLV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 29 
                 QVQLVESGGGLVQPGGSLRLSCAASGFLLRSNTMGWY 
                 R11-H4 
               
               
                   
                 RQAPGKERELVAFIRPSGLTNYNDAVQGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCHTRPPFQRDSWGQGTLV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 30 
                 QVQLVESGGGLVQPGGSLRLSCAGPGFLLRSNTMGWY 
                 R11-H5 
               
               
                   
                 RQAPGKERELVAFIRPSGLTNYNDAVQGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCHTRPPFQRDSWGQGTLV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 31 
                 QVQLVESGGGLVQPGGSLRLSCAGPGFLLRSNTMGWY 
                 R11-H6 
               
               
                   
                 RQAPGKERELVAFIRPSGLTNYNDAVQGRFTISRDNAK 
                   
               
               
                   
                 NTVYLQMNSLRAEDTAVYYCHTRPPFQRDSWGQGTLV 
                   
               
               
                   
                 TVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 32 
                 QVQLVESGGGLVQAGGSLRLSCVASGRTFIAYAMGWF 
                 R28 sdAb 
               
               
                   
                 RQAPGKEREFVAAITNFAGGTTYYADSVKGRFTISRDNA 
                   
               
               
                   
                 KTTVYLQMNSLKPEDTALYYCAADRSAQTMRQVRPVLP 
                   
               
               
                   
                 YWGQGTQVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 33 
                 EVQLVESGGGLVQPGGSLRLSCAASGRTFIAYAMGWV 
                 R28-H0 
               
               
                   
                 RQAPGKGLEWVSAITNFAGGTTYYADSVKGRFTISRDN 
                   
               
               
                   
                 SKNTLYLQMNSLRAEDTAVYYCAADRSAQTMRQVRPV 
                   
               
               
                   
                 LPYWGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 34 
                 EVQLVESGGGLVQPGGSLRLSCAASGRTFIAYAMGWF 
                 R28-H1 
               
               
                   
                 RQAPGKGLEFVSAITNFAGGTTYYADSVKGRFTISRDNS 
                   
               
               
                   
                 KNTLYLQMNSLRAEDTAVYYCAADRSAQTMRQVRPVL 
                   
               
               
                   
                 PYWGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 35 
                 EVQLVESGGGLVQPGGSLRLSCAASGRTFIAYAMGWF 
                 R28-H2 
               
               
                   
                 RQAPGKEREFVSAITNFAGGTTYYADSVKGRFTISRDNS 
                   
               
               
                   
                 KNTLYLQMNSLRAEDTAVYYCAADRSAQTMRQVRPVL 
                   
               
               
                   
                 PYWGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 36 
                 QVQLVESGGGLVQPGGSLRLSCAASGRTFIAYAMGWF 
                 R28-H3 
               
               
                   
                 RQAPGKEREFVAAITNFAGGTTYYADSVKGRFTISRDNS 
                   
               
               
                   
                 KNTLYLQMNSLRAEDTAVYYCAADRSAQTMRQVRPVL 
                   
               
               
                   
                 PYWGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 37 
                 QVQLVESGGGLVQPGGSLRLSCAASGRTFIAYAMGWF 
                 R28-H4 
               
               
                   
                 RQAPGKEREFVAAITNFAGGTTYYADSVKGRFTISRDNA 
                   
               
               
                   
                 KTTLYLQMNSLRAEDTAVYYCAADRSAQTMRQVRPVLP 
                   
               
               
                   
                 YWGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 38 
                 QVQLVESGGGLVQPGGSLRLSCAASGRTFIAYAMGWF 
                 R28-H5 
               
               
                   
                 RQAPGKEREFVAAITNFAGGTTYYADSVKGRFTISRDNA 
                   
               
               
                   
                 KTTVYLQMNSLRAEDTAVYYCAADRSAQTMRQVRPVL 
                   
               
               
                   
                 PYWGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 39 
                 QVQLVESGGGFVQAGGSLRLSCAASGRTFDNYVMAWF 
                 M75 sdAb 
               
               
                   
                 RQAPGKEREFVASISGSGSITNYANSVKDRFTISRDSAK 
                   
               
               
