Modified single-domain antibodies

EP4713364A1Pending Publication Date: 2026-03-25ODYSSEY THERAPEUTICS INC
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Patent Information

Authority / Receiving Office
EP · EP
Patent Type
Applications
Current Assignee / Owner
Filing Date
2024-05-16
Publication Date
2026-03-25

AI Technical Summary

Technical Problem

Drug-induced immunogenicity, particularly the formation of anti-drug antibodies (ADAs), leads to reduced efficacy and adverse events in biological drugs, with pre-existing ADAs against single-domain antibodies like VHHs binding to their carboxy-terminus, causing inactivation and enhanced clearance.

Method used

Modification of single-domain antibodies at the carboxy-terminus with specific amino acid sequences, such as V, VX, VXiX2, or VXiX2G, to reduce binding of pre-existing ADAs, including substitutions at positions 11, 13, 87, 88, 89, and 108, resulting in reduced ADA binding compared to unmodified antibodies.

Benefits of technology

The modified single-domain antibodies exhibit at least 80% reduced ADA binding to their C-terminus, as measured by ELISA, compared to unmodified counterparts, thereby enhancing the stability and efficacy of biological drugs.

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Abstract

The present disclosure provides single-domain antibodies comprising one or more modifications, e.g., at the carboxy-terminus (C-terminus), which can reduce binding by anti-drug antibodies to the carboxy-terminus of the single-domain antibodies as compared to an unmodified single-domain antibody. The application also provides fusion proteins and conjugates comprising the single-domain antibodies, polynucleotides and recombinant vectors encoding the single-domain antibodies, compositions and kits comprising same, as well as host cells and methods for preparing the single-domain antibodies.
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Description

MODIFIED SINGLE-DOMAIN ANTIBODIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 467,208, filed May 17,2023, the disclosure of which is herein incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 30,2024, is named 260525_000044_SL.xml and is 257,846 bytes in size.FIELD OF THE INVENTION

[0003] The present disclosure relates to single-domain antibodies comprising one or more modifications, e.g., at the carboxy-terminus (C-terminus), which can reduce binding of anti-drug antibodies to the carboxy-terminus of the single-domain antibody as compared to an unmodified singledomain antibody, methods for their preparation, and uses thereof.BACKGROUND OF THE INVENTION

[0004] Drug-induced immunogenicity is a major challenge in the development of biological drugs, which can be associated with reduced efficacy and adverse events in a clinical setting. It is important to evaluate the immunogenic potential of candidate biological drugs during clinical development and, in particular, the degree to which such drugs can trigger immune responses that lead to the formation of anti-drug antibodies, or ADAs. From a mechanistic standpoint, ADAs can inactivate the drug and promote enhanced clearance of ADA-drug complexes and / or loss of targeting, thereby leading to suboptimal exposure and loss of efficacy. Pre-existing ADAs against antibody fragments including, e.g., fragment antigen-binding (Fab) fragments, human heavy chain variable domains (VHs), and singledomain antibodies (e.g., VHHs), were first reported in drug-naive individuals more than 10 years ago. Up to 50% of healthy humans were reported to have pre-existing anti-VHH ADAs, yet the origin of these ADAs remains unknown. It is established, however, that the VHH carboxy-terminus (C-terminus) outside of the IgG context can be a dominant epitope of these ADAs. Accordingly, there is a need in the art todevelop non-immunogenic VHHs, or functional fragments thereof, engineered to abolish or impede binding of pre-existing ADAs to the VHH such as by way of its C-terminus.SUMMARY OF THE INVENTION

[0005] As mentioned in the background section above, there is an unmet need in the art to develop non-immunogenic VHHs, or functional fragments thereof, which are engineered to abolish or impede binding of pre-existing ADA to the VHH, e.g., via its C-terminus. This application provides compositions and methods to address this and other related needs.

[0006] In one aspect, the present disclosure provides a single-domain antibody which is modified to comprise, at the carboxy-terminus starting from position 111 according to Chothia, an amino acid sequence selected from(1) V,(2) VX1;(3) VXiX2,(4) VXiX2G, or(5) VXiX2P; wherein Xi is selected from amino acids Ala (A), Asp (D), Glu (E), Gly (G), lie (I), Lys (K), Leu (L), Asn (N), Pro (P), Arg (R), Ser (S), Thr (T), and Vai (V), andX2is selected from amino acids Ala (A), Asp (D), Glu (E), Gly (G), He (I), Lys (K), Leu (L), Asn (N), Pro (P), Gin (Q), Arg (R), Ser (S), Thr (T), and Vai (V).

[0007] In some embodiments, Xi is selected from Ala (A), Gly (G), Pro ( P), Asp (D), and Leu (L); and / or X2is selected from amino acids Ala (A), Gly (G), Pro (P), Asp (D), Gin (Q), and Leu (L).

[0008] In some embodiments, Xi is selected from Ala (A), Gly (G), and Pro (P); and / or X2is selected from amino acids Ala (A), Gly (G), Gin (Q), and Pro (P).

[0009] In some embodiments, the amino acid sequence is not one selected from VSS, VE, VEG, VEP, VEPG (SEQ ID NO: 35), VK, VKS, VKG, VKP, VKPG (SEQ ID NO: 294), VQS, VS, VSE, VSEG (SEQ ID NO: 135), VSK, VSKG (SEQ ID NO: 141), VRP, VRPG (SEQ ID NO: 126), VDP, VDPG (SEQ ID NO: 25), VSSP (SEQ ID NO: 295), and VSSG (SEQ ID NO: 286).

[0010] In some embodiments, the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, an amino acid sequence select-ed from(1) V;(2) VA, VD, VE, VG, VI, VK, VL, VN, VP, VR, VS, VT, or VV;(3) VAA, VAD, VAE, VAG, VAI, VAK, VAL, VAN, VAP, VAQ, VAR, VAS, VAT, VAV, VDA, VDD, VDE, VDG, VDI, VDK, VDL, VDN, VDP, VDQ, VDR, VDS, VDT, VDV, VEA, VED, VEE, VEG, VEI, VEK, VEL, VEN, VEP, VEQ, VER, VES, VET, VEV, VGA, VGD, VGE, VGG, VGI, VGK, VGL, VGN, VGP, VGQ, VGR, VGS, VGT, VGV, VIA, VID, VIE, VIG, VII, VIK, VIL, VIN, VIP, VIQ, VIR, VIS, VIT, VIV, VLA, VLD, VLE, VLG, VLI, VLK, VLL, VLN, VLP, VLQ, VLR, VLS, VLT, VLV, VNA, VND, VNE, VNG, VNI, VNK, VNL, VNN, VNP, VNQ, VNR, VNS, VNT, VNV, VPA, VPD, VPE, VPG, VPI, VPK, VPL, VPN, VPP, VPQ, VPR, VPS, VPT, VPV, VRA, VRD, VRE, VRG, VRI, VRK, VRL, VRN, VRP, VRQ, VRR, VRS, VRT, VRV, VSA, VSD, VSE, VSG, VSI, VSK, VSL, VSN, VSP, VSQ, VSR, VST, VSV, VTA, VTD, VTE, VTG, VTI, VTK, VTL, VTN, VTP, VTQ, VTR, VTS, VTT, VTV, VVA, VVD, WE, VVG, VVI, WK, WL, WN, WP, VVQ, WR, WS, WT, or VW;(4) VADG (SEQ ID NO: 1), VAEG (SEQ ID NO: 3), VAGG (SEQ ID NO: 5), VAKG (SEQ ID NO: 7), VANG (SEQ ID NO: 9), VAPG (SEQ ID NO: 11), VAQG (SEQ ID NO: 13), VARG (SEQ ID NO: 15), VASG (SEQ ID NO: 17), VATG (SEQ ID NO: 19), VDAG (SEQ ID NO: 21), VDGG (SEQ ID NO: 23), VDPG (SEQ ID NO: 25), VDSG (SEQ ID NO: 27), VDTG (SEQ ID NO: 29), VEAG (SEQ ID NO: 31), VEGG (SEQ ID NO: 33), VEPG (SEQ ID NO: 35), VESG (SEQ ID NO: 37), VETG (SEQ ID NO: 39), VGAG (SEQ ID NO: 41), VGDG (SEQ ID NO: 43), VGEG (SEQ ID NO: 45), VGGG (SEQ ID NO: 47), VGIG (SEQ ID NO: 49), VGKG (SEQ ID NO: 51), VGLG (SEQ ID NO: 53), VGNG (SEQ ID NO: 55), VGPG (SEQ ID NO: 57), VGQG (SEQ ID NO: 59), VGRG (SEQ ID NO: 61), VGSG (SEQ ID NO: 63), VGTG (SEQ ID NO: 65), VGVG (SEQ ID NO: 67), VIGG (SEQ ID NO: 69), VIPG (SEQ ID NO: 71), VISG (SEQ ID NO: 73), VITG (SEQ ID NO: 75), VLGG (SEQ ID NO: 77), VLPG (SEQ ID NO: 79), VLSG (SEQ ID NO: 283), VLTG (SEQ ID NO: 82), VNAG (SEQ ID NO: 84), VNGG (SEQ ID NO: 86), VNPG (SEQ ID NO: 88), VNSG (SEQ ID NO: 90), VNTG (SEQ ID NO: 92), VPAG (SEQ ID NO: 94), VPDG (SEQ ID NO: 96), VPEG (SEQ ID NO: 98), VPGG (SEQ ID NO: 100), VPIG (SEQ ID NO: 102), VPKG (SEQ ID NO: 104), VPLG (SEQ ID NO: 106), VPNG (SEQ ID NO: 108), VPPG (SEQ ID NO: 110), VPQG (SEQ ID NO: 112), VPRG (SEQ ID NO: 114), VPSG (SEQ ID NO: 116), VPTG (SEQ ID NO: 118), VPVG (SEQ ID NO: 120), VRAG (SEQ ID NO: 122), VRGG (SEQ ID NO: 124), VRPG (SEQ ID NO: 126), VRSG (SEQ ID NO: 128), VRTG (SEQ ID NO: 130), VSAG (SEQ ID NO: 284), VSDG (SEQ ID NO: 133), VSEG (SEQ ID NO: 135), VSGG (SEQ ID NO: 137), VSIG (SEQ ID NO: 139), VSKG (SEQ ID NO: 141), VSLG (SEQ ID NO: 143), VSNG (SEQ ID NO: 145), VSPG (SEQ ID NO: 147), VSQG (SEQ ID NO: 149), VSRG (SEQ ID NO: 151), VSTG (SEQ ID NO: 153), VSVG (SEQ ID NO: 285), VTAG (SEQ ID NO: 156), VTDG (SEQ ID NO: 158), VTEG (SEQ ID NO: 160), VTGG (SEQ ID NO: 162), VTIG (SEQ ID NO: 164), VTKG (SEQ ID NO: 166), VTLG (SEQ ID NO: 168), VTNG (SEQ ID NO: 170), VTPG (SEQ ID NO: 172), VTQG (SEQ ID NO: 174), VTRG (SEQ ID NO: 176), VTSG (SEQ ID NO: 178), VTTG (SEQ ID NO: 180), VTVG (SEQ ID NO: 182), WGG (SEQ ID NO: 184), WPG (SEQ ID NO: 186), WSG (SEQ ID NO: 188), or WTG (SEQ ID NO: 190); or(5) VADP (SEQ ID NO: 2), VAEP (SEQ ID NO: 4), VAGP (SEQ ID NO: 6), VAKP (SEQ ID NO: 8), VANP(SEQ ID NO: 10), VAPP (SEQ ID NO: 12), VAQP (SEQ ID NO: 14), VARP (SEQ ID NO: 16), VASP (SEQ ID NO:18), VATP (SEQ ID NO: 20), VDAP (SEQ ID NO: 22), VDGP (SEQ ID NO: 24), VDPP (SEQ ID NO: 26), VDSP (SEQ ID NO: 28), VDTP (SEQ ID NO: 30), VEAP (SEQ ID NO: 32), VEGP (SEQ ID NO: 34), VEPP (SEQ ID NO: 36), VESP (SEQ ID NO: 38), VETP (SEQ ID NO: 40), VGAP (SEQ ID NO: 42), VGDP (SEQ ID NO: 44), VGEP (SEQ ID NO: 46), VGGP (SEQ ID NO: 48), VGIP (SEQ ID NO: 50), VGKP (SEQ ID NO: 52), VGLP (SEQ ID NO: 54), VGNP (SEQ ID NO: 56), VGPP (SEQ ID NO: 58), VGQP (SEQ ID NO: 60), VGRP (SEQ ID NO: 62), VGSP (SEQ ID NO: 64), VGTP (SEQ ID NO: 66), VGVP (SEQ ID NO: 68), VIGP (SEQ ID NO: 70), VIPP (SEQ ID NO: 72), VISP (SEQ ID NO: 74), VITP (SEQ ID NO: 76), VLGP (SEQ ID NO: 78), VLPP (SEQ ID NO: 80), VLSP (SEQ ID NO: 81), VLTP (SEQ ID NO: 83), VNAP (SEQ ID NO: 85), VNGP (SEQ ID NO: 87), VNPP (SEQ ID NO: 89), VNSP (SEQ ID NO: 91), VNTP (SEQ ID NO: 93), VPAP (SEQ ID NO: 95), VPDP (SEQ ID NO: 97), VPEP (SEQ ID NO: 99), VPGP (SEQ ID NO: 101), VPIP (SEQ ID NO: 103), VPKP (SEQ ID NO: 105), VPLP (SEQ ID NO: 107), VPNP (SEQ ID NO: 109), VPPP (SEQ ID NO: 111), VPQP (SEQ ID NO: 113), VPRP (SEQ ID NO: 115), VPSP (SEQ ID NO: 117), VPTP (SEQ ID NO: 119), VPVP (SEQ ID NO: 121), VRAP (SEQ ID NO: 123), VRGP (SEQ ID NO: 125), VRPP (SEQ ID NO: 127), VRSP (SEQ ID NO: 129), VRTP (SEQ ID NO: 131), VSAP (SEQ ID NO: 132), VSDP (SEQ ID NO: 134), VSEP (SEQ ID NO: 136), VSGP (SEQ ID NO: 138), VSIP (SEQ ID NO: 140), VSKP (SEQ ID NO: 142), VSLP (SEQ ID NO: 144), VSNP (SEQ ID NO: 146), VSPP (SEQ ID NO: 148), VSQP (SEQ ID NO: 150), VSRP (SEQ ID NO: 152), VSTP (SEQ ID NO: 154), VSVP (SEQ ID NO: 155), VTAP (SEQ ID NO: 157), VTDP (SEQ ID NO: 159), VTEP (SEQ ID NO: 161), VTGP (SEQ ID NO: 163), VTIP (SEQ ID NO: 165), VTKP (SEQ ID NO: 167), VTLP (SEQ ID NO: 169), VTNP (SEQ ID NO: 171), VTPP (SEQ ID NO: 173), VTQP (SEQ ID NO: 175), VTRP (SEQ ID NO: 177), VTSP (SEQ ID NO: 179), VTTP (SEQ ID NO: 181), VTVP (SEQ ID NO: 183), VVGP (SEQ ID NO: 185), WPP (SEQ ID NO: 187), VVSP (SEQ ID NO: 189), or VVTP (SEQ ID NO: 191).

[0011] In some embodiments, the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, an amino acid sequence select-ed from VR, VG, VP, VA, VPG, VDG, VPQ, VPA, VAGG (SEQ ID NO: 5), VAPG (SEQ ID NO: 11), VAQP (SEQ ID NO: 14), VPAG (SEQ ID NO: 94), VPGG (SEQ ID NO: 100), VPPG (SEQ ID NO: 110), VPQG (SEQ ID NO: 112), VPAP (SEQ ID NO: 95), VPGP (SEQ ID NO: 101), VPLP (SEQ ID NO: 107), VGP, VGAG (SEQ ID NO: 41), VGGG (SEQ ID NO: 47), VGQG (SEQ ID NO: 59), VDGG (SEQ ID NO: 23), or VDAP (SEQ ID NO: 22).

[0012] In some embodiments, the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, an amino acid sequence selected from VAGG (SEQ ID NO: 5), VAPG (SEQ ID NO: 11), VAQP (SEQ ID NO: 14), VPA, VPAG (SEQ ID NO: 94), VPGG (SEQ ID NO: 100), VPPG(SEQ ID NO: 110), VPQ, VPQG (SEQ ID NO: 112), VGAG (SEQ ID NO: 41), VGGG (SEQ ID NO: 47), VGQG (SEQ ID NO: 59), VDGG (SEQ ID NO: 23), or VDAP (SEQ ID NO: 22).

[0013] In some embodiments, the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, the amino acid sequence VAGG (SEQ ID NO: 5) or VPAG (SEQ ID NO: 94).

[0014] In some embodiments, the single-domain antibody further comprises one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108.

[0015] In some embodiments, the Leu (L) at position 11 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Gin (Q) at position 13 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y); the Gly (G) at position 88 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Vai (V) or lie (I) at position 89 is mutated to Ala (A), Asp (D), Glu (E), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), or Tyr (Y); and / or the Leu (L) or Gin (Q) at position 108 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

[0016] In some embodiments, the single-domain antibody comprises at least an amino acid substitution at position 87.

[0017] In some embodiments, the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y).

[0018] In some embodiments, the Thr (T) at position 87 is mutated to Ala (A), Ser (S), or Vai (V).