                   
                 NAIYLQMNSLKPEDTALYYCAAGSRRTYYREPKFYPSW 
                   
               
               
                   
                 GQGTQVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 40 
                 EVQLVESGGGLVQPGGSLRLSCAASGRTFDNYVMAWV 
                 M75-H0 
               
               
                   
                 RQAPGKGLEWVSSISGSGSITNYANSVKDRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCAAGSRRTYYREPKFYPS 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 41 
                 EVQLVESGGGLVQPGGSLRLSCAASGRTFDNYVMAWF 
                 M75-H1 
               
               
                   
                 RQAPGKGLEFVSSISGSGSITNYANSVKDRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCAAGSRRTYYREPKFYPS 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 42 
                 EVQLVESGGGLVQPGGSLRLSCAASGRTFDNYVMAWF 
                 M75-H2 
               
               
                   
                 RQAPGKEREFVSSISGSGSITNYANSVKDRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCAAGSRRTYYREPKFYPS 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 43 
                 QVQLVESGGGLVQPGGSLRLSCAASGRTFDNYVMAWF 
                 M75-H3 
               
               
                   
                 RQAPGKEREFVASISGSGSITNYANSVKDRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCAAGSRRTYYREPKFYPS 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 44 
                 QVQLVESGGGLVQPGGSLRLSCAASGRTFDNYVMAWF 
                 M75-H4 
               
               
                   
                 RQAPGKEREFVASISGSGSITNYANSVKDRFTISRDSSK 
                   
               
               
                   
                 NALYLQMNSLRAEDTAVYYCAAGSRRTYYREPKFYPS 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 45 
                 QVQLVESGGGLVQPGGSLRLSCAASGRTFDNYVMAWF 
                 M75-H5 
               
               
                   
                 RQAPGKEREFVASISGSGSITNYANSVKDRFTISRDSAK 
                   
               
               
                   
                 NAIYLQMNSLRAEDTAVYYCAAGSRRTYYREPKFYPSW 
                   
               
               
                   
                 GQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 46 
                 QVKLEESGGGLVQAGGSLKLSCAASGSTFSSSSVGWY 
                 M79 sdAb 
               
               
                   
                 RQAPGQQRELVAAITSGGSTNTADSVKGRFTMSRDNA 
                   
               
               
                   
                 KNTVYLQMRDLKPEDTAVYYCNVAGRNWVPISRYSPG 
                   
               
               
                   
                 PYWGQGTQVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 47 
                 EVQLVESGGGLVQPGGSLRLSCAASGSTFSSSSVGWV 
                 M79-H0 
               
               
                   
                 RQAPGKGLEWVSAITSGGSTNTADSVKGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCAVAGRNWVPISRYSPGPY 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 48 
                 EVQLVESGGGLVQPGGSLRLSCAASGSTFSSSSVGWY 
                 M79-H1 
               
               
                   
                 RQAPGKGLELVSAITSGGSTNTADSVKGRFTISRDNSKN 
                   
               
               
                   
                 TLYLQMNSLRAEDTAVYYCAVAGRNWVPISRYSPGPY 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 49 
                 EVQLVESGGGLVQPGGSLRLSCAASGSTFSSSSVGWY 
                 M79-H2 
               
               
                   
                 RQAPGKGLELVSAITSGGSTNTADSVKGRFTISRDNSKN 
                   
               
               
                   
                 TLYLQMNSLRAEDTAVYYCNVAGRNWVPISRYSPGPY 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 50 
                 EVQLVESGGGLVQPGGSLRLSCAASGSTFSSSSVGWY 
                 M79-H3 
               
               
                   
                 RQAPGKQRELVSAITSGGSTNTADSVKGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCNVAGRNWVPISRYSPGPY 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 51 
                 QVQLVESGGGLVQPGGSLRLSCAASGSTFSSSSVGWY 
                 M79-H4 
               
               
                   
                 RQAPGQQRELVAAITSGGSTNTADSVKGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCNVAGRNWVPISRYSPGPY 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 52 
                 QVQLVESGGGLVQPGGSLRLSCAASGSTFSSSSVGWY 
                 M79-H5 
               
               
                   
                 RQAPGQQRELVAAITSGGSTNTADSVKGRFTISRDNAK 
                   
               
               