[0019] In another aspect, the present disclosure provides a single-domain antibody which is modified to comprise one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108, wherein the Leu (L) at position 11 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Gin (Q) at position 13 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N),Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu ( E), He (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y); the Gly (G) at position 88 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Vai (V) or lie (I) at position 89 is mutated to Ala (A), Asp (D), Glu (E), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), or Tyr (Y); and / or the Leu (L) or Gin (Q) at position 108 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Asn (N), Arg ( R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

[0020] In some embodiments, the single-domain antibody comprises at least an amino acid substitution at position 87.

[0021] In some embodiments, the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys ( K), Leu (L), Asn ( N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y).

[0022] In some embodiments, the Thr (T) at position 87 is mutated to Ala (A), Ser (S), or Vai (V).

[0023] In some embodiments, the single-domain antibody has reduced binding by anti-drug antibodies (ADAs) to its C-terminus as compared to an unmodified single-domain antibody.

[0024] In some embodiments, the unmodified single-domain antibody comprises VSS at the C- terminus.

[0025] In some embodiments, the single-domain antibody has at least about 80% reduced ADA binding to its C-terminus as compared to an unmodified single-domain antibody.

[0026] In some embodiments, the single-domain antibody has at least about 85% reduced ADA binding to its C-terminus as compared to an unmodified single-domain anti-body.

[0027] In some embodiments, the single-domain antibody has at least about 90% ADA binding to its C- terminus as compared to an unmodified single-domain antibody.

[0028] In some embodiments, the ADA binding is measured using an enzyme-linked immunosorbent assay (ELISA).

[0029] In some embodiments, the single-domain antibody is a VHH, or a VH domain.

[0030] In some embodiments, the single-domain antibody is a camelid VHH.

[0031] In some embodiments, the single-domain antibody is a humanized VHH.

[0032] In some embodiments, the single-domain antibody is a camelized VH.

[0033] In another aspect, the present disclosure provides a fusion protein comprising one or more of a single-domain antibody described herein, wherein at least one single-domain antibody is at the carboxyterminus of the fusion protein.

[0034] In another aspect, the present disclosure provides a conjugate comprising a single-domain antibody described herein or a fusion protein described herein, wherein the single-domain antibody or fusion protein is conjugated to a second moiety.

[0035] In another aspect, the present disclosure provides a polynucleotide molecule encoding a singledomain antibody described herein or a fusion protein described herein.

[0036] In another aspect, the present disclosure provides a recombinant vector comprising a polynucleotide molecule described herein.

[0037] In another aspect, the present disclosure provides a host cell comprising a polynucleotide molecule described herein, or a recombinant vector described herein.

[0038] In another aspect, the present disclosure provides a kit comprising a single-domain antibody described herein, a fusion protein described herein, a conjugate described herein, a polynucleotide molecule described herein, or a recombinant vector described herein, and optionally, instructions and / or packaging for the same.

[0039] In another aspect, the present disclosure provides a method for producing a single-domain antibody described herein or a fusion protein described herein comprising expressing a polynucleotide sequence described herein or a recombinant vector described herein in a host cell.

[0040] In another aspect, the present disclosure provides a composition comprising a single-domain antibody described herein, a fusion protein described herein, a conjugate of described herein, a polynucleotide molecule described herein, or a recombinant vector described herein, and at least one carrier, diluent or excipient.

[0041] In another aspect, the present disclosure provides a method of modifying a single-domain antibody, the method comprising mutating the amino acid sequence, at the carboxy-terminus of the single-domain antibody starting from position 111 according to Chothia, to an amino acid sequence selected from(1) V,(2) VXi,(3) VXiX2,(4) VXiX2G, or(5) VXiX2P; wherein Xi is selected from amino acids Ala (A), Asp (D), Glu (E), Gly (G), lie (I), Lys (K), Leu (L),Asn (N), Pro (P), Arg (R), Ser (S), Thr (T), and Vai (V), andXz is selected from amino acids Ala (A), Asp (D), Glu (E), Gly (G), He (I), Lys (K), Leu (L), Asn (N),Pro (P), Gin (Q), Arg (R), Ser (S), Thr (T), and Vai (V).

[0042] In some embodiments, Xi is selected from Ala (A), Gly (G), Pro ( P), Asp ( D), and Leu (L); and / or X2is selected from amino acids Ala (A), Gly (G), Pro (P), Asp (D), Gin (Q), and Leu (L).

[0043] In some embodiments, Xi is selected from Ala (A), Gly (G), and Pro (P); and / or X2is selected from amino acids Ala (A), Gly (G), Gin (Q), and Pro (P).

[0044] In some embodiments, the amino acid sequence is not one selected from VSS, VE, VEG, VEP, VEPG (SEQ ID NO: 35), VK, VKS, VKG, VKP, VKPG (SEQ ID NO: 294), VQS, VS, VSE, VSEG (SEQ ID NO: 135), VSK, VSKG (SEQ ID NO: 141), VRP, VRPG (SEQ ID NO: 126), VDP, VDPG (SEQ ID NO: 25), VSSP (SEQ ID NO: 295), and VSSG (SEQ ID NO: 286).

[0045] In some embodiments, the amino acid sequence is selected from(1) V;(2) VA, VD, VE, VG, VI, VK, VL, VN, VP, VR, VS, VT, or VV;(3) VAA, VAD, VAE, VAG, VAI, VAK, VAL, VAN, VAP, VAQ, VAR, VAS, VAT, VAV, VDA, VDD, VDE, VDG, VDI, VDK, VDL, VDN, VDP, VDQ, VDR, VDS, VDT, VDV, VEA, VED, VEE, VEG, VEI, VEK, VEL, VEN, VEP, VEQ, VER, VES, VET, VEV, VGA, VGD, VGE, VGG, VGI, VGK, VGL, VGN, VGP, VGQ, VGR, VGS, VGT, VGV, VIA, VID, VIE, VIG, VII, VIK, VIL, VIN, VIP, VIQ, VIR, VIS, VIT, VIV, VLA, VLD, VLE, VLG, VLI, VLK, VLL, VLN, VLP, VLQ, VLR, VLS, VLT, VLV, VNA, VND, VNE, VNG, VNI, VNK, VNL, VNN, VNP, VNQ, VNR, VNS, VNT, VNV, VPA, VPD, VPE, VPG, VPI, VPK, VPL, VPN, VPP, VPQ, VPR, VPS, VPT, VPV, VRA, VRD, VRE, VRG, VRI, VRK, VRL, VRN, VRP, VRQ, VRR, VRS, VRT, VRV, VSA, VSD, VSE, VSG, VSI, VSK, VSL, VSN, VSP, VSQ, VSR, VST, VSV, VTA, VTD, VTE, VTG, VTI, VTK, VTL, VTN, VTP, VTQ, VTR, VTS, VTT, VTV, VVA, VVD, WE, VVG, VVI, WK, WL, WN, WP, WQ, WR, WS, WT, or VW;(4) VADG (SEQ ID NO: 1), VAEG (SEQ ID NO: 3), VAGG (SEQ ID NO: 5), VAKG (SEQ ID NO: 7), VANG (SEQ ID NO: 9), VAPG (SEQ ID NO: 11), VAQG (SEQ ID NO: 13), VARG (SEQ ID NO: 15), VASG (SEQ ID NO: 17), VATG (SEQ ID NO: 19), VDAG (SEQ ID NO: 21), VDGG (SEQ ID NO: 23), VDPG (SEQ ID NO: 25), VDSG (SEQ ID NO: 27), VDTG (SEQ ID NO: 29), VEAG (SEQ ID NO: 31), VEGG (SEQ ID NO: 33), VEPG (SEQ ID NO: 35), VESG (SEQ ID NO: 37), VETG (SEQ ID NO: 39), VGAG (SEQ ID NO: 41), VGDG (SEQ ID NO: 43), VGEG (SEQ ID NO: 45), VGGG (SEQ ID NO: 47), VGIG (SEQ ID NO: 49), VGKG (SEQ ID NO: 51), VGLG (SEQ ID NO: 53), VGNG (SEQ ID NO: 55), VGPG (SEQ ID NO: 57), VGQG (SEQ ID NO: 59), VGRG (SEQ ID NO: 61), VGSG (SEQ ID NO: 63), VGTG (SEQ ID NO: 65), VGVG (SEQ ID NO: 67), VIGG (SEQ ID NO: 69), VIPG (SEQ ID NO: 71), VISG (SEQ ID NO: 73), VITG (SEQ ID NO: 75), VLGG (SEQ ID NO: 77), VLPG (SEQ ID NO: 79), VLSG (SEQ ID NO: 283), VLTG (SEQ ID NO: 82), VNAG (SEQ ID NO: 84), VNGG (SEQ ID NO: 86), VNPG (SEQ IDNO: 88), VNSG (SEQ ID NO: 90), VNTG (SEQ ID NO: 92), VPAG (SEQ ID NO: 94), VPDG (SEQ ID NO: 96), VPEG (SEQ ID NO: 98), VPGG (SEQ ID NO: 100), VPIG (SEQ ID NO: 102), VPKG (SEQ ID NO: 104), VPLG (SEQ ID NO: 106), VPNG (SEQ ID NO: 108), VPPG (SEQ ID NO: 110), VPQG (SEQ ID NO: 112), VPRG (SEQ ID NO: 114), VPSG (SEQ ID NO: 116), VPTG (SEQ ID NO: 118), VPVG (SEQ ID NO: 120), VRAG (SEQ ID NO: 122), VRGG (SEQ ID NO: 124), VRPG (SEQ ID NO: 126), VRSG (SEQ ID NO: 128), VRTG (SEQ ID NO: 130), VSAG (SEQ ID NO: 284), VSDG (SEQ ID NO: 133), VSEG (SEQ ID NO: 135), VSGG (SEQ ID NO: 137), VSIG (SEQ ID NO: 139), VSKG (SEQ ID NO: 141), VSLG (SEQ ID NO: 143), VSNG (SEQ ID NO: 145), VSPG (SEQ ID NO: 147), VSQG (SEQ ID NO: 149), VSRG (SEQ ID NO: 151), VSTG (SEQ ID NO: 153), VSVG (SEQ ID NO: 285), VTAG (SEQ ID NO: 156), VTDG (SEQ ID NO: 158), VTEG (SEQ ID NO: 160), VTGG (SEQ ID NO: 162), VTIG (SEQ ID NO: 164), VTKG (SEQ ID NO: 166), VTLG (SEQ ID NO: 168), VTNG (SEQ ID NO: 170), VTPG (SEQ ID NO: 172), VTQG (SEQ ID NO: 174), VTRG (SEQ ID NO: 176), VTSG (SEQ ID NO: 178), VTTG (SEQ ID NO: 180), VTVG (SEQ ID NO: 182), VVGG (SEQ ID NO: 184), WPG (SEQ ID NO: 186), WSG (SEQ ID NO: 188), or VVTG (SEQ ID NO: 190); or(5) VADP (SEQ ID NO: 2), VAEP (SEQ ID NO: 4), VAGP (SEQ ID NO: 6), VAKP (SEQ ID NO: 8), VANP (SEQ ID NO: 10), VAPP (SEQ ID NO: 12), VAQP (SEQ ID NO: 14), VARP (SEQ ID NO: 16), VASP (SEQ ID NO: 18), VATP (SEQ ID NO: 20), VDAP (SEQ ID NO: 22), VDGP (SEQ ID NO: 24), VDPP (SEQ ID NO: 26), VDSP (SEQ ID NO: 28), VDTP (SEQ ID NO: 30), VEAP (SEQ ID NO: 32), VEGP (SEQ ID NO: 34), VEPP (SEQ ID NO: 36), VESP (SEQ ID NO: 38), VETP (SEQ ID NO: 40), VGAP (SEQ ID NO: 42), VGDP (SEQ ID NO: 44), VGEP (SEQ ID NO: 46), VGGP (SEQ ID NO: 48), VGIP (SEQ ID NO: 50), VGKP (SEQ ID NO: 52), VGLP (SEQ ID NO: 54), VGNP (SEQ ID NO: 56), VGPP (SEQ ID NO: 58), VGQP (SEQ ID NO: 60), VGRP (SEQ ID NO: 62), VGSP (SEQ ID NO: 64), VGTP (SEQ ID NO: 66), VGVP (SEQ ID NO: 68), VIGP (SEQ ID NO: 70), VIPP (SEQ ID NO: 72), VISP (SEQ ID NO: 74), VITP (SEQ ID NO: 76), VLGP (SEQ ID NO: 78), VLPP (SEQ ID NO: 80), VLSP (SEQ ID NO: 81), VLTP (SEQ ID NO: 83), VNAP (SEQ ID NO: 85), VNGP (SEQ ID NO: 87), VNPP (SEQ ID NO: 89), VNSP (SEQ ID NO: 91), VNTP (SEQ ID NO: 93), VPAP (SEQ ID NO: 95), VPDP (SEQ ID NO: 97), VPEP (SEQ ID NO: 99), VPGP (SEQ ID NO: 101), VPIP (SEQ ID NO: 103), VPKP (SEQ ID NO: 105), VPLP (SEQ ID NO: 107), VPNP (SEQ ID NO: 109), VPPP (SEQ ID NO: 111), VPQP (SEQ ID NO: 113), VPRP (SEQ ID NO: 115), VPSP (SEQ ID NO: 117), VPTP (SEQ ID NO: 119), VPVP (SEQ ID NO: 121), VRAP (SEQ ID NO: 123), VRGP (SEQ ID NO: 125), VRPP (SEQ ID NO: 127), VRSP (SEQ ID NO: 129), VRTP (SEQ ID NO: 131), VSAP (SEQ ID NO: 132), VSDP (SEQ ID NO: 134), VSEP (SEQ ID NO: 136), VSGP (SEQ ID NO: 138), VSIP (SEQ ID NO: 140), VSKP (SEQ ID NO: 142), VSLP (SEQ ID NO: 144), VSNP (SEQ ID NO: 146), VSPP (SEQ ID NO: 148), VSQP (SEQ ID NO: 150), VSRP (SEQ ID NO: 152), VSTP (SEQ ID NO: 154), VSVP (SEQ ID NO: 155), VTAP (SEQ ID NO: 157), VTDP (SEQ ID NO: 159), TEP (SEQ ID NO: 161), VTGP (SEQ ID NO: 163), VTIP (SEQ ID NO: 165),VTKP (SEQ ID NO: 167), VTLP (SEQ ID NO: 169), VTNP (SEQ ID NO: 171), VTPP (SEQ ID NO: 173), VTQP (SEQ ID NO: 175), VTRP (SEQ ID NO: 177), VTSP (SEQ ID NO: 179), VTTP (SEQ ID NO: 181), VTVP (SEQ ID NO: 183), VVGP (SEQ ID NO: 185), WPP (SEQ ID NO: 187), VVSP (SEQ ID NO: 189), or VVTP (SEQ ID NO: 191).

[0046] In some embodiments, the amino acid sequence is selected from VR, VG, VP, VA, VPG, VDG, VPQ, VPA, VAGG (SEQ ID NO: 5), VAPG (SEQ ID NO: 11), VAQP (SEQ ID NO: 14), VPAG (SEQ ID NO: 94), VPGG (SEQ ID NO: 100), VPPG (SEQ ID NO: 110), VPQG (SEQ ID NO: 112), VPAP (SEQ ID NO: 95), VPGP (SEQ ID NO: 101), VPLP (SEQ ID NO: 107), VGP, VGAG (SEQ ID NO: 41), VGGG (SEQ ID NO: 47), VGQG (SEQ ID NO: 59), VDGG (SEQ ID NO: 23), or VDAP (SEQ ID NO: 22).

[0047] In some embodiments, the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, an amino acid sequence selected from VAGG (SEQ ID NO: 5), VAPG (SEQ ID NO: 11), VAQP (SEQ ID NO: 14), VPA, VPAG (SEQ ID NO: 94), VPGG (SEQ ID NO: 100), VPPG (SEQ ID NO: 110), VPQ, VPQG (SEQ ID NO: 112), VGAG (SEQ ID NO: 41), VGGG (SEQ ID NO: 47), VGQG (SEQ ID NO: 59), VDGG (SEQ ID NO: 23), or VDAP (SEQ ID NO: 22).

[0048] In some embodiments, the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, the amino acid sequence VAGG (SEQ ID NO: 5) or VPAG (SEQ ID NO: 94).

[0049] In some embodiments, the method comprises introducing into the single-domain antibody one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108.

[0050] In some embodiments, the Leu (L) at position 11 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Gin (Q) at position 13 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y); the Gly (G) at position 88 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Vai (V) or lie (I) at position 89 is mutated to Ala (A), Asp (D), Glu (E), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), or Tyr (Y); and / or the Leu (L) or Gin (Q) at position 108 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Asn(N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

[0051] In some embodiments, the single-domain antibody comprises at least an amino acid substitution at position 87.

[0052] In some embodiments, the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys ( K), Leu ( L), Asn ( N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y).

[0053] In some embodiments, the Thr (T) at position 87 is mutated to Ala (A), Ser (S), or Vai (V).

[0054] In another aspect, the present disclosure provides a method of modifying a single-domain antibody, the method comprising introducing into the single-domain antibody one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108, wherein the Leu (L) at position 11 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Gin (Q) at position 13 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Arg ( R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y); the Gly (G) at position 88 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y); the Vai (V) or lie (I) at position 89 is mutated to Ala (A), Asp (D), Glu (E), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), or Tyr (Y); and / or the Leu (L) or Gin (Q) at position 108 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Asn( N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

[0055] In some embodiments, the single-domain antibody comprises at least an amino acid substitution at position 87.