                   
                 NTVYLQMNSLRAEDTAVYYCNVAGRNWVPISRYSPGP 
                   
               
               
                   
                 YWGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 53 
                 QVQLVESGGGLVQAGGSLRLSCTASGSIESINRMAWYR 
                 H18 sdAb 
               
               
                   
                 QAPGQQREFVARISKGGSTNYPDSVKGRFTISRDNAKN 
                   
               
               
                   
                 TVYLQMNSLKPEDTAVYYCAAGPVWEQFWGQGTQVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 SEQ ID NO: 54 
                 EVQLVESGGGLVQPGGSLRLSCAASGSIESINRMAWVR 
                 H18-H0 
               
               
                   
                 QAPGKGLEWVSRISKGGSTNYPDSVKGRFTISRDNSKN 
                   
               
               
                   
                 TLYLQMNSLRAEDTAVYYCAAGPVWEQFWGQGTLVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 SEQ ID NO: 55 
                 EVQLVESGGGLVQPGGSLRLSCAASGSIESINRMAWYR 
                 H18-H1 
               
               
                   
                 QAPGKGLEFVSRISKGGSTNYPDSVKGRFTISRDNSKN 
                   
               
               
                   
                 TLYLQMNSLRAEDTAVYYCAAGPVWEQFWGQGTLVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 SEQ ID NO: 56 
                 EVQLVESGGGLVQPGGSLRLSCAASGSIESINRMAWYR 
                 H18-H2 
               
               
                   
                 QAPGKQREFVSRISKGGSTNYPDSVKGRFTISRDNSKN 
                   
               
               
                   
                 TLYLQMNSLRAEDTAVYYCAAGPVWEQFWGQGTLVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 SEQ ID NO: 57 
                 QVQLVESGGGLVQPGGSLRLSCAASGSIESINRMAWYR 
                 H18-H3 
               
               
                   
                 QAPGKQREFVARISKGGSTNYPDSVKGRFTISRDNSKN 
                   
               
               
                   
                 TLYLQMNSLRAEDTAVYYCAAGPVWEQFWGQGTLVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 SEQ ID NO: 58 
                 QVQLVESGGGLVQPGGSLRLSCAASGSIESINRMAWYR 
                 H18-H4 
               
               
                   
                 QAPGQQREFVARISKGGSTNYPDSVKGRFTISRDNSKN 
                   
               
               
                   
                 TLYLQMNSLRAEDTAVYYCAAGPVWEQFWGQGTLVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 SEQ ID NO: 59 
                 QVQLVESGGGLVQPGGSLRLSCAASGSIESINRMAWYR 
                 H18-H5 
               
               
                   
                 QAPGQQREFVARISKGGSTNYPDSVKGRFTISRDNAKN 
                   
               
               
                   
                 TVYLQMNSLRAEDTAVYYCAAGPVWEQFWGQGTLVTV 
                   
               
               
                   
                 SS 
                   
               
               
                   
               
               
                 SEQ ID NO: 60 
                 QVKLEESGGGLVQAGGSLRLSCAASGRTISLYAVGWFR 
                 Rh34 sdAb 
               
               
                   
                 RAPGKEREFVAAISWTDSSTYYADSVKGRFTISRDNAK 
                   
               
               
                   
                 NTVYLGMNSLNPEDTAVYYCAADVSIRGLQKYEYDYWG 
                   
               
               
                   
                 QGTQVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 61 
                 EVQLVESGGGLVQPGGSLRLSCAASGRTISLYAVGWVR 
                 Rh34-H0 
               
               
                   
                 QAPGKGLEWVSAISWTDSSTYYADSVKGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCAADVSIRGLQKYEYDYWG 
                   
               
               
                   
                 QGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 62 
                 EVQLVESGGGLVQPGGSLRLSCAASGRTISLYAVGWFR 
                 Rh34-H1 
               
               
                   
                 QAPGKGLEFVSAISWTDSSTYYADSVKGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCAADVSIRGLQKYEYDYWG 
                   
               
               
                   
                 QGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 63 
                 EVQLVESGGGLVQPGGSLRLSCAASGRTISLYAVGWFR 
                 Rh34-H2 
               
               
                   
                 QAPGKEREFVSAISWTDSSTYYADSVKGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCAADVSIRGLQKYEYDYWG 
                   
               
               
                   
                 QGTLVTVS 
                   
               
               
                   
               
               
                 SEQ ID NO: 64 
                 QVQLVESGGGLVQPGGSLRLSCAASGRTISLYAVGWF 
                 Rh34-H3 
               
               
                   
                 RQAPGKEREFVAAISWTDSSTYYADSVKGRFTISRDNS 
                   
               
               
                   
                 KNTLYLQMNSLRAEDTAVYYCAADVSIRGLQKYEYDYW 
                   
               
               
                   
                 GQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 65 
                 QVQLVESGGGLVQPGGSLRLSCAASGRTISLYAVGWF 
                 Rh34-H4 
               
               
                   
                 RRAPGKEREFVAAISWTDSSTYYADSVKGRFTISRDNS 
                   
               
               
                   
                 KNTLYLQMNSLRAEDTAVYYCAADVSIRGLQKYEYDYW 
                   
               
               
                   
                 GQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 66 
                 QVQLVESGGGLVQPGGSLRLSCAASGRTISLYAVGWF 
                 Rh34-H5 
               
               
                   
                 RRAPGKEREFVAAISWTDSSTYYADSVKGRFTISRDNA 
                   
               
               
                   
                 KNTVYLQMNSLRAEDTAVYYCAADVSIRGLQKYEYDYW 
                   
               
               
                   
                 GQGTLVTVS 
                   
               
               
                   
               
               
                 SEQ ID NO: 67 
                 QVKLEESGGGLVQAGGSLRLSCTASTRTFSSYIMGWFR 
                 Rh46 sdAb 
               
               
                   
                 QAPGKERELVAAISWSGRMTHYADSVKGRFAISRDNAK 
                   
               
               
                   
                 NTVYLQMNVLKPEDTAIYSCAADRTTAWGAPRSQYDS 
                   
               
               
                   
                 WGQGTQVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 68 
                 EVQLVESGGGLVQPGGSLRLSCAASTRTFSSYIMGWV 
                 Rh46-H0 
               
               
                   
                 RQAPGKGLEWVSAISWSGRMTHYADSVKGRFTISRDN 
                   
               
               
                   
                 SKNTLYLQMNSLRAEDTAVYYCAADRTTAWGAPRSQY 
                   
               
               
                   
                 DSWGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 69 
                 EVQLVESGGGLVQPGGSLRLSCAASTRTFSSYIMGWFR 
                 Rh46-H1 
               
               
                   
                 QAPGKGLELVSAISWSGRMTHYADSVKGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCAADRTTAWGAPRSQYDS 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 70 
                 EVQLVESGGGLVQPGGSLRLSCAASTRTFSSYIMGWFR 
                 Rh46-H2 
               
               
                   
                 QAPGKERELVSAISWSGRMTHYADSVKGRFTISRDNSK 
                   
               
               
                   
                 NTLYLQMNSLRAEDTAVYYCAADRTTAWGAPRSQYDS 
                   
               
               
                   
                 WGQGTLVTVS 
                   
               
               
                   
               
               
                 SEQ ID NO: 71 
                 QVQLVESGGGLVQPGGSLRLSCAASTRTFSSYIMGWF 
                 Rh46-H3 
               
               
                   
                 RQAPGKERELVAAISWSGRMTHYADSVKGRFTISRDNS 
                   
               
               
                   
                 KNTLYLQMNSLRAEDTAVYYCAADRTTAWGAPRSQYD 
                   
               
               
                   
                 SWGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 72 
                 QVQLVESGGGLVQPGGSLRLSCAASTRTFSSYIMGWF 
                 Rh46-H4 
               
               
                   
                 RQAPGKERELVAAISWSGRMTHYADSVKGRFTISRDNS 
                   
               
               
                   
                 KNTLYLQMNSLRAEDTAIYSCAADRTTAWGAPRSQYDS 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 73 
                 QVQLVESGGGLVQPGGSLRLSCAASTRTFSSYIMGWF 
                 Rh46-H5 
               
               
                   