[0056] In some embodiments, the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys ( K), Leu ( L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y).

[0057] In some embodiments, the Thr (T) at position 87 is mutated to Ala (A), Ser (S), or Vai (V).

[0058] In some embodiments, the single-domain antibody has reduced binding by anti-drug antibodies (ADAs) to its C-terminus as compared to an unmodified single-domain antibody.

[0059] In some embodiments, the unmodified single-domain antibody comprises VSS at the C- terminus.

[0060] In some embodiments, the single-domain antibody has at least about 80% reduced ADA binding to its C-terminus as compared to an unmodified single-domain antibody.

[0061] In some embodiments, the single-domain antibody has at least about 85% reduced ADA binding to its C-terminus as compared to an unmodified single-domain antibody.

[0062] In some embodiments, the single-domain antibody has at least about 90% ADA binding to its C- terminus as compared to an unmodified single-domain antibody.

[0063] In some embodiments, the ADA binding is measured using an enzyme-linked immunosorbent assay (ELISA).

[0064] In some embodiments, the single-domain antibody is a VHH, or a VH domain.

[0065] In some embodiments, the single-domain antibody is a camelid VHH.

[0066] In some embodiments, the single-domain antibody is a humanized VHH.

[0067] In some embodiments, the single-domain antibody is a camelized VH.

[0068] In some embodiments, the single-domain antibody is present in a fusion protein, and the singledomain antibody is at the carboxy-terminus of the fusion protein.BRIEF DESCRIPTION OF DRAWINGS

[0069] Figure 1 shows the location of engineering activities on the C-terminus and in the frameworks of VHHs. Engineering on the C-terminus was focused on residues H112-113. Engineering of the framework was performed on residues Hll, H13, H87-89 and H108. Respective residues are shown in stick representation (pdb: 7LVU).

[0070] Figure 2A-2E depict Kabat- and Chothia-based annotation of a VHH sequence. Kabat- and Chothia-based sequence annotation using H-numbering is shown for the human DP47 germline VH sequence and an exemplary camelid VHH sequence. Framework regions 1-4 and CDR regions 1-3 are labeled. Positions that were used for engineering activities are highlighted in black. Figure discloses SEQ. ID NO: 206 and 207, respectively, in order of appearance.

[0071] Figure 3 shows a bar graph of binding of pre-anti-drug antibodies (pre-ADAs) (human IgG) to purified V-bodies. Four-hundred and fifty-two (452) engineered V-bodies were incubated with human intravenous IgG ( I Vlg) and binding of human IgG was detected. The original V-body sequence without engineering of the framework or C-terminal sequence is labeled as WT (wild-type) (black bar, dotted line). Based on the WT absorption value, 90% of signal reduction was calculated and depicted as 90% reduction of pre-ADA binding (dashed line). Phosphate buffered saline (PBS) was used as a background control. All values shown are blank subtracted.

[0072] Figure 4A-4W show a summary of binding of pre-ADAs (human IgG) to purified V-bodies normalized to control VHH ODY-349. Four-hundred and fifty-two (452) purified, engineered V-bodieswere incubated with IVIg and binding of human IgG was detected. PBS was used as background control, all values shown are blank subtracted. Absorption of the unmodified V-body (ODY-349) was set to 1, all values were normalized accordingly.

[0073] Figures 5A-5D depict a summary of pre-ADA-binding (human IgG) to purified V-bodies. The panels show the normalized enzyme-linked immunosorbent assay (ELISA) values (WT sequence = 1.000; PBS control subtracted) and their corresponding C-terminal sequence. Values exceeding WT level, were capped at 100% (1.000). Along the vertical and horizontal axis, amino acids at position H112 and H113 are highlighted, respectively. The C-terminal-most residue is H113 for VXX and framework mutations (Figure 5A), glycine H114 for VXXG (Figure 5B), or proline H114 for VXXP (Figure 5C). N / A depicts V- bodies below the concentration threshold for this experiment. Crossed-out V-bodies were not tested. Grayscale is from white (0.000) to medium gray (0.500) to dark gray (1.000).

[0074] Figure 6 shows a heat map presenting the optical density (OD) values of pre-existing ADA binding analysis for 24 V-body variants screened against 50 individual sera from human healthy donors. Grayscale code is based on the mean OD value per donor, on a logarithmic scale. Grayscale shows labels on log scale (values in brackets represent the original scale). Intensity of grayscale represents the detected level of pre-existing ADA binding to purified V-bodies. Figure discloses SEQ ID NO: 5, 11, 23, 41, 47, 59, 94, 100, 110, 112, 14, 22, 95, 101, and 107, respectively, in order of appearance.

[0075] Figures 7A-7B illustrate that C-terminal modification of tetravalent V-body agonists targeting a hepatocyte membrane protein significantly reduced hepatotoxicity. The relative viability of the primary hepatocytes as measured by CellTiter-Glo assay (CTG) on the y-axis is plotted against the indicated concentration of wild-type V-body agonist (wt-Vtetra3) and modified Vtetra3-VPAG in the presence and absence of human intravenous IgG (IVIg) after 24 hours of incubation (x-axis) (Figure 7A). The relative viability of the primary hepatocytes as measured by CellTiter-Glo assay (CTG) on the y-axis is plotted against the indicated concentration of wild-type V-body agonist (wt-Vtetra3) and modified Vtetra3- VAGG in the presence and absence of human IVIg after 24 hours of incubation (x-axis) (Figure 7B).Dashed horizontal lines highlight the viability of the hepatocytes for the highest concentration of agonist tested in the absence of IVIg for comparison. Structural models of the V-body agonists and position of the modifications (very C-terminus) are also included at the bottom of the figure panels.DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0076] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.

[0077] As used herein, the term "about," when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 5%. For example, as used herein, the expression "about 100" includes 95 and 105 and all values in between (e.g., 96, 97, 98, 99, etc.).

[0078] The term "antigen" encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof) that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. In various embodiments of the present disclosure, the antigen described herein may be a human, cynomolgus, and / or mouse antigen.

[0079] The term "epitope" can refer to an antigenic determinant on the surface of an antigen to which an antibody molecule binds. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects (e.g., agnostic or antagonistic effects). Epitopes may be either conformational or linear. A conformational epitope is formed by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include non-peptidic moieties on the antigen, such as saccharides, phosphoryl groups, or sulfonyl groups.

[0080] The term "antibody" and "immunoglobulin" or "Ig" are used interchangeably herein, and is used in the broadest sense and encompasses, for example, individual monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies (e.g., VHH), single chain antibodies, intrabodies, anti-idiotypic (anti-ld) antibodies, andantigen-binding fragments of antibodies, as described below. An antibody can be human, humanized, camelized, recombinantly produced, chimeric, synthetic, affinity de-matured and / or affinity matured as well as an antibody from other species, for example mouse, camel, llama, rabbit, etc. An "antigenbinding fragment" generally refers a portion of an antibody heavy and / or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Nonlimiting examples of antigen-binding fragments include single-domain antibody (e.g., VHH), single-chain Fvs (scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulf ide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody, or a chemically modified derivative thereof. Such antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. EnzymoL, 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2), or any subclass (e.g., lgG2a and lgG2b) of immunoglobulin molecule.

[0081] The term "single-domain antibody" or "sdAb" as used herein, refers to an antibody or antibody fragment containing a single antibody variable domain that is able to bind to a specific antigen alone, without the requirement of another antibody variable domain. The complementary determining regions (CDRs) of a single-domain antibody are part of a single antibody variable domain. Examples of singledomain antibodies include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered antibodies, variable domains derived from the aforementioned antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, shark, goat, rabbit, and / or bovine. In some embodiments, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. For clarity reasons, the variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four-chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, e.g., camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain, which are also within the scope of the invention. For example, cartilaginous fishes such as sharks can produce immunoglobulin-like structures known as VNAR. In some embodiments, a single-domain antibody maybe obtained from a Camelidae VH domain. In some embodiments, a single-domain antibody may be obtained from human VH by camelization. See Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608, the disclosure of which being incorporated by reference, for review of single-domain antibodies.

[0082] The term "isolated" when used in the context of single-domain antibodies, polypeptides, polynucleotides, and vectors, means the antibodies, polypeptides, polynucleotides and vectors are at least partially free of other biological molecules from the cells or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated single-domain antibody may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term "isolated" is not intended to refer to a complete absence of such biological molecules (e.g., minor or insignificant amounts of impurity may remain) or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the single-domain antibodies.

[0083] The term "operably linked" as used herein can refer to a functional relationship between two or more regions of a polypeptide chain in which the two or more regions are linked so as to produce a functional polypeptide.

[0084] As used herein, the term "variant", "derivative" or "derived from" in the context of proteins or polypeptides (e.g., single-domain antibodies or domains thereof) refer to: (a) a polypeptide that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the polypeptide it is a variant or derivative of; (b) a polypeptide encoded by a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity to a nucleotide sequence encoding the polypeptide it is a variant or derivative of; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions and / or substitutions) relative to the polypeptide it is a variant or derivative of; (d) a polypeptide encoded by nucleic acids can hybridize under high, moderate or typical stringency hybridization conditions to nucleic acids encoding the polypeptide it is a variant or derivative of; (e) a polypeptide encoded by a nucleotide sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleotide sequence encoding a fragment of the polypeptide, it is a variant or derivative of, of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, orat least 150 contiguous amino acids; or (f) a fragment of the polypeptide it is a variant or derivative of. The terms also encompass a fusion protein or polypeptide comprising the polypeptide it is a variant or derivative of.

[0085] The term "substantial identity" or "substantially identical ' when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FAST A, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0086] As applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur- containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamateaspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM 250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0087] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each herein incorporated by reference.

[0088] The terms "enhance" or "promote," or "increase," or "expand," or "improve" refer generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. A measurable physiological response may include an increase in immune cell expansion, activation, effector function, persistence, and / or an increase in tumor cell death killing ability, among others apparent from the understanding in the art and the description herein. In certain embodiments, an "increased" or "enhanced" amount can be a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition.

[0089] The terms "decrease" or "lower," or "lessen," or "reduce," or "abate", or "impede" refer generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. In certain embodiments, a "decrease" or "reduced" amount can be a "statistically significant" amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle or a control composition.

[0090] The terms "treat" or "treatment" of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub- clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0091] The terms "effective amount" or "therapeutically effective amount" refer to a quantity and / or concentration of a composition containing an active ingredient (e.g., a single-domain antibody, or a fusion protein or conjugate thereof) that when administered into a patient either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a significant decrease in disease progression as, for example, by ameliorating or eliminating symptoms and / or the cause of the disease. An effective amount may be an amount that relieves, lessens, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents progression of the disease or disorder, or improves physical functioning of the patient. A therapeutically effective amount of a composition containing an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the active agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic and / or prophylactic result.

[0092] The terms "individual", "subject" and "patient" are used interchangeably herein to refer to an animal; for example a mammal. The terms include human and veterinary subjects. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. The subjectcan be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, a subject can be a subject in need of treatment for a disease or disorder. In particular embodiments, the subject is a human.Single-domain Antibodies

[0093] In one aspect, the present disclosure provides a single-domain antibody (also termed as "sdAb") which is modified, e.g., at the carboxy-terminus, to reduce ADA binding. The single-domain antibodies of the present disclosure can be derived from numerous sources, including but not limited to VHHs, VNARs, or VH domains (naturally occurring or engineered VH domains). VHHs can be generated from camelid heavy chain only antibodies and libraries thereof. VNARs can be generated from cartilaginous fish heavy chain only antibodies and libraries thereof. Various methods have been implemented to generate monomeric sdAbs from conventionally heterodimeric VH and VL domains, including interface engineering and selection of specific germline families. In some embodiments, the sdAb of the present invention are human or humanized.

[0094] In some embodiments, a single-domain antibody described herein is a VHH fragment (also known as a nanobody). VHH fragments are also referred to as "V-bodies" in the present disclosure. In some embodiments, the VHH is a camelid VHH, a humanized VHH or a camelized VH. In some embodiments, a single-domain antibody described herein is a VH domain. In some embodiments, a single-domain antibody described herein is a naturally occurring VH domain or engineered VH domain.

[0095] The variable domain of a single-domain antibody of the present disclosure comprises at least three complementarity determining regions (CDRs) which determine its binding specificity. Preferably, in a variable domain, the CDRs are distributed between framework regions (FRs). The variable domain typically contains 4 framework regions interspaced by 3 CDR regions, resulting in the following typical antibody variable domain structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. CDRs and / or FRs of the single domain antibody of the invention may be fragments or derivatives from a naturally occurring antibody variable domain or may be synthetic.

[0096] The present disclosure provides, in particular, single-domain antibodies (e.g., VHHs) comprising one or more modifications. In certain aspects, single-domain antibodies (e.g., VHHs) of the present disclosure are modified by one or more changes to the carboxy-terminus (C-terminus), i.e., the singledomain antibodies may be modified to comprise a C-terminal modification(s). Without wishing to be bound by theory, it has been described that the exposed C-terminus of an immunoglobulin variable domain may form, e.g., epitopes such as B-cell epitopes, which can induce and / or interact withemerging and / or pre-existing antibodies, e.g., anti-drug antibodies (ADAs). In some embodiments, a single-domain antibody which has been modified at the C-terminus may have, e.g., reduced ADA binding to the C-terminus as compared to an unmodified single-domain antibody. Single-domain antibodies may be modified, in particular, to reduce binding of the single-domain antibodies (e.g., VHH) described herein by ADAs (or other aspecific proteins) found in human blood including whole blood, serum, and / or plasma (or other bodily fluids such as, but not limited to, bronchoalveolar fluid, ocular fluid, cerebrospinal fluid, and mucus) as compared to an unmodified (e.g., "wild-type") single-domain antibody.

[0097] In some embodiments, a C-terminal modification described herein may comprise a change to, e.g., Framework 4 (FR4) of a single-domain antibody (e.g., VHH) described herein. In some embodiments, a C-terminal modification may comprise a change to an amino acid(s) at any of positions 111, 112, 113 or 114 (numbering according to Chothia) of the C-terminus, or combination thereof (see, e.g., Figures 1-2).

[0098] In some embodiments, a single-domain antibody (e.g., VHH) described herein may comprise one or more amino acid mutations. Non-limiting examples of amino acid mutations comprise amino acid substitutions, insertions, additions, and / or deletions. Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position. Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent to one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent to another inserted amino acid residue. In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure may be modified to comprise one or more amino acid mutations (e.g., substitution mutation(s)) at any of various amino acid position(s) described herein. In some embodiments, a single domain antibody (e.g., VHH) described herein may be modified to comprise at least one substitution mutation. In some embodiments, a single domain antibody (e.g., VHH) described herein may be modified to comprise one substitution mutation.

[0099] In some embodiments, a single-domain antibody (e.g., VHH) described herein may comprise one or more amino acid mutations, e.g., within the C-terminus (e.g., at any of residues at positions 111-114 (numbering according to Chothia), or combination thereof) of the single-domain antibody (see, e.g., Figures 1-2).

[0100] In some embodiments, a single-domain antibody (e.g., VHH) described herein may comprise one or more amino acid mutations within a framework region (e.g., FR1-4, or combination thereof) of the single-domain antibody (see, e.g., Figures 1-2).

[0101] In some embodiments, a single-domain antibody (e.g., VHH) described herein may comprise one or more amino acid mutations within FR1. In some embodiments, FR1 of a single-domain antibody described herein may be modified to comprise, for example, without limitation, a mutation of an amino acid at positions 11 and / or 13 (numbering according to Chothia). In some embodiments, FR2 of a singledomain antibody described herein may be modified to comprise, for example, without limitation, a mutation of an amino acid at any of positions 87, 88, or 89 (numbering according to Chothia), or combination thereof. In some embodiments, FR4 of a single-domain antibody described herein may be modified to comprise, for example, without limitation, a mutation of an amino acid at any of positions 108, 111, 112, 113, or 114 (numbering according to Chothia), or combination thereof. In some embodiments, an FR region (e.g., FR1-4, or combination thereof) may be modified to comprise a mutation of an amino acid(s) at any of positions 11, 13, 87, 88, 89, 108, 111, 112, 113, or 114 (numbering according to Chothia), or combination thereof. In some embodiments, a mutation within any of FR1-4, or combination thereof, may comprise one or more of a substitution mutation(s).

[0102] In some embodiments, a single domain antibody may comprise one or more C-terminal modifications described herein or one or more of any of various other mutation(s) (e.g., framework mutations) described herein, or combination thereof. In some embodiments, a single-domain antibody described herein may comprise one or more of a C-terminal modification(s) and one or more of a framework mutation(s) (e.g., a substitution mutation(s)) described herein. In some embodiments, a single-domain antibody described herein may comprise one or more of a C-terminal modification(s) or one or more of a framework mutation(s) (e.g., substitution mutation(s)) described herein.

[0103] In some embodiments, a single-domain antibody described herein may comprise at the C- terminus, starting from position 111 (numbering according to Chothia), any of various modifications disclosed herein. In some embodiments, a single-domain antibody may comprise any of various modifications, for example, at positions 111, 112, 113, or 114 (numbering according to Chothia), or combination thereof.

[0104] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure may be modified to comprise one or more mutations, e.g., within Framework regions FR1, FR3 and / or FR4, or combination thereof (i.e., framework mutation(s)). In some embodiments, such mutations (e.g., a substitution mutation(s)) may comprise, one or more of a mutation at any of amino acid position 11, 13, 87, 88, 89, or 108 (numbering according to Chothia), or combination thereof.

[0105] In some embodiments, the modified single-domain antibody has at least about 60%, 65%, 70%, 72%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%, or about 60%-70%, about 70%-80%, about 80%-85%, about 85%-90%, about 90%-95%, or about 90%-100% reduced ADA binding as compared to an unmodified single-domain antibody.

[0106] In some embodiments, the single-domain antibody has about 80% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 81% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 82% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 83% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 84% reduced ADA binding as compared to an unmodified singledomain antibody. In some embodiments, the single-domain antibody has 85% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 86% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 87% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 88% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 89% reduced ADA binding as compared to an unmodified singledomain antibody. In some embodiments, the single-domain antibody has about 90% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the singledomain antibody has about 91% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 92% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 93% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 94% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has 95% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 96% reduced ADA binding as compared to an unmodified singledomain antibody. In some embodiments, the single-domain antibody has about 97% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the singledomain antibody has about 98% reduced ADA binding as compared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 99% reduced ADA binding ascompared to an unmodified single-domain antibody. In some embodiments, the single-domain antibody has about 100% reduced ADA binding as compared to an unmodified single-domain antibody.

[0107] In some embodiments, ADA binding to a single-domain antibody described herein is measured using an enzyme-linked immunosorbent assay (ELISA) assay. There are different assays, methods and techniques for performing ADA assays, including, but not limited to, ligand binding assays (e.g. ELISA, electrochemiluminescence immunoassay (ECLIA)) in different formats such as (i) bridging format; (ii) direct format; (iii) indirect format; (iv) radio immuno-precipitation assay (RIP); or (v) surface plasmon resonance. Reference is made, e.g., to Table 1 in the review by Mire-Sluis et al., J. Immunol. Meth. 289 (2004), 1-16, Table 1 in the 2010 review by Wadwha and Thorpe, Bioanalysis (2010), 2(6), 1073-1084 and Table 2 in the 2006 article by Wadwha and Thorpe, Journal of Immunotoxicology, 3:115-121, 2006 (each of which is incorporated herein by reference in its entirety). A non-limiting example of an antidrug antibody (ADA) assay is described in Example 2 below.

[0108] In some embodiments, ADA assays of the present disclosure may be performed in accordance with any of the methods and / or techniques described in, for example, without limitation, International Publication Number WO 2016 / 118733, Ackaert et al., Front Immunol. 2021 Mar 9;12:632687, and / or Jordan and Staack Bioanalysis. 2020 Jul;12(14):1021-1031, each of which is incorporated herein by reference in its entirety.C-terminal modifications

[0109] In various embodiments, single-domain antibodies (VHHs) described herein comprise one or more modifications at the C-terminus, i.e., C-terminal modification(s). In some embodiments, a singledomain antibody of the present disclosure may be modified to comprise, at the C-terminus (starting from position 111 according to Chothia), an amino acid sequence selected from(1) V,(2) VX(3) VXiX2,(4) VX1X2G, or(5) VXiX2P.

[0110] In various embodiments, Xi is selected from amino acids Ala (A), Asp (D), Glu (E), Gly (G), He (I), Lys (K), Leu (L), Asn (N), Pro (P), Arg (R), Ser (S), Thr (T), and Vai (V).

[0111] In various embodiments, X2is selected from amino acids Ala (A), Asp (D), Glu (E), Gly (G), He (I), Lys (K), Leu (L), Asn (N), Pro (P), Gin (Q), Arg (R), Ser (S), Thr (T), and Vai (V).

[0112] In various embodiments, the single-domain antibody has reduced ADA binding to the C- terminus as compared to an unmodified (e.g., wild-type) single-domain antibody.

[0113] In some embodiments, the unmodified (e.g., wild-type) single-domain antibody comprises VSS at the carboxy-terminus.

[0114] In some embodiments, Xi is selected from Ala (A), Gly (G), Pro (P), Asp (D), and Leu (L).

[0115] In some embodiments, X2is selected from amino acids Ala (A), Gly (G), Pro (P), Asp (D), Gin (Q), and Leu (L).

[0116] In some embodiments, Xi is selected from Ala (A), Gly (G), Pro (P), Asp (D), and Leu (L); and X2is selected from amino acids Ala (A), Gly (G), Pro (P), Asp (D), Gin (Q), and Leu (L).

[0117] In some embodiments, Xi is selected from Ala (A), Gly (G), and Pro (P).

[0118] In some embodiments, X2is selected from amino acids Ala (A), Gly (G), Gin (Q) and Pro (P).

[0119] In some embodiments, Xi is selected from Ala (A), Gly (G), and Pro (P); and X2is selected from amino acids Ala (A), Gly (G), Gin (Q), and Pro (P).

[0120] In some embodiments, the single-domain antibody comprises at the carboxy-terminus (starting from position 111 according to Chothia) an amino acid sequence selected from(1) V;(2) VA, VD, VE, VG, VI, VK, VL, VN, VP, VR, VS, VT, or VV;(3) VAA, VAD, VAE, VAG, VAI, VAK, VAL, VAN, VAP, VAQ, VAR, VAS, VAT, VAV, VDA, VDD, VDE, VDG, VDI, VDK, VDL, VDN, VDP, VDQ, VDR, VDS, VDT, VDV, VEA, VED, VEE, VEG, VEI, VEK, VEL, VEN, VEP, VEQ, VER, VES, VET, VEV, VGA, VGD, VGE, VGG, VGI, VGK, VGL, VGN, VGP, VGQ, VGR, VGS, VGT, VGV, VIA, VID, VIE, VIG, VII, VIK, VIL, VIN, VIP, VIQ, VIR, VIS, VIT, VIV, VLA, VLD, VLE, VLG, VLI, VLK, VLL, VLN, VLP, VLQ, VLR, VLS, VLT, VLV, VNA, VND, VNE, VNG, VNI, VNK, VNL, VNN, VNP, VNQ, VNR, VNS, VNT, VNV, VPA, VPD, VPE, VPG, VPI, VPK, VPL, VPN, VPP, VPQ, VPR, VPS, VPT, VPV, VRA, VRD, VRE, VRG, VRI, VRK, VRL, VRN, VRP, VRQ, VRR, VRS, VRT, VRV, VSA, VSD, VSE, VSG, VSI, VSK, VSL, VSN, VSP, VSQ, VSR, VST, VSV, VTA, VTD, VTE, VTG, VTI, VTK, VTL, VTN, VTP, VTQ, VTR, VTS, VTT, VTV, VVA, VVD, WE, VVG, VVI, WK, WL, WN, WP, VVQ, WR, WS, WT, or VW;(4) VADG (SEQ ID NO: 1), VAEG (SEQ ID NO: 3), VAGG (SEQ ID NO: 5), VAKG (SEQ ID NO: 7), VANG (SEQ ID NO: 9), VAPG (SEQ ID NO: 11), VAQG (SEQ ID NO: 13), VARG (SEQ ID NO: 15), VASG (SEQ ID NO: 17), VATG (SEQ ID NO: 19), VDAG (SEQ ID NO: 21), VDGG (SEQ ID NO: 23), VDPG (SEQ ID NO: 25), VDSG (SEQ ID NO: 27), VDTG (SEQ ID NO: 29), VEAG (SEQ ID NO: 31), VEGG (SEQ ID NO: 33), VEPG (SEQ ID NO: 35), VESG (SEQ ID NO: 37), VETG (SEQ ID NO: 39), VGAG (SEQ ID NO: 41), VGDG (SEQ ID NO: 43), VGEG (SEQ ID NO: 45), VGGG (SEQ ID NO: 47), VGIG (SEQ ID NO: 49), VGKG (SEQ ID NO: 51), VGLG (SEQID NO: 53), VGNG (SEQ ID NO: 55), VGPG (SEQ ID NO: 57), VGQG (SEQ ID NO: 59), VGRG (SEQ ID NO: 61), VGSG (SEQ ID NO: 63), VGTG (SEQ ID NO: 65), VGVG (SEQ ID NO: 67), VIGG (SEQ ID NO: 69), VIPG (SEQ ID NO: 71), VISG (SEQ ID NO: 73), VITG (SEQ ID NO: 75), VLGG (SEQ ID NO: 77), VLPG (SEQ ID NO: 79), VLSG (SEQ ID NO: 283), VLTG (SEQ ID NO: 82), VNAG (SEQ ID NO: 84), VNGG (SEQ ID NO: 86), VNPG (SEQ ID NO: 88), VNSG (SEQ ID NO: 90), VNTG (SEQ ID NO: 92), VPAG (SEQ ID NO: 94), VPDG (SEQ ID NO: 96), VPEG (SEQ ID NO: 98), VPGG (SEQ ID NO: 100), VPIG (SEQ ID NO: 102), VPKG (SEQ ID NO: 104), VPLG (SEQ ID NO: 106), VPNG (SEQ ID NO: 108), VPPG (SEQ ID NO: 110), VPQG (SEQ ID NO: 112), VPRG (SEQ ID NO: 114), VPSG (SEQ ID NO: 116), VPTG (SEQ ID NO: 118), VPVG (SEQ ID NO: 120), VRAG (SEQ ID NO: 122), VRGG (SEQ ID NO: 124), VRPG (SEQ ID NO: 126), VRSG (SEQ ID NO: 128), VRTG (SEQ ID NO: 130), VSAG (SEQ ID NO: 284), VSDG (SEQ ID NO: 133), VSEG (SEQ ID NO: 135), VSGG (SEQ ID NO: 137), VSIG (SEQ ID NO: 139), VSKG (SEQ ID NO: 141), VSLG (SEQ ID NO: 143), VSNG (SEQ ID NO: 145), VSPG (SEQ ID NO: 147), VSQG (SEQ ID NO: 149), VSRG (SEQ ID NO: 151), VSTG (SEQ ID NO: 153), VSVG (SEQ ID NO: 285), VTAG (SEQ ID NO: 156), VTDG (SEQ ID NO: 158), VTEG (SEQ ID NO: 160), VTGG (SEQ ID NO: 162), VTIG (SEQ ID NO: 164), VTKG (SEQ ID NO: 166), VTLG (SEQ ID NO: 168), VTNG (SEQ ID NO: 170), VTPG (SEQ ID NO: 172), VTQG (SEQ ID NO: 174), VTRG (SEQ ID NO: 176), VTSG (SEQ ID NO: 178), VTTG (SEQ ID NO: 180), VTVG (SEQ ID NO: 182), VVGG (SEQ ID NO: 184), WPG (SEQ ID NO: 186), WSG (SEQ ID NO: 188), or WTG (SEQ ID NO: 190); or(5) VADP (SEQ ID NO: 2), VAEP (SEQ ID NO: 4), VAGP (SEQ ID NO: 6), VAKP (SEQ ID NO: 8), VANP (SEQ ID NO: 10), VAPP (SEQ ID NO: 12), VAQP (SEQ ID NO: 14), VARP (SEQ ID NO: 16), VASP (SEQ ID NO: 18), VATP (SEQ ID NO: 20), VDAP (SEQ ID NO: 22), VDGP (SEQ ID NO: 24), VDPP (SEQ ID NO: 26), VDSP (SEQ ID NO: 28), VDTP (SEQ ID NO: 30), VEAP (SEQ ID NO: 32), VEGP (SEQ ID NO: 34), VEPP (SEQ ID NO: 36), VESP (SEQ ID NO: 38), VETP (SEQ ID NO: 40), VGAP (SEQ ID NO: 42), VGDP (SEQ ID NO: 44), VGEP (SEQ ID NO: 46), VGGP (SEQ ID NO: 48), VGIP (SEQ ID NO: 50), VGKP (SEQ ID NO: 52), VGLP (SEQ ID NO: 54), VGNP (SEQ ID NO: 56), VGPP (SEQ ID NO: 58), VGQP (SEQ ID NO: 60), VGRP (SEQ ID NO: 62), VGSP (SEQ ID NO: 64), VGTP (SEQ ID NO: 66), VGVP (SEQ ID NO: 68), VIGP (SEQ ID NO: 70), VIPP (SEQ ID NO: 72), VISP (SEQ ID NO: 74), VITP (SEQ ID NO: 76), VLGP (SEQ ID NO: 78), VLPP (SEQ ID NO: 80), VLSP (SEQ ID NO: 81), VLTP (SEQ ID NO: 83), VNAP (SEQ ID NO: 85), VNGP (SEQ ID NO: 87), VNPP (SEQ ID NO: 89), VNSP (SEQ ID NO: 91), VNTP (SEQ ID NO: 93), VPAP (SEQ ID NO: 95), VPDP (SEQ ID NO: 97), VPEP (SEQ ID NO: 99), VPGP (SEQ ID NO: 101), VPIP (SEQ ID NO: 103), VPKP (SEQ ID NO: 105), VPLP (SEQ ID NO: 107), VPNP (SEQ ID NO: 109), VPPP (SEQ ID NO: 111), VPQP (SEQ ID NO: 113), VPRP (SEQ ID NO: 115), VPSP (SEQ ID NO: 117), VPTP (SEQ ID NO: 119), VPVP (SEQ ID NO: 121), VRAP (SEQ ID NO: 123), VRGP (SEQ ID NO: 125), VRPP (SEQ ID NO: 127), VRSP (SEQ ID NO: 129), VRTP (SEQ ID NO: 131), VSAP (SEQ ID NO:132), VSDP (SEQ ID NO: 134), VSEP (SEQ ID NO: 136), VSGP (SEQ ID NO: 138), VSIP (SEQ ID NO: 140), VSKP (SEQ ID NO: 142), VSLP (SEQ ID NO: 144), VSNP (SEQ ID NO: 146), VSPP (SEQ ID NO: 148), VSQP (SEQ ID NO: 150), VSRP (SEQ ID NO: 152), VSTP (SEQ ID NO: 154), VSVP (SEQ ID NO: 155), VTAP (SEQ ID NO: 157), VTDP (SEQ ID NO: 159), VTEP (SEQ ID NO: 161), VTGP (SEQ ID NO: 163), VTIP (SEQ ID NO: 165), VTKP (SEQ ID NO: 167), VTLP (SEQ ID NO: 169), VTNP (SEQ ID NO: 171), VTPP (SEQ ID NO: 173), VTQP (SEQ ID NO: 175), VTRP (SEQ ID NO: 177), VTSP (SEQ ID NO: 179), VTTP (SEQ ID NO: 181), VTVP (SEQ ID NO: 183), VVGP (SEQ ID NO: 185), WPP (SEQ ID NO: 187), VVSP (SEQ ID NO: 189), or VVTP (SEQ ID NO: 191).

[0121] In some embodiments, the single-domain antibody comprises at the carboxy-terminus (starting from position 111 according to Chothia) an amino acid sequence selected from VR, VG, VP, VA, VPG, VDG, VPQ, VPA, VAGG (SEQ ID NO: 5), VAPG (SEQ ID NO: 11), VAQP (SEQ ID NO: 14), VPAG (SEQ ID NO: 94), VPGG (SEQ ID NO: 100), VPPG (SEQ ID NO: 110), VPQG (SEQ ID NO: 112), VPAP (SEQ ID NO: 95), VPGP (SEQ ID NO: 101), VPLP (SEQ ID NO: 107), VGP, VGAG (SEQ ID NO: 41), VGGG (SEQ ID NO: 47), VGQG (SEQ ID NO: 59), VDGG (SEQ ID NO: 23), or VDAP (SEQ ID NO: 22).

[0122] In some embodiments, the single-domain antibody comprises at the carboxy-terminus (starting from position 111 according to Chothia) an amino acid sequence selected from VAGG (SEQ ID NO: 5), VAPG (SEQ ID NO: 11), VAQP (SEQ ID NO: 14), VPA, VPAG (SEQ ID NO: 94), VPGG (SEQ ID NO: 100), VPPG (SEQ ID NO: 110), VPQ, VPQG (SEQ ID NO: 112), VGAG (SEQ ID NO: 41), VGGG (SEQ ID NO: 47), VGQG (SEQ ID NO: 59), VDGG (SEQ ID NO: 23), or VDAP (SEQ ID NO: 22).

[0123] In some embodiments, the single-domain antibody does not comprise at the carboxy-terminus (starting from position 111 according to Chothia) an amino acid sequence selected from VSS, VE, VEG, VEP, VEPG (SEQ ID NO: 35), VK, VKS, VKG, VKP, VKPG (SEQ ID NO: 294), VQS, VS, VSE, VSEG (SEQ ID NO: 135), VSK, VSKG (SEQ ID NO: 141), VRP, VRPG (SEQ ID NO: 126), VDP, VDPG (SEQ ID NO: 25), VSSP (SEQ ID NO: 295), and VSSG (SEQ ID NO: 286).

[0124] In some embodiments, the single-domain antibody comprises at the carboxy-terminus (starting from position 111 according to Chothia) the amino acid sequence VAGG (SEQ ID NO: 5) or VPAG (SEQ ID NO: 94),

[0125] In various embodiments, a single domain antibody described herein may comprise, Vai (V), Ser (S), and another Ser (S), (i.e., VSS), at the C-terminus, starting from amino acid position 111 (according to Chothia), such that Vai (V) is at position 111, Ser (S) is at position 112, and another Ser (S) is at position 113 for the VSS sequence. A VSS sequence comprising Vai (V) at position 111, Ser (S) at position 112, and Ser (S) at position 113 may be referred to herein as an "unmodified" VSS sequence (or a "wild-type" VSSsequence). In various embodiments, the wild-type VSS sequence may be modified in accordance with any of various modifications described herein.