                 RQAPGKERELVAAISWSGRMTHYADSVKGRFTISRDNA 
                   
               
               
                   
                 KNTVYLQMNSLRAEDTAIYSCAADRTTAWGAPRSQYDS 
                   
               
               
                   
                 WGQGTLVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 74 
                 QVQLVESGGGLVQAGGSLRLSCAASGLTFSRYVMGWF 
                 B39 
               
               
                   
                 RQAPGKEREFVAAITWGGTPNYADSVKGRFTISRDNSK 
                   
               
               
                   
                 NTQYLQMNSLKPEDTAVYYCAAGLGWDSRYSQSYNY 
                   
               
               
                   
                 WGQGTQVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 75 
                 QVQLVESGGGLAQAGGSLRLSCAASGRTFSMDPMAW 
                 A20 
               
               
                   
                 FRQPPGKEREFVAAGSSTGRTTYYADSVKGRFTISRDN 
                   
               
               
                   
                 AKNTVYLQMNSLKPEDTAVYYCAAAPYGANWYRDEYA 
                   
               
               
                   
                 YWGQGTQVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 76 
                 QVKLEESGGGLVQAGGSLRLSCAASERTFSRYPVAWF 
                 A26 
               
               
                   
                 RQAPGAEREFVAVISSTGTSTYYADSVKGRFTISRDNAK 
                   
               
               
                   
                 VTVYLQMNNLKREDTAVYFCAVNSQRTRLQDPNEYDY 
                   
               
               
                   
                 WGQGTQVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 77 
                 KGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTM 
                 CIBP2 
               
               
                   
                 RLPDERGPLEHLYSLHIPNCDKHGLYNLKQCKMSLNGQ 
                   
               
               
                   
                 RGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEAR 
                   
               
               
                   
                 GVHTQRMQ 
                   
               
               
                   
               
               
                 SEQ ID NO: 78 
                 EVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWF 
                 FC5 
               
               
                   
                 RQAPGKEREFVSRITWGGDNTFYSNSVKGRFTISRDNA 
                   
               
               
                   
                 KNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYW 
                   
               
               
                   
                 GKGTQVTVSS 
                   
               
               
                   
               
               
                 SEQ ID NO: 79 
                 KTFKTRKASAQASLASKDKTPKSKSKKRNSTQLKSRVK 
                 ABP 
               
               
                   
                 NI 
                   
               
               
                   
               
               
                 SEQ ID NO: 80 
                 SETQANSTTDALNVLLIIVDDLRPSLGCYGDKLVRSPNID 
                 IDS 
               
               
                   
                 QLASHSLLFQNAFAQQAVCAPSRVSFLTGRRPDTTRLY 
                   
               
               
                   
                 DFNSYWRVHAGNFSTIPQYFKENGYVTMSVGKVFHPGI 
                   
               
               
                   
                 SSNHTDDSPYSWSFPPYHPSSEKYENTKTCRGPDGEL 
                   
               
               
                   
                 HANLLCPVDVLDVPEGTLPDKQSTEQAIQLLEKMKTSAS 
                   
               
               
                   
                 PFFLAVGYHKPHIPFRYPKEFQKLYPLENITLAPDPEVPD 
                   
               
               
                   
                 GLPPVAYNPWMDIRQREDVQALNISVPYGPIPVDFQRKI 
                   
               
               
                   
                 RQSYFASVSYLDTQVGRLLSALDDLQLANSTIIAFTSDH 
                   
               
               
                   
                 GWALGEHGEWAKYSNFDVATHVPLIFYVPGRTASLPEA 
                   
               
               
                   
                 GEKLFPYLDPFDSASQLMEPGRQSMDLVELVSLFPTLA 
                   
               
               
                   
                 GLAGLQVPPRCPVPSFHVELCREGKNLLKHFRFRDLEE 
                   
               
               
                   
                 DPYLPGNPRELIAYSQYPRPSDIPQWNSDKPSLKDIKIM 
                   
               
               
                   
                 GYSIRTIDYRYTVWVGFNPDEFLANFSDIHAGELYFVDS 
                   
               
               
                   
                 DPLQDHNMYNDSQGGDLFQLLMP 
               
               
                   
               
            
           
         
       
     
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