[0126] In some embodiments any of positions 111, 112, 113, or 114, or any combination thereof, may be modified in accordance with any of the modifications described herein.

[0127] In some embodiments, a single-domain antibody of the present disclosure comprises Vai (V) at position 111. In some embodiments, a single-domain antibody of the present disclosure comprises Pro (P) at position 111.

[0128] In some embodiments, a single-domain antibody described herein may comprise, without limitation, Ser (S), Ala (A), Asp (D), Glu (E), Gly (G), He (I), Leu (L), Asn (N), Pro (P), Arg (R), Thr (T), Vai (V), or Lys (K) at position 112. In some embodiments, an amino acid at position 112 may be absent (e.g., deleted) such that position 112 is not occupied by an amino acid. A deletion of one or more amino acid from a given amino acid position described herein may be indicated by a dash, i.e., in lieu of the deleted amino acid(s). As an example, a C-terminal sequence comprising amino acids which have been deleted from positions 112 and 113 may comprise the sequence "V - and the V is at position 111.

[0129] In some embodiments, a single-domain antibody described herein may comprise, without limitation, a Ser (S), Asp (D), Glu (E), Gly (G), Lys (K), Asn (N), Pro (P), Gin (Q), Arg (R), Thr (T), Ala (A), He (I), Leu (L) or Vai (V) at position 113. In some embodiments, an amino acid at position 113 may be absent (e.g., deleted) such that position 113 is not occupied by an amino acid.

[0130] In some embodiments, a single-domain antibody described herein may comprise a C-terminal sequence which is longer than the unmodified VSS sequence (i.e., 3 amino acids long). In some embodiments, without limitation, a C-terminal sequence comprising an amino acid addition may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more, amino acids in length. In some embodiments, a single-domain antibody disclosed herein may comprise one or more amino acids which have been added to, or inserted at, the C-terminal end of a sequence comprising any of various amino acids at positions 111-113, such that position 114 (or higher) can be occupied , e.g., by an amino acid such as, but not limited to, a Gly (G) and / or a Pro (P). In some embodiments, a single-domain antibody described herein may comprise, without limitation, a Gly (G) or a Pro (P) at position 114. In some embodiments, the sequence comprising any of various amino acids at positions 111-113 may comprise one or more amino acid additions to (or insertions at) the C-terminal end. In some embodiments, an amino acid at position 114 may be absent (e.g., deleted) such that position 114 is not occupied by an amino acid. Non-limiting examples of C-terminal addition modifications to a singledomain antibody are set forth in Table 1-1.

[0131] In some embodiments, a single-domain antibody described herein may comprise a C-terminal sequence which is shortened in length as compared to the wild-type VSS sequence (i.e., 3 amino acids long). As a non-limiting example, when the C-terminal domain a single-domain antibody described herein is shortened, positions 112 and / or 113 may lack an amino acid. Non-limiting examples of shortened C-terminal amino acid sequences (starting from position 111 according to Chothia) include, e.g., PP -, V - -, VA -, VD -, VE -, VG -, VI -, VK -, VL -, VN -, VP -, VR -, VS -, VT -, and VV -. Non-limiting examples of C-terminal shortening modifications to a single-domain antibody are set forth in Table 1-1.

[0132] In some embodiments, a single-domain antibody described herein may comprise a C-terminal sequence which is the same length as the wild-type VSS sequence (i.e., 3 amino acids long) and is a variant of the wild-type VSS sequence, i.e., differs from the wild-type VSS sequence by at least one amino acid. For example, a C-terminal sequence comprising a C-terminal variation may comprise a Vai (V) at position 111 in combination with any amino acid at positions 112 and 113, apart from the combination of Ser (S) at position 112 and Ser (S) at position 113. In some embodiments, a singledomain antibody comprising a C-terminal variant sequence (i.e., a C-terminal sequence comprising a C- terminal variation) comprises a Ala (A), Asp (D), Glu (E), Gly (G), He (I), Leu (L), Asn (N), Pro (P), Arg (R), Ser (S), Thr (T), or Vai (V) at position 112 In some embodiments, a single-domain antibody comprising a C-terminal variant sequence (i.e., a C-terminal sequence comprising a C-terminal variation) comprises Ala (A), Asp (D), Glu (E), Gly (G), He (I), Lys (K), Leu (L), Asn (N), Pro (P), Gin (Q), Arg (R), Ser (S), Thr (T), or Vai (V) at position 113. Non-limiting examples of C-terminal variant modifications to a single-domain antibody are set forth in Table 1-1.Table 1-1. Exemplary C-terminal modificationsFramework mutations

[0133] In various embodiments, single-domain antibodies (e.g., VHHs) described herein comprise one or more modifications comprising amino acid mutation(s) in one or more positions within any offramework regions FR1-FR4, or combination thereof. The one or more amino acid mutation(s) may comprise any of various mutation(s) described herein. In some embodiments, the amino acid mutation(s) comprise, without limitation, amino acid substitution(s), insertion(s), or deletion(s), or any combination thereof. In some embodiments, the single-domain antibodies (e.g., VHHs) described herein may comprise one or more amino acid substitutions. Non-limiting examples of exemplary amino acid substitutions are set forth in Table 1-2. In some embodiments, a single-domain antibody described herein may comprise one or more amino acid mutation, e.g., framework mutation described herein, paired with (i.e., in combination with) one or more C-terminal modifications described herein.

[0134] Exemplary framework mutations contemplated herein are set forth in Table 1-2.Table 1-2. Exemplary Framework Mutations

[0135] In some embodiments, the one or more amino acid substitutions may be any of various conservative amino acid substitution(s) described herein. As an example, without limitation, the one or more amino acid substitution(s) may be a conservative amino acid substitution(s) within at least one of FR1, FR2, FR3, and FR4. In some embodiments, FR1, FR2, FR3 and / or FR4 may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more, conservative amino acid substitutions.

[0136] In some embodiments, a single-domain antibody (e.g., VHH) described herein may comprise one or more amino acid mutations within Framework Region 1 (FR1). The one or more mutations within FR1 may comprise any mutation(s) described herein at any of amino acid positions 1-25 (numbering according to Chothia), or combination thereof (see, e.g., Figure 2). In some embodiments, FR1 of a single-domain antibody described herein may be modified to comprise, for example, without limitation, a mutation of an amino acid at positions 11 and / or 13 (numbering according to Chothia).

[0137] In some embodiments, a single-domain antibody (e.g., VHH) described herein may comprise one or more amino acid mutations within Framework Region 2 (FR2). The one or more mutations within FR2 may comprise any mutation(s) described herein at any of amino acid positions 33-51 (numbering according to Chothia), or combination thereof (see, e.g., Figure 2).

[0138] In some embodiments, a single-domain antibody (e.g., VHH) described herein may comprise one or more amino acid mutations within Framework Region 3 (FR3). The one or more mutations within FR3 may comprise any mutation(s) described herein at any of amino acid positions 57-81, 82, 82A, 82B, 82C, or 83-94 (numbering according to Chothia), or combination thereof (see, e.g., Figure 2). In some embodiments, FR3 of a single-domain antibody described herein may be modified to comprise, for example, without limitation, a mutation of an amino acid at any of positions 87, 88, or 89 (numbering according to Chothia), or combination thereof.

[0139] In some embodiments, a single-domain antibody (e.g., VHH) described herein may comprise one or more amino acid mutations within Framework Region 4 (FR4). The one or more mutations within FR4 may comprise any mutation(s) described herein at any of amino acid positions 103-113 (numbering according to Chothia), or combination thereof (see, e.g., Figure 2). In some embodiments, FR4 of a single-domain antibody described herein may be modified to comprise, for example, without limitation, a mutation of an amino acid at any of positions 108, 111, 112, or 113 (numbering according to Chothia), or combination thereof.

[0140] In some embodiments, a single-domain antibody (e.g., VHH) described herein may comprise an amino acid mutation at position 114 (numbering according to Chothia). In some embodiments, a single-domain antibody described herein may be modified to comprise a mutation of an amino acid(s) at any of positions 11, 13, 87, 88, 89, 108, 111, 112, 113, or 114, or combination thereof.

[0141] In some embodiments, a FR (e.g., FR1-4, or combination thereof) of a single-domain antibody (e.g., VHH) described herein may be modified to comprise a mutation of an amino acid(s) at any of positions 11, 13, 87, 88, 89, 108, 111, 112, 113, or 114, or combination thereof.

[0142] In some embodiments, a single-domain antibody (e.g., VHHs) of the present disclosure may be modified by one or more framework mutations (e.g., a substitution mutation(s)) at any of amino acid position 11, 13, 87, 88, 89, or 108 (numbering according to Chothia)), or combination thereof.

[0143] In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may comprise a Leu (L) at amino acid position 11 (Leull), e.g., within FR1. In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may comprise a Gin (Q) at amino acid position 13 (Glnl3), e.g., within FR1. In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may comprise a Thr (T) at amino acid position 87 (Thr87), e.g., of FR3. In some embodiments, the unmodified (i.e., wild-type) single-domain antibody comprises a Gly (G) at amino acid position 88 (Gly88), e.g., within FR3. In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may comprise a Vai (V) or an lie (I) at amino acid position 89 (Val89 or Ile89, respectively), e.g., of FR3. In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may comprise a Gin (Q) or Leu (L) at position 108 (Glnl08 or Leul08, respectively, e.g., within FR4.

[0144] In some embodiments, the single-domain antibody described herein may be modified at position Leull within Framework Region 1 (FR1). In some embodiments, the single-domain antibody described herein may be modified at position Glnl3 within Framework Region 1 (FR1). In some embodiments, the single-domain antibody described herein may be modified at position Thr87 within Framework Region 3 (FR3). In some embodiments, the single-domain antibody described herein may be modified at position Gly88 within Framework Region 3 (FR3). In some embodiments, the single-domain antibody described herein may be modified at position Val89 or Ile89 within Framework Region 3 (FR3). In some embodiments, the single-domain antibody described herein may be modified at position Glnl08 or Leul08 within Framework Region 4 (FR4).

[0145] In some embodiments, a single-domain antibody (e.g., VHH) described herein may be modified at position Leull within Framework Region 1 (FR1), Glnl3 within Framework Region 1 (FR1), Thr87 within Framework Region 3 (FR3), Gly88 within Framework Region 3 (FR3), Val89 and / or Ile89 within Framework Region 3 (FR3), or Glnl08 and / or Leul08 within Framework Region 4 (FR4), or any combination thereof.

[0146] In some embodiments, the single-domain antibody comprises one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108.

[0147] In some embodiments, the Leu (L) at position 11 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys ( K), Asn ( N ), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

[0148] In some embodiments, the Gin (Q) at position 13 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

[0149] In some embodiments, the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn ( N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y). In some embodiments, the Thr (T) at position 87 is mutated to Ala (A), Ser (S), or Vai (V).

[0150] In some embodiments, the Gly (G) at position 88 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys ( K), Leu ( L), Asn ( N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

[0151] In some embodiments, the Vai (V) or lie (I) at position 89 is mutated to Ala (A), Asp (D), Glu (E), Lys (K), Leu (L), Asn ( N), Gin (Q), Arg (R), Ser (S), Thr (T), or Tyr (Y).

[0152] In some embodiments, the Leu (L) or Gin (Q) at position 108 is mutated to Ala (A), Asp (D), Glu ( E), He (I), Lys (K), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

[0153] In some embodiments, a single-domain antibody of the present disclosure may comprise one or more substitution mutations. Non-limiting examples of substitution mutations include (a) LeullAsp (L11D), LeullGlu (L11E), LeullLys (L11K), LeullAsn (L11N), LeullArg (L11R), LeullSer (L11S), LeullThr (L11T), LeullVal (L11V), LeullAla (L11A), Leulllle (Llll), LeullGIn (L11Q), or LeullTyr (L11Y); (b) Glnl3Asn (Q13N), Glnl3Ser (Q13S), Glnl3Thr (Q13T), Glnl3Ala (Q13A), Glnl3Asp (Q13D), Glnl3Glu (Q13E), Glnl3lle (Q.13I), Glnl3Lys (Q.13K), Glnl3Leu (Q13L), Glnl3Arg (Q13R), Glnl3Val (Q13V), or Glnl3Tyr (Q13Y); (c) Thr87Asn (T87N), Thr87Ser (T87S), Thr87Ala (T87A), Thr87Asp (T87D), Thr87Glu (T87E), Thr87lle (T87I), Thr87Lys (T87K), Thr87Leu (T87L), Thr87Gln (T87Q), Thr87Arg (T87R), Thr87Val (T87V), or Thr87Tyr (T87Y); (d) Gly88Asp (G88D), Gly88Glu (G88E), Gly88Lys (G88K), Gly88Arg (G88R), Gly88Ala (G88A), Gly88lle (G88I), Gly88Leu (G88L), Gly88Asn (G88N), Gly88Gln (G88Q), Gly88Ser (G88S), Gly88Thr (G88T), Gly88Val (G88V), or Gly88Tyr (G88Y); (e) Val89Leu (V89L), Val89Asn (V89N), Val89Ser (V89S), Val89Thr (V89T), Val89Ala (V89A), Val89Asp (V89D), Val89Glu (V89E), Val89Lys (V89K), Val89Gln (V89Q), Val89Arg (V89R), Val89Tyr (V89Y), He89Leu (I89L), He89Asn (I89N), He89Ser (I89S), He89Thr (I89T), He89Ala (I89A), He89Asp (I89D), He89Glu (I89E), lle89Lys (I89K), lle89Gln (I89Q), He89Arg (I89R), or He89Tyr (I89Y); and / or (f) LeulO8Asn (L108N), LeulO8Ser (L108S), LeulO8Thr (L108T), LeulO8Ala (L108A), LeulO8Asp (L108D), Leul08Glu (L108E), LeulO8lle (L108I), LeulO8Lys (L108K), LeulO8Arg (L108R), LeulO8Val (L108V), LeulO8Tyr (L108Y), GlnlO8Asn (Q108N), GlnlO8Ser (Q108S), GlnlO8Thr(Q108T), GlnlO8Ala (Q108A), GlnlO8Asp (Q108D), GlnlO8Glu (Q108E), Glnl08lle (Q108I), GlnlO8Lys (Q108K), GlnlO8Arg (Q108R), GlnlO8Val (Q108V), GlnlO8Tyr (Q108Y), or any combination thereof.

[0154] In some embodiments, a single-domain antibody of the present disclosure may comprise a substitution mutation selected from LeullLys (L11K), LeullArg (L11R), LeullAsp (L11D), or LeullGlu (L11E).

[0155] In some embodiments, a single domain antibody of the present disclosure may comprise a substitution mutation selected from Ala88Glu (A88E), Ala88Asp (A88D), Ala88Arg (A88R), or Ala88Lys (A88K).

[0156] In some embodiments, a single-domain antibody described herein may be modified to comprise one or more substitution mutation(s) within FR1. In some embodiments, the substitution mutation may comprise a mutation at amino acid position 11 or amino acid position 13, or combination thereof, within FR1. In some embodiments, the substitution mutation(s) at amino acid position 11 may comprise, without limitation, any of LeullAsp ( Lil D), LeullGlu ( LI IE), LeullLys (L11K), LeullAsn (L11N), LeullArg (L11R), LeullSer (L11S), LeullThr (L11T), LeullVal (L11V), LeullAla (L11A), Leulllle (Llll), LeullGIn (L11Q), or LeullTyr (L11Y). In some embodiments, the substitution mutation(s) at amino acid position 13 within FR1 may comprise, without limitation, any of Glnl3Asn (Q13N), Glnl3Ser (Q13S), Glnl3Thr (Q13T), Glnl3Ala (Q13A), Glnl3Asp (Q13D), Glnl3Glu (Q13E), Glnl3lle (Q13I), Glnl3Lys (Q13K), Glnl3Leu (Q13L), Glnl3Arg (Q13R), Glnl3Val (Q13V), or Glnl3Tyr (Q13Y).

[0157] In some embodiments, a single-domain antibody may comprise an amino acid at position 11 (numbering according to Chothia) which is mutated to Ser (S) or Vai (V).

[0158] In some embodiments, a single-domain antibody may comprise an amino acid at position 11 (numbering according to Chothia) which is mutated to Asp (D), Glu (E), Lys (K), Asn (N), Arg (R), Ser (S), Thr (T), or Vai (V). In some embodiments, mutations at amino acid position 11 which may be present within the single-domain antibodies of the present disclosure may include any of those described in US 10,526,397, US 11,306,139, US 2022 / 0324951, US 2022 / 0119497, US 11,220,539, US 11,312,765, US 11,485,777, US 10,858,418, US 11,192,937, EP 3143042, US 2017 / 0121399, US 2022 / 0332807, and EP 3693386, each of which is incorporated by reference in its entirety.

[0159] In some embodiments, a single-domain antibody may comprise an amino acid at position 13 (numbering according to Chothia) which is mutated to Asn (N), Ser (S), or Thr (T). In some embodiments, mutations at amino acid position 13 which may be present within the single-domain antibodies of the present disclosure may include any of those described in US 11,306,139 and US 2022 / 0324951, each of which is incorporated by reference in its entirety.

[0160] In some embodiments, a single-domain antibody described herein may be modified to comprise Vai (V) at position 11 and Leu (L) at position 89. In some embodiments, when a single-domain antibody may comprise Vai (V) at position 11 and Leu (L) at position 89, the amino acid at position 110 can be, e.g., Thr (T), He (I), Ala (A), Lys (K) or Gin (Q); and / or (ii) the amino acid residue at position 112 can be, e.g., Ser (S), Phe (F), Lys (K) or Gin (Q).

[0161] In some embodiments, a single-domain antibody described herein may be modified to comprise one or more substitution mutation(s) within FR3. In some embodiments, the substitution mutation may comprise a mutation at any of amino acid position 87, 88, or 89, or combination thereof, within FR3. In some embodiments, the substitution mutation at amino acid position 87 may comprise, without limitation, any of Thr87Asn (T87N), Thr87Ser (T87S), Thr87Ala (T87A), Thr87Asp (T87D), Thr87Glu (T87E), Thr87lle (T87I), Thr87Lys (T87K), Thr87Leu (T87L), Thr87Gln (T87Q), Thr87Arg (T87R), Thr87Val (T87V), or Thr87Tyr (T87Y). In some embodiments, the substitution mutation at amino acid position 87 may comprise Thr87Ala (T87A), Thr87Ser (T87S) or Thr87Val (T87V). In some embodiments, the substitution mutation at amino acid position 88 may comprise, without limitation, any of Gly88Asp (G88D), Gly88Glu (G88E), Gly88Lys (G88K), Gly88Arg (G88R), Gly88Ala (G88A), Gly88lle (G88I), Gly88Leu (G88L), Gly88Asn (G88N), Gly88Gln (G88Q), Gly88Ser (G88S), Gly88Thr (G88T), Gly88Val (G88V), or Gly88Tyr (G88Y). In some embodiments, the substitution mutation at amino acid position 89 may comprise, without limitation, any of Val89Leu (V89L), Val89Asn (V89N), Val89Ser (V89S), Val89Thr (V89T), Val89Ala (V89A), Val89Asp (V89D), Val89Glu (V89E), Val89Lys (V89K), Val89Gln (V89Q), Val89Arg (V89R), Val89Tyr (V89Y), He89Leu (I89L), lle89Asn (I89N), He89Ser (I89S), He89Thr (I89T), He89Ala (I89A), He89Asp (I89D), He89Glu (I89E), He89Lys (I89K), He89Gln (I89Q), lle89Arg (I89R), or He89Tyr (I89Y).

[0162] In some embodiments, a single-domain antibody may comprise an amino acid at position 87 (numbering according to Chothia) which is mutated to Asn (N) or Ser (S). In some embodiments, mutations at amino acid position 87 which may be present within the single-domain antibodies of the present disclosure may include any of those described in US 11,306,139 and US 2022 / 0324951, each of which are incorporated by reference in its entirety.

[0163] In some embodiments, a single-domain antibody may comprise an amino acid at position 88 (numbering according to Chothia) which is mutated to Asp (D), Glu (E), Lys (K), or Arg (R). In some embodiments, mutations at amino acid position 88 which may be present within the single-domain antibodies of the present disclosure may include any of those described in US 2020 / 0140525, which is incorporated by reference in its entirety.

[0164] In some embodiments, a single-domain antibody may comprise an amino acid at position 88 (numbering according to Chothia) which is mutated to Lys (L), Asn (N), Ser (S), or Thr (T). In some embodiments, mutations at amino acid position 88 which may be present within the single-domain antibodies of the present disclosure may include any of those described in US 11,220,539, US 11,306,139, US 11,485,777, US 2022 / 0324951, US 11,312,765, US 2017 / 0121399, US 10,030,068, US 2022 / 0332807, US 11,014,977, EP 3143042, EP 3066120, EP 3693386, US 2021 / 0347855, and EP 3447068, each of which are incorporated by reference in its entirety.

[0165] In some embodiments, a single-domain antibody described herein may be modified to comprise one or more substitution mutation(s) within FR4. In some embodiments, the substitution mutation may comprise, for example, a mutation at amino acid position 108 within FR4. In some embodiments, the substitution mutation at amino acid position 108 may comprise, without limitation, any of LeulO8Asn (L108N), LeulO8Ser (L108S), LeulO8Thr (L108T), Leul08Ala (L108A), LeulO8Asp (L108D), LeulO8Glu (L108E), Leul08lle (L108I), LeulO8Lys (L108K), LeulO8Arg (L108R), Leul08Val (L108V), LeulO8Tyr (L108Y), GlnlO8Asn (Q108N), GlnlO8Ser (Q108S), GlnlO8Thr (Q108T), GlnlO8Ala (Q108A), GlnlO8Asp (Q108D), Glnl08Glu (Q108E), Glnl08lle (Q.108I), GlnlO8Lys (Q108K), GlnlO8Arg (Q108R), GlnlO8Val (Q108V), or GlnlO8Tyr (Q108Y).

[0166] In some embodiments, the modified single-domain antibody comprises Gin (Q) or Leu (L) at position 108 (numbering according to Chothia).

[0167] In some embodiments, a single-domain antibody may comprise an amino acid at position 108 (numbering according to Chothia) which is mutated to Asn (N), Ser (5), or Thr (T). In some embodiments, mutations at amino acid position 88 which may be present within the single-domain antibodies of the present disclosure may include any of those described in US 11,306,139, EP 3271391, and US 2022 / 0324951, each of which are incorporated by reference in its entirety.

[0168] In some embodiments, a single-domain antibody described herein may comprise a modification at amino acid position 110 (numbering according to Chothia). In some embodiments, the amino acid at position 110 is Lys (K) or Gin (Q). In some embodiments, when the amino acid at position 110 is Lys (K) or Gin (Q), the amino acid at position 11 can be Vai (V) or the amino acid at Kabat position 89 can be Leu (L), or a combination thereof. In some embodiments of any of the above-described modifications, the amino acid at position 108 can be Gin (Q) or Leu (L). In some embodiments, when the amino acid at position 110 is Lys (K) or Gin (Q), the amino acid at Chothia position 11 may be Vai (V) and / or the amino acid at position 89 may be Leu (L).

[0169] In some embodiments, a single-domain antibody described herein may comprise an amino acid at position 112 (numbering according to Chothia) which is Lys (K) or Gin (Q). In some embodiments, when a single-domain antibody comprises an amino acid at position 112 which is Lys (K) or Gin (Q), the amino acid at position 11 may be, e.g., Leu ( L), Glu (E), Lys (K), Vai (V), or Tyr (Y). In some embodiments, when a single-domain antibody comprises an amino acid at position 112 which is Lys (K) or Gin (Q), the amino acid at position 13 may be, e.g., Ser (S), Thr (T), Ala (A), Leu (L), Pro (P), Phe ( F), Glu (E), or Vai (V).

[0170] In some embodiments, a single-domain antibody described herein may be modified to comprise any number of various modifications, e.g., mutations including substitution mutations, as described in US 2022 / 0332807, US 2022 / 0332806, US 2022 / 0324951, US 2022 / 0119497, US 2021 / 0403536, US 2021 / 0388062 , US 2021 / 0347855, US 2021 / 0341490, US 2020 / 0325221, US 2020 / 0140525, US 2018 / 0355031, US 2018 / 0009888, US 2017 / 0121399, US 2015 / 0050266, US 9,150,640, US 11,485,777, US 11,426,468, US 11,312,765, US 11,312,764, US 11,306,139, US 11,220,539, US 11,192,938, US 11,192,937, US 11,021,544, US 11,014,977, US 11,009,511, US 10,858,418, US 10,526,397 , US 10,526,397, US 10,030,068, EP 3693386, EP 3447068, EP 3339322, EP 3271391, EP 3143042, EP 3066120, and EP 2987806, each of which is incorporated by reference in its entirety.

[0171] In some embodiments, a single-domain antibody described herein may comprise any mutation within any of FR1-4 described herein in combination with any other mutation within any of FR1-4 described herein. In some embodiments, any amino acid mutation(s) within FR1 may be combined with any other amino acid mutation(s) within FR1. In some embodiments, any amino acid mutation(s) within FR2 may be combined with any other amino acid mutation(s) within FR2. In some embodiments, any amino acid mutation(s) within FR3 may be combined with any other amino acid mutation(s) within FR3. In some embodiments, any amino acid mutation(s) within FR4 may be combined with any other amino acid mutation(s) within FR4. In some embodiments, any amino acid mutation(s) within FR1 described herein may be combined with any amino mutation(s) within FR2, FR3, and / or FR4, or combination thereof, described herein. In some embodiments, amino acid mutation(s) within FR2 described herein may be combined with any amino mutation(s) within FR1, FR3, and / or FR4, or combination thereof, described herein. In some embodiments, any amino acid mutation(s) within FR3 described herein may be combined with any amino mutation(s) within FR1, FR2, and / or FR4, or combination thereof, described herein. In some embodiments, any amino acid mutation(s) within FR4 described herein may be combined with any amino mutation(s) within FR1, FR2, and / or FR3, or combination thereof, described herein.

[0172] It is also contemplated within the present disclosure that any combination of any amino acid mutation(s) within FR1, FR2, FR3, and / or FR4 described herein, or combination thereof, may be paired with (i.e., combined with) one or more of any C-terminal modifications described herein, or combination thereof. As a non-limiting example, any of the amino acid mutations set forth in Table 1-2 may be combined with any of the C-terminal modifications set forth in Table 1-1.

[0173] In some embodiments, any of the amino acid mutations selected from (a) LeullAsp (L11D), LeullGlu (L11E), LeullLys (L11K), LeullAsn (L11N), LeullArg (L11R), LeullSer (L11S), LeullThr (L11T), LeullVal (L11V), LeullAla (L11A), Leulllle (Llll), LeullGIn (L11Q), or LeullTyr (L11Y); (b) Glnl3Asn (Q13N), Glnl3Ser (Q13S), Glnl3Thr (Q13T), Glnl3Ala (Q13A), Glnl3Asp (Q13D), Glnl3Glu (Q13E), Gin 13lle (Q.13I), Glnl3Lys (Q13K), Glnl3Leu (Q13L), Glnl3Arg (Q13R), Glnl3Val (Q13V), or Glnl3Tyr (Q13Y); (c) Thr87Asn (T87N), Thr87Ser (T87S), Thr87Ala (T87A), Thr87Asp (T87D), Thr87Glu (T87E), Thr87lle (T87I), Thr87Lys (T87K), Thr87Leu (T87L), Thr87Gln (T87Q), Thr87Arg (T87R), Thr87Val (T87V), Thr87Tyr (T87Y); (d) Gly88Asp (G88D), Gly88Glu (G88E), Gly88Lys (G88K), Gly88Arg (G88R), Gly88Ala (G88A), Gly88lle (G88I), Gly88Leu (G88L), Gly88Asn (G88N), Gly88Gln (G88Q), Gly88Ser (G88S), Gly88Thr (G88T), Gly88Val (G88V), Gly88Tyr (G88Y); (e) Val89Leu (V89L), Val89Asn (V89N), Val89Ser (V89S), Val89Thr (V89T), Val89Ala (V89A), Val89Asp (V89D), Val89Glu (V89E), Val89Lys (V89K), Val89Gln (V89Q), Val89Arg (V89R), Val89Tyr (V89Y), He89Leu (I89L), lle89Asn (I89N), He89Ser (I89S), He89Thr (I89T), He89Ala (I89A), lle89Asp (I89D), He89Glu (I89E), He89Lys (I89K), He89Gln (I89Q), He89Arg (I89R), or He89Tyr (I89Y); or (f) Leul08Asn (L108N), LeulO8Ser (L108S), LeulO8Thr (L108T), LeulO8Ala (L108A), LeulO8Asp (L108D), LeulO8Glu (L108E), Leul08lle (L108I), LeulO8Lys (L108K), LeulO8Arg (L108R), Leul08Val (L108V), LeulO8Tyr (L108Y), Glnl08Asn (Q108N), GlnlO8Ser (Q108S), GlnlO8Thr (Q108T), Glnl08Ala (Q108A), GlnlO8Asp (Q108D), Glnl08Glu (Q108E), Glnl08lle (Q.108I), GlnlO8Lys (Q108K), GlnlO8Arg (Q108R), GlnlO8Val (Q108V), or GlnlO8Tyr (Q108Y), or a combination thereof, may be combined with any of the C-terminal sequences (starting from position 111 according to Chothia) selected from VSS (wild-type), VADG (SEQ ID NO: 1), VADP (SEQ ID NO: 2), VAEG (SEQ ID NO: 3), VAEP (SEQ ID NO: 4), VAGG (SEQ ID NO: 5), VAGP (SEQ ID NO: 6), VAKG (SEQ ID NO: 7), VAKP (SEQ ID NO: 8), VANG (SEQ ID NO: 9), VANP (SEQ ID NO: 10), VAPG (SEQ ID NO: 11), VAPP (SEQ ID NO: 12), VAQG (SEQ ID NO: 13), VAQP (SEQ ID NO: 14), VARG (SEQ ID NO: 15), VARP (SEQ ID NO: 16), VASG (SEQ ID NO: 17), VASP (SEQ ID NO: 18), VATG (SEQ ID NO: 19), VATP (SEQ ID NO: 20), VDAG (SEQ ID NO: 21), VDAP (SEQ ID NO: 22), VDGG (SEQ ID NO: 23), VDGP (SEQ ID NO: 24), VDPG (SEQ ID NO: 25), VDPP (SEQ ID NO: 26), VDSG (SEQ ID NO: 27), VDSP (SEQ ID NO: 28), VDTG (SEQ ID NO: 29), VDTP (SEQ ID NO: 30), VEAG (SEQ ID NO: 31), VEAP (SEQ ID NO: 32), VEGG (SEQ ID NO: 33), VEGP (SEQ ID NO: 34), VEPG (SEQ ID NO: 35),VEPP (SEQ ID NO: 36), VESG (SEQ ID NO: 37), VESP (SEQ ID NO: 38), VETG (SEQ ID NO: 39), VETP (SEQ ID NO: 40), VGAG (SEQ ID NO: 41), VGAP (SEQ ID NO: 42), VGDG (SEQ ID NO: 43), VGDP (SEQ ID NO: 44), VGEG (SEQ ID NO: 45), VGEP (SEQ ID NO: 46), VGGG (SEQ ID NO: 47), VGGP (SEQ ID NO: 48), VGIG (SEQ ID NO: 49), VGIP (SEQ ID NO: 50), VGKG (SEQ ID NO: 51), VGKP (SEQ ID NO: 52), VGLG (SEQ ID NO: 53), VGLP (SEQ ID NO: 54), VGNG (SEQ ID NO: 55), VGNP (SEQ ID NO: 56), VGPG (SEQ ID NO: 57), VGPP (SEQ ID NO: 58), VGQG (SEQ ID NO: 59), VGQP (SEQ ID NO: 60), VGRG (SEQ ID NO: 61), VGRP (SEQ ID NO: 62), VGSG (SEQ ID NO: 63), VGSP (SEQ ID NO: 64), VGTG (SEQ ID NO: 65), VGTP (SEQ ID NO: 66), VGVG (SEQ ID NO: 67), VGVP (SEQ ID NO: 68), VIGG (SEQ ID NO: 69), VIGP (SEQ ID NO: 70), VIPG (SEQ ID NO: 71), VIPP (SEQ ID NO: 72), VISG (SEQ ID NO: 73), VISP (SEQ ID NO: 74), VITG (SEQ ID NO: 75), VITP (SEQ ID NO: 76), VLGG (SEQ ID NO: 77), VLGP (SEQ ID NO: 78), VLPG (SEQ ID NO: 79), VLPP (SEQ ID NO: 80), VLSP (SEQ ID NO: 81), VLTG (SEQ ID NO: 82), VLTP (SEQ ID NO: 83), VNAG (SEQ ID NO: 84), VNAP (SEQ ID NO: 85), VNGG (SEQ ID NO: 86), VNGP (SEQ ID NO: 87), VNPG (SEQ ID NO: 88), VNPP (SEQ ID NO: 89), VNSG (SEQ ID NO: 90), VNSP (SEQ ID NO: 91), VNTG (SEQ ID NO: 92), VNTP (SEQ ID NO: 93), VPAG (SEQ ID NO: 94), VPAP (SEQ ID NO: 95), VPDG (SEQ ID NO: 96), VPDP (SEQ ID NO: 97), VPEG (SEQ ID NO: 98), VPEP (SEQ ID NO: 99), VPGG (SEQ ID NO: 100), VPGP (SEQ ID NO: 101), VPIG (SEQ ID NO: 102), VPIP (SEQ ID NO: 103), VPKG (SEQ ID NO: 104), VPKP (SEQ ID NO: 105), VPLG (SEQ ID NO: 106), VPLP (SEQ ID NO: 107), VPNG (SEQ ID NO: 108), VPNP (SEQ ID NO: 109), VPPG (SEQ ID NO: 110), VPPP (SEQ ID NO: 111), VPQG (SEQ ID NO: 112), VPQP (SEQ ID NO: 113), VPRG (SEQ ID NO: 114), VPRP (SEQ ID NO: 115), VPSG (SEQ ID NO: 116), VPSP (SEQ ID NO: 117), VPTG (SEQ ID NO: 118), VPTP (SEQ ID NO: 119), VPVG (SEQ ID NO: 120), VPVP (SEQ ID NO: 121), VRAG (SEQ ID NO: 122), VRAP (SEQ ID NO: 123), VRGG (SEQ ID NO: 124), VRGP (SEQ ID NO: 125), VRPG (SEQ ID NO: 126), VRPP (SEQ ID NO: 127), VRSG (SEQ ID NO: 128), VRSP (SEQ ID NO: 129), VRTG (SEQ ID NO: 130), VRTP (SEQ ID NO: 131), VSAP (SEQ ID NO: 132), VSDG (SEQ ID NO: 133), VSDP (SEQ ID NO: 134), VSEG (SEQ ID NO: 135), VSEP (SEQ ID NO: 136), VSGG (SEQ ID NO: 137), VSGP (SEQ ID NO: 138), VSIG (SEQ ID NO: 139), VSIP (SEQ ID NO: 140), VSKG (SEQ ID NO: 141), VSKP (SEQ ID NO: 142), VSLG (SEQ ID NO: 143), VSLP (SEQ ID NO: 144), VSNG (SEQ ID NO: 145), VSNP (SEQ ID NO: 146), VSPG (SEQ ID NO: 147), VSPP (SEQ ID NO: 148), VSQG (SEQ ID NO: 149), VSQP (SEQ ID NO: 150), VSRG (SEQ ID NO: 151), VSRP (SEQ ID NO: 152), VSTG (SEQ ID NO: 153), VSTP (SEQ ID NO: 154), VSVP (SEQ ID NO: 155), VTAG (SEQ ID NO: 156), VTAP (SEQ ID NO: 157), VTDG (SEQ ID NO: 158), VTDP (SEQ ID NO: 159), VTEG (SEQ ID NO: 160), VTEP (SEQ ID NO: 161), VTGG (SEQ ID NO: 162), VTGP (SEQ ID NO: 163), VTIG (SEQ ID NO: 164), VTIP (SEQ ID NO: 165), VTKG (SEQ ID NO: 166), VTKP (SEQ ID NO: 167), VTLG (SEQ ID NO: 168), VTLP (SEQ ID NO: 169), VTNG (SEQ ID NO: 170), VTNP (SEQ ID NO: 171), VTPG (SEQ ID NO: 172), VTPP (SEQ ID NO: 173), VTQG (SEQ ID NO: 174), VTQP (SEQ ID NO: 175),VTRG (SEQ ID NO: 176), VTRP (SEQ ID NO: 177), VTSG (SEQ ID NO: 178), VTSP (SEQ ID NO: 179), VTTG (SEQ ID NO: 180), VTTP (SEQ ID NO: 181), VTVG (SEQ ID NO: 182), VTVP (SEQ ID NO: 183), VVGG (SEQ ID NO: 184), WGP (SEQ ID NO: 185), WPG (SEQ ID NO: 186), WPP (SEQ ID NO: 187), WSG (SEQ ID NO: 188), WSP (SEQ ID NO: 189), WTG (SEQ ID NO: 190), WTP (SEQ ID NO: 191), VLSG (SEQ ID NO: 283), VSAG (SEQ ID NO: 284), VSVG (SEQ ID NO: 285), PP - , V - - , VA - , VD - , VE - , VG - , VI -, VK - , VL - , VN - , VP - , VR - , VS - , VT - , W - , VAA, VAD, VAE, VAG, VAI, VAK, VAL, VAN, VAP, VAQ, VAR, VAS, VAT, VAV, VDA, VDD, VDE, VDG, VDI, VDK, VDL, VDN, VDP, VDQ, VDR, VDS, VDT, VDV, VED, VEE, VEG, VEI, VEK, VEL, VEN, VEP, VEQ, VER, VES, VEV, VGA, VGD, VGE, VGG, VGI, VGK, VGL, VGN, VGP, VGQ, VGR, VGS, VGT, VGV, VIA, VID, VIE, VIG, VII, VIK, VIL, VIN, VIP, VIQ, VIR, VIS, VIT, VIV, VLA, VLD, VLE, VLG, VLI, VLK, VLL, VLN, VLP, VLQ, VLR, VLS, VLT, VLV, VNA, VNE, VNG, VNI, VNK, VNL, VNN, VNP, VNQ, VNR, VNS, VNT, VNV, VPA, VPD, VPE, VPG, VPI, VPK, VPL, VPN, VPP, VPQ, VPR, VPS, VPT, VPV, VRA, VRD, VRE, VRG, VRI, VRK, VRL, VRN, VRP, VRQ, VRR, VRS, VRT, VRV, VSA, VSD, VSE, VSG, VSI, VSK, VSL, VSN, VSP, VSQ, VSR, VST, VSV, VTA, VTD, VTE, VTG, VTI, VTK, VTL, VTN, VTP, VTQ, VTR, VTS, VTT, VTV, WA, WD, WE, WG, Wl, WK, WL, WN, WP, WQ, WR, WS, WT, VW, VEA, VET, or VND, or a combination thereof.

[0174] It is also contemplated within the present disclosure that any combination of any amino acid mutation(s) within FR1, FR2, FR3, and / or or FR4 described herein, or combination thereof, may be paired with (i.e., combined with) one or more of any C-terminal modification(s) described herein, or combination thereof, and may be further, optionally, combined with one or more of any modifications (e.g., mutations such as but not limited to those set forth in Table 1-4) to any amino acid position(s) within any of CDR1, CDR2 and / or or CDR3 described herein, or a combination thereof.

[0175] In some embodiments, the amino acid at one or more of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 112, 113 or 114 (numbering according to Chothia), or any combination thereof, of a single-domain antibody described herein may comprise a glycosylation site which is or can be glycosylated. For example, without limitation, the amino acid at one or more of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 112, 113 or 114, or any combination thereof, may be Asp (D) which may comprise a glycosylation site which is or can be N-glycosylated. In some embodiments, position 11 may be Vai (V) or Lys (K) and the amino acid residue at one or more of positions 10, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 110, 112, 113 or 114, or any combination thereof, may comprise a glycosylation site which is or can be glycosylated. In some embodiments, position 89 may be Thr (T) or Leu (L) and the amino acid residue at one or more of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 108, 110, 112, 113 or 114, or any combination thereof, may comprise a glycosylation site which is or can be glycosylated.

[0176] A single-domain antibody (e.g., VHH) can be obtained by immunization of dromedaries, camels, llamas, alpacas, or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens. By reverse transcription and polymerase chain reaction (PCR), a gene library of single-domain antibodies containing several million clones can be produced. Screening techniques such as phage display, yeast display, and ribosome display help to identify the clones binding the antigen. Methods generation of heavy-chain antibody fragments are described in e.g., WO 94 / 04678; Hamers-Casterman et al. 1993; Muyldermans et al. 2001; and Arbabi Ghahroudi, M. et al. (1997). FEBS Letters 414 (3): 521-526, each of which is incorporated herein by reference in its entirety.

[0177] A different method may use gene libraries from animals that have not been previously immunized. Such naive libraries usually contain only antibodies with low affinity to the desired antigen, making it necessary to apply affinity maturation by random mutagenesis as an additional step. See e.g., Saerens, D.; et al. (2008). "Single-domain antibodies as building blocks for novel therapeutics". Current Opinion in Pharmacology 8 (5): 600-608.

[0178] Affinity maturation strategies can be categorized as either targeted / rational approaches or untargeted / random approaches. For targeted approaches information about the VHH of interest is needed, such as hot spots for affinity maturation or structural information on the VHH:antigen complex, whereas for untargeted approaches no prior information is needed. Targeted approaches that may be applied for affinity maturation of VHHs include site-directed in-vitro mutagenesis and in- s / 7 / co / computational approaches. Common untargeted approaches used for affinity maturation of VHHs include random in-vitro mutagenesis, CDR swapping and autonomous hypermutation yeast surface display, with the latter two being novel, emerging and very time efficient techniques. Most of these strategies have in common, that after applying a certain randomization strategy to generate a mutational library, the resulting library can be screened by employing standard display techniques such as yeast, phage or ribosome display to select for the best binders. The choice of the display system is often guided by the library size to be displayed, with yeast display being able to handle library sizes of ~107- 109, phage display ~1O8-1O10and ribosome display ~1O12-1O13(Chan and Groves, 2021). Notably, during affinity maturation the number of highly interactive residues such as aromatic amino acids usually increase in the CDR regions. The selected affinity matured clones may be further evaluated by a developability assessment to test for undesired properties, such as unspecific binding to off-targets or VHH instability.

[0179] For targeted in vitro mutagenesis, a set of selected residues within the CDRs of a VHH may be mutated (Tiller et al., 2017; Yau et aL, 2005). Pre-selection of these residues can be either performed using alanine scanning to identify hot spot residues for mutation or by using structural data of the antigen:VHH complex to identify positions to be mutated. These sites can then be either submitted to saturating mutagenesis to substitute a specific site with all possible amino acids or specific amino acid substitutions yielding several smaller libraries. After mutagenesis binders can be displayed to select the best matured candidate. Usually, several rounds of targeted mutagenesis are performed with separate sub-libraries to obtain combinations of individual mutations that cooperatively result in increased binding affinity.

[0180] Computer-aided / / n silico methods are often used to guide targeted in vitro mutagenesis. Using homology modeling of the target:VHH complex or docking, hotspots for mutations can be identified that are then submitted to in vitro mutagenesis (Bert Schepens et al., 2021; Cheng et aL, 2019; Inoue et aL, 2013; Mahajan et aL, 2018). Further, in silico methods can search all designed variants in a virtual library (~1O40members) in a rather short amount of time to identify a feasible number of promising candidates to be tested experimentally. These techniques can be especially valuable if structural data on the drugtarget interaction are available.

[0181] Untargeted / random affinity maturation strategies that can be applied to affinity mature VHHs include random in vitro mutagenesis, CDR shuffling / swapping and in vivo affinity maturation via yeast display. For random in vitro mutagenesis the sequence of either the entire VHH or only the CDRs are mutated randomly (Chen et aL, 2021; Ye et aL, 2021; Zupancic et aL, 2021). The most commonly used technique is error prone PCR employing a DNA polymerase that lacks proof reading activity and PCR conditions that increase the polymerase error rate even further. This technique can be applied without further structural knowledge or information on the importance of residues that contribute to antigen:VHH interaction. The resulting mutational library can then be displayed to select the best matured candidate. This technique may also be combined with NGS sequencing of the display elutions to get an in-depth readout of all obtained candidates, enabling the identification of low abundant but still promising clones (Chen et aL, 2021).

[0182] In some embodiments, CDR shuffling or swapping is applied for VHH affinity maturation, such as described in Zupancic et aL, 2021. For CDR swapping, enriched libraries can be used as input material for a PCR reaction to individually amplify the CDR of the VHHs. The PCR products can then be mixed and reassembled using overlapping PCR to generate the entire plasmid for further rounds of display to selectfor the best matured binder. One limitation of this approach is that it can only be used for VHHs comprising the same framework as it is the case for synthetic libraries.

[0183] In some embodiments, in vivo affinity maturation via yeast display is applied for VHH affinity maturation, such as described in Wellner et al., 2021. The method is based on an autonomous hypermutation yeast surface display (AHEAD), which imitates somatic hypermutation during VHH selection using engineered yeast strains. The yeast's error prone orthogonal DNA replication system can generate new variants during plasmid replication by randomly introducing mutations. The new variants can then be displayed and selected using yeast surface display to identify the best binders. This enables the production of high affinity clones in very little time (about 2 weeks), which is significantly faster than classical affinity maturation procedures. The method can be applied using synthetic or immune libraries using unenriched libraries enriched libraries or a subset of preselected clones.

[0184] In case binders with medium affinity are required, as it is the case for the V-bodies and the affinity of the identified candidates need to be decreased, very similar techniques can be applied. For example, mutations that are aiming at lowering the affinity can be introduced using the same targeted or untargeted approaches as described for the affinity maturation. The selection afterwards can be adapted accordingly. If larger libraries are generated that need to be screened via a display technique, the selection strategy can be adapted to enrich medium affinity binders while excluding high affinity candidates. This could, for example be a pre-panning in phage display with low antigen concentration to remove all higher affinity candidates, followed by a selection with high antigen concentration to obtain medium affinity VHHs. For library sizes of up to 1000 candidates a kinetic off-rate characterization can be used to get immediate information about the kinetic behavior of the candidates.

[0185] When the most potent clones have been identified, their DNA sequence can be optimized, for example to improve their stability towards enzymes. Another goal is humanization to prevent immunological reactions of the human organism against the antibody. Humanization can be achieved based on the homology between camelid VHH and human VH fragments, which is described in further detail below. Finally, the optimized single-domain antibody can be translated and expressed in suitable organisms such as E. coli or Saccharomyces cerevisiae.

[0186] Single-domain antibodies can also be derived from conventional antibodies. In some embodiments, single-domain antibodies can be made from conventional murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naive donors and display techniques for identification of the most specific antigens. However, the binding region of a conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because oftheir lipophilicity. Monomerization can be accomplished by replacing lipophilic by hydrophilic amino acids. (See e.g., Borrebaeck, C. A. K.; Ohlin, M. (2002). "Antibody evolution beyond Nature". Nature Biotechnology 20 (12): 1189-90.) If affinity can be retained after monomerization, the single-domain antibodies can likewise be produced in E. coli, S. cerevisiae or other suitable organisms.

[0187] A "humanized antibody" refers to a chimeric, genetically engineered, antibody in which the amino acid sequences (typically CDRs) from an antibody (donor antibody), e.g., a camelid antibody, are grafted onto a human antibody (acceptor antibody). Thus, a humanized antibody typically comprises CDRs from a donor antibody and variable region framework and constant regions, when present, from a human antibody. Accordingly, a "humanized VHH" comprises CDRs that corresponds to the CDRs of a naturally occurring VHH domain (e.g., a camelid VHH), but that has been "humanized". Humanized VHH may be prepared by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (particularly in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4- chain human antibody. Such humanized VHHs can be obtained in any suitable manner known to a skilled person in the art and thus not strictly limited to methods described herein.

[0188] Humanization of VHHs can achieved using resurfacing or CDR grafting. Resurfacing strategies have been described in e.g., Conrath et al., 2005 J Mol Biol; Kazemi-Lomedasht et aL, 2018; Vincke et al., 2009 J Biol Chem, and CDR grafting strategies have been described in e.g., ben Abderrazek et aL, 2011; van Faassen et aL, 2020 FASEB; Li et aL, 2018; Vaneycken et aL, 2010; Vincke et aL, 2009 J Biol Chem; and Yu et aL, 2017, each of which is incorporated herein by reference in its entirety.

[0189] To humanize a camelid VHH using a resurfacing approach, a human germline reference that is most similar to the camelid germline sequence of the selected VHH may be identified. Most of the isolated camelid VHHs in literature belong to the camelid IGHV3 subfamily 2 (Nguyen et aL, 2000, EMBO J) with DP-47 / VH3-23 from the IGHV3 family commonly used as human reference. The framework of the camelid VHH can then be compared to the human reference sequence. Surface exposed residues are substituted to their human counterpart as it is assumed that their contribution to protein stability is rather low. Buried residues however remain of camelid origin, as they likely contribute to the overall VHH stability. Humanization of framework regions 1, 3 and 4 usually does not impact the physicochemical properties of the VHHs, whereas a general humanization of framework 2 would significantly increase local hydrophobicity. Residues H37, H44, H45 and H47 (Chothia numbering) in framework 2, the so called tetrade or hallmark residues, have a rather hydrophobic nature in human VHs (VGLW) as they are partially buried and involved in VH / VL paring, while in camelid VHHs theseresidues are partially charged (FERG), which significantly increases VHH solubility and inhibits paring of camelid VL (Soler et al., 2021, Biomolecules, Conrath et al., 2005 J Mol BiolJ. Further, residues H37 and H47 are known to interact with the CDR-H3 loop in many VHHs, stabilizing its conformation and thereby contributing to antigen binding affinity. In addition, a significant number of VHHs use framework 2 residues H44, H45 and H47 for antigen binding (Zavrtanik et aL, 2018, J Mol Biol). A full humanization of these residues hence frequently results in reduced solubility or aggregation of the VHHs and a reduced or complete loss of binding affinity for the target antigen (van Faassen et al., 2020, Vincke et al., 2009). In consequence, all or at least some of these hallmark residues in framework 2 remain of camelid origin when humanizing VHHs.

[0190] Another approach that may be applied to humanize VHHs is CDR grafting. CDRs of the selected VHHs can be transplanted onto a universal VHH framework that has been partially or fully humanized (Saerens et al., 2009 J Biol Chem, Soler et al., 2021, Vincke et al., 2009 J Biol Chem). CDR grafting has been successfully used in some cases but failed for several others, with VHHs frequently losing their potential to bind to the desired antigen and / or becoming structurally instable with a high tendency to aggregate (van Faassen et al., 2020, FASEB). This is mostly attributed to interactions of CDR3 with specific residues in framework 2 that are important for CDR3 conformation, general VHH stability and overall hydrophobicity, which are impaired by this approach. Sometimes camelid backmutations are introduced into the framework to compensate for these effects (van Faassen et al., 2020, FASEB).

[0191] An alternative strategy to mitigate the need of humanizing the selected VHH sequences is to use fully or partially humanized synthetic VHH libraries instead of camelid immune libraries for VHH discovery (Moutel et al. 2016, eLife; McMahon, 2018, NSMB; Zimmermann et aL, 2018, eLife). In many of these libraries the hallmark residues are still of camelid origin for reasons discussed above.

[0192] Other suitable humanizing substitutions are described in WO 09 / 138519 and WO 08 / 020079, as well as Tables A-3 to A-8 from WO 08 / 020079 (which are lists showing possible humanizing substitutions), each of which is incorporated herein by reference in its entirety. Non-limiting examples of such humanizing substitutions include Q108L and A14P. Such humanizing substitutions may also be suitably combined with one or more other mutations as described herein (such as with one or more mutations that reduce binding by pre-existing antibodies).

[0193] In some embodiments, humanized VHH sequences still retain the residues that are relevant for protein A binding. In some embodiments, the engineering activities during humanization may be applied to engineer protein A binding properties into a VHH that did previously not interact with protein A (Graille et aL, 2000, PNAS).

[0194] Like a "humanized antibody", a "camelized antibody" refers to an antibody having amino acid sequences (typically CDRs) from a donor antibody, e.g., a human antibody, and variable region framework and constant regions, when present, from a camelid antibody. Accordingly, a "camelized VH" comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been "camelized". Camelized VH may be prepared by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner, for example as described in WO 2008 / 020079. Such "camelizing" substitutions are usually inserted at amino acid positions that form and / or are present at the VH— VL interface, and / or at the so-called Camelidae hallmark residues, e.g., F37, E44, R45 and F47 (see for example WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). In one embodiment, the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, or the VH sequence of a human antibody. However, such camelized VH can be obtained in any suitable manner known to a skilled person in the art and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.

[0195] The amino acid residues of a single-domain antibody can be numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), as applied to VHH domains from Camelids described in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for example FIG. 2 of this publication). The total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. For example, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. As a result, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain is usually in the range of from 110 to 120, often between 112 and 115. However, smaller and longer sequences may also be suitable for the purposes described herein.

[0196] Determination of CDR regions in a single-domain antibody may be accomplished using different methods, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numberingscheme); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et aL, J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745." ("Contact" numbering scheme); Lefranc M P et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 January; 27(l):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun. 8; 309(3):657-70, ("Aho" numbering scheme); and Martin et aL, "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272, ("AbM" numbering scheme), each reference cited herein is incorporated by reference in its entirety.

[0197] The boundaries of a given CDR or framework (FR) may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a," and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular's AbM antibody modeling software.

[0198] In some embodiments, the amino acid residues of a single domain antibody (e.g., VHH) described herein are numbered according to the Chothia scheme (see Figure 2).

[0199] In some embodiments, CDRs can be defined in accordance with any of the Kabat numbering scheme, the Chothia numbering scheme, a combination of Kabat and Chothia, the AbM numbering scheme, and / or the Contact numbering scheme. A VHH typically comprises three CDRs, designated CDR1, CDR2, and CDR3. Table 1-3, below, lists exemplary position boundaries of CDR-H1, CDR-H2, CDR- H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-H1 located before CDR-H1, FR-H2 located between CDR-H1 and CDR-H2, FR- H3 located between CDR-H2 and CDR-H3 and FR-H4 is located after CDR-H3. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.Table 1-3. CDRs definitions according to various numbering schemes.2Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD;2AI-Lazikani et al., (1997) JMB 273, 927-948

[0200] Thus, unless otherwise specified, a "CDR" or "complementary determining region," or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) CDR as defined by any of the above-mentioned schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VHH amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the VHH, as defined by any of the above-mentioned schemes. In some embodiments, specific CDR sequences are specified. CDR sequences of may be described using various numbering schemes (see e.g., Table 1-3), although it is understood that an antibody can include CDRs as described according to any of the other above-mentioned numbering schemes or other numbering schemes known to a person of ordinary skill in the art.

[0201] In a single-domain antibody sequence of the present disclosure, the framework sequences may be any suitable framework sequences. For example, the framework sequences may be framework sequences derived from a heavy chain variable domain (e.g., a VH sequence or VHH sequence). In some embodiments, the framework sequences are either framework sequences that have been derived from a VHH sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences (in which said framework sequences may optionally have been partially or fully camelized).

[0202] Antigen-binding fragments (or combinations of fragments) of any of single-domain antibodies described herein, such as fragments that contain one or more CDR sequences, suitably flanked byand / or linked via one or more framework sequences, are also encompassed within the present disclosure.

[0203] It should be noted, however, that the present disclosure is not limited to the origin of the singledomain antibody (or of the nucleotide sequence used to express it), nor to the way that the singledomain antibody or nucleotide sequence is generated or obtained. Thus, a single-domain antibody of the present disclosure may comprise naturally occurring sequences (from a suitable species), recombinant sequences, or synthetic or semi-synthetic sequences. Similarly, nucleotide sequences encoding single-domain antibodies of the present disclosure may comprise naturally occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (for example, sequences that are prepared by PCR or isolated from a library).

[0204] In some embodiments, a single-domain antibody described herein binds to a therapeutic target, e.g., an antigen. Binding affinity of a molecular interaction between two molecules can be measured via various techniques, such as surface plasmon resonance (SPR), bio-layer interferometry (BLI), enzyme- linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescent-activated cell sorting (FACS), or flow cytometry binding assays and the like. Surface plasmon resonance is a biosensor technique that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions (see e.g., Ober et al. 2001, Intern. Immunology 13: 1551-1559). SPR can for example be performed using the BIACORE® system or Carterra LSA system. Another biosensor technique that can be used to determine affinities of biomolecular interactions is bio-layer interferometry (BLI) (see e.g., Abdiche et aL 2008, Anal. Biochem. 377: 209-217). Bio-layer Interferometry is a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam). A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since the interactions can be measured in real-time, association and dissociation rates and affinities can be determined. BLI can for example be performed using the Octet® Systems. Alternatively, affinities can be measured in Kinetic Exclusion Assay (KinExA) (see e.g., Drake et al. 2004, Anal.Biochem., 328: 35-43), which is a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules. Equilibrated solutions of an antibody / antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen)to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).

[0205] Single-domain antibodies of the present disclosure may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domains as compared to the exemplary antibody sequences provided herein. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The antigen-binding molecules of the present disclosure may comprise antigen-binding domains which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as "germline mutations"). A person of ordinary skill in the art, starting with the heavy chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VHH domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived).

[0206] Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen-binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved or enhanced biological properties (e.g., agonisticeffect), reduced immunogenicity, etc. Single-domain antibodies comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.

[0207] Contemplated herein are single-domain antibodies comprising variants of any of the VHH and / or CDR amino acid sequences having one or more amino acid substitutions. For example, the present disclosure includes single-domain antibodies having VHH and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2, or 1 amino acid substitutions relative to any VHH and / or CDR amino acid sequences of an original germline sequence. Amino acid substitutions may be introduced into a single-domain antibody of interest and the resultant variants can be screened for a desired activity, for example, retained / improved antigen binding, decreased immunogenicity, or reduced ADCC or CDC.

[0208] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. In some embodiments, an amino acid substitution is a conservative substitution, meaning exchanging an amino acid with another amino acid of the same class. In some embodiments, amino acid substitutions may also include a non-conservative substitution, meaning exchanging an amino acid with an amino acid of a different class. Other exemplary amino acid substitutions are shown in Table 1-4.Table 1-4. Exemplary amino acid substitutions

[0209] In some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure are modified to enhance binding to staphylococcal protein A (SpA) or streptococcal protein G (SpG). Binding of SpA and SpG to antibodies or antibody fragments can be useful in the manufacturing process of the antibodies or antibody fragments. The high-affinity interaction of the IgG Fc region with SpA and SpG has been extensively exploited and became the gold standard for monoclonal antibody purification (Bjorck and Kronvall, 1984). Other non-Fc containing antibody fragments, such as VHHs and Fabs do not have the capacity to bind to SpA or SpG via their Fc regions. However, sequence-dependent interaction with SpA has been demonstrated for these non-Fc containing antibody fragments(Graille et aL, 2000; Henry et al., 2016). This characteristic circumvents potential use of affinity tags fused to the drug candidate for affinity chromatography that have the disadvantage as being regarded as a sequence liability, as it may impact protein immunogenicity as well as protein structure and stability and could compromise functionality. The interaction of the single-domain antibodies (e.g., VHH) to SpA relies on an alternative binding mode, with a 1-5 pM affinity, which is comparable to the 0.2 -3 pM measured for VH-SpA interactions (To et al., JBC, 2005; Henry et aL, Pios One, 2016).

[0210] In some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure have, or are modified to have a SpA-binding motif. For example, The VHH-SpA interface has been mapped to thirteen residues, which cluster within the framework at the back side of the V-body, distant to the CDRs (Grail le et al., 2000, Henry et al., 2016). In the absence of a VHH-SpA co-structure, superposition of a SpA-Fab crystal structure and a VHH allows for visualizing the binding mode. Based on a structural and functional analysis, the thirteen residues of the VHH-SpA interface have been characterized to be intolerant to substitutions (residues Glyl5, Argl9, Tyr59, Gly65, and Arg66), tolerant to specific substitutions (residues Thr / Lys / Arg57, Thr68, Gln81, Asn82a, and Ser82b) or generally tolerant to a variety of substitutions (residues Serl7, Lys64, and Ser70) (all residue positions refer to Kabat numbering) (Henry et aL, Pios One, 2016). Thus, a SpA-binding motif included in a single-domain antibody (e.g., VHH) of the present disclosure may include one or more, or all of the thirteen residues.Fusion Proteins and Conjugates

[0211] In one aspect, provided herein are fusion proteins and conjugates comprising one or more of the single-domain antibodies described herein. In some embodiments, at least one single-domainantibody is located at the carboxy-terminus of the fusion protein. In some embodiments, the one or more of the single-domain antibodies may be linked, directly or indirectly, to one or more additional domains or moieties. In some embodiments, the one or more of the single-domain antibodies may be conjugated to a second moiety. In some embodiments, the fusion protein or conjugate of the present disclosure comprises a single polypeptide. In other embodiments, the fusion protein or conjugate of the present disclosure comprises more than one polypeptide. In some embodiments, the fusion protein or conjugate of the present disclosure comprises two polypeptides.

[0212] In some embodiments, the fusion protein or conjugate of the present disclosure comprises at least one single-domain antibody described herein. In some embodiments, the fusion protein or conjugate is multivalent. For example, the fusion protein or conjugate of the present disclosure may be at least bivalent, but can also be e.g., trivalent, tetravalent, pentavalent, hexavalent, etc. The terms "bivalent", "trivalent", "tetravalent", "pentavalent", or "hexavalent" all fall under the term "multivalent" and indicate the presence of two, three, four, five or six binding units (e.g., VHHs), respectively.

[0213] In certain embodiments, the fusion protein or conjugate is multispecific. For example, in some cases, the one or more additional domain or moieties may be one or more additional binding domain that binds to one or more further antigen or protein. The fusion protein or conjugate of the present disclosure may be, for example, bispecific, trispecific, tetraspecific, pentaspecific, etc. The terms "bispecific", "trispecific", "tetraspecific", "pentaspecific", etc., all fall under the term "multispecific" and refer to binding to two, three, four, five, etc., different target molecules, respectively.

[0214] When two or more single-domain antibodies are included in a fusion protein or conjugate, the two or more single-domain antibodies may comprise the same sequence or may comprise different sequences. In such embodiments, the two or more single-domain antibodies may bind to the same epitope on a target antigen or different epitopes on a target antigen. For example, a fusion protein or conjugate of the present disclosure may be biparatopic, e.g., if two VHHs bind two different epitopes on a given target antigen.Fusion or Conjugation to Fc regions

[0215] In some embodiments, a fusion protein or conjugate of the present disclosure comprises at least one single-domain antibody provided herein operably linked to an immunoglobulin Fc region. An immunoglobulin Fc region may be linked indirectly or directly to the at least one single-domain antibody. In some embodiments, a fusion protein or conjugate of the present disclosure comprises one,two, three, four, five, six or more single-domain antibodies provided herein operably linked to an Fc region.

[0216] A "Fc region" as used herein refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, an Fc region comprises a hinge, CH2, and CH3. In various embodiments, when an Fc region comprises a hinge, the hinge can mediate dimerization between two Fc-containing polypeptides. In various embodiments, an Fc region included in a fusion protein or conjugate of the present disclosure is a human immunoglobulin Fc region or is derived from a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is of IgG, IgE, IgM, IgD, IgA or IgY isotype. In some embodiments, the immunoglobulin Fc region is an IgG isotype, such as IgGl, lgG2, lgG3, or lgG4 subclass. The immunoglobulin Fc region may comprise a variant or fragment of a native IgG Fc region.

[0217] A native Fc region typically possesses an effector function, including but not limited to, Fc receptor binding; Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (for example B-cell receptor); and B-cell activation, etc. Such effector functions generally require the Fc region to be combined with a binding domain (for example, an antibody variable domain) and can be assessed using various assays.

[0218] In some embodiments, a fusion protein or conjugate of the present disclosure can comprise a dimer of Fc regions. In some embodiments, an Fc region mediates dimerization of the antigen-binding units at physiological conditions, such as when expressed from a cell, such that a dimer is formed that doubles the number of antigen-binding units. For example, a fusion polypeptide comprising one VHH domain that binds an antigen and an Fc region is monovalent as a monomer, but the Fc region can mediate dimerization; as a result, the fusion protein is bivalent (i.e., having two VHH domains per molecule). Similarly, in some embodiments, two VHH domains (2x) are fused to an IgG Fc region and as a result of dimerization, the fusion protein is tetravalent (i.e., having four VHH domains per molecule). In some embodiments, three VHH domain (3x) are fused to an IgG Fc region and as a result of dimerization, the fusion protein is hexavalent (i.e., having six VHH domains per molecule).

[0219] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure: (VHH)n-Linker-Fc- (VHH)m, wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc.). When n>2 or m>2, each VHH may be optionally operably linked to another VHH via a linker.

[0220] For example, a fusion protein or conjugate of the present disclosure may be a tetravalent fusion protein or conjugate comprising two polypeptide chains, each polypeptide chain having the followingstructure: (VHH)-Linker-Fc-Linker-(VHH). The multiple linkers used in the fusion protein are not necessarily the same.

[0221] As another example, a fusion protein or conjugate of the disclosure may be a hexavalent fusion protein or conjugate comprising two polypeptide chains, each polypeptide chain having the following structure: (VHH)-Linker-(VHH)-Linker-Fc-linker-(VHH), or (VHH)-Linker-Fc-Linker-(VHH)-Linker-(VHH). The multiple linkers used in the fusion protein are not necessarily the same.

[0222] In some embodiments, the CH3 domain of the Fc region can be used as homodimerization domain, such that the resulting fusion protein may be formed from two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated to enable heterodimerization. For example, a heterodimerization domain can be incorporated into the fusion protein such that the construct is a heterodimeric fusion protein.

[0223] When a dimer of Fc regions is used in a fusion protein or conjugate of the present disclosure, the first and second Fc regions may be of the same IgG isotype such as, e.g., IgGl / IgGl, lgG2 / lgG2, lgG4 / lgG4. Alternatively, the first and second Fc regions may be of different IgG isotypes such as, e.g., lgGl / lgG2, lgGl / lgG4, lgG2 / lgG4, etc.

[0224] In some embodiments, the Fc region included in a fusion protein or conjugate of the present disclosure can be mutated or modified. In some embodiments, the mutations include one or more amino acid substitutions to reduce an effector function of the Fc region. Various examples of mutations to Fc regions to alter, such as reduce, effector function are known, including any as described below. In general, the numbering of the residues in an immunoglobulin heavy chain or portion thereof, such as an Fc region, is according to the EU index as in Kabat et aL, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0225] In some embodiments, the human IgG Fc region is modified to alter antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Non-limiting examples of amino acid modifications that can alter ADCC and / or CDC are described in Alegre et al, 1992 J Immunol, 148: 3461-3468; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Shields et aL, 2001 JBC, 276(9): 6591-6604; Lazar et aL, 2006 PNAS, 103(11): 4005-4010; Stavenhagen et aL, 2007 Cancer Res, 67(18): 8882-8890;Natsume et aL, 2008 Cancer Res, 68(10): 3863-72; Stavenhagen et aL, 2008 Advan. Enzyme ReguL, 48: 152-164; Moore et aL, 2010 mAbs, 2(2): 181-189; and Kaneko and Niwa, 2011 Biodrugs, 25( 1) : 1-11, each of which is incorporated herein by reference in its entirety.

[0226] In some embodiments, an Fc region included in a fusion protein or conjugate of the present disclosure exhibits reduced effector functions (such as CDC and ADCC). Various in vitro and / or in vivocytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the fusion protein construct and / or cleaved components thereof lack FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC are NK cells which express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in e.g., US 5,500,362; US 5,821,337; Hellstrom. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986); and Hellstrom et al., Proc. Nat'l Acad. Sci.USA 82:1499-1502 (1985); Bruggemann. et al., J. Exp. Med. 166:1351-1361 (1987). Alternatively, nonradioactive assay methods may be employed, such as ACTI™ non-radioactive cytotoxicity assay for flow cytometry or CytoTox96™ non-radioactive cytotoxicity assay. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq binding assays may also be carried out to confirm that the fusion protein construct or cleaved components thereof is unable to bind Clq and hence lacks CDC activity (see, e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402). To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano- Santoro et aL, J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0227] Examples of mutations that enhance ADCC include modification at Ser239 and Ile332, for example Ser239Asp and He332Glu (S239D, 1332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of these positions, for example Lys326Ala and / or Glu333Ala (K326A and E333A) using the Kabat numbering system.

[0228] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: Leu 234 (L234), Leu235 (L235), Gly236 (G236), Met252 (M252), Ser254 (S254), Asp265 (D265), Asp270 (D270), Ser298 (S298), Asn297 (N297), Asn325 (N325) or Ala327 (A327) or Pro329 (P329). For example, Leu234Ala (L234A), Leu234Gly (L234G), Leu234Ser (L234S), Leu234Thr (L234T), Leu234Ala (L234A), Leu235Ala (L235A), Leu235Glu (L235E), Leu235Ser (L235S), Leu235Thr (L235T), Leu235Val (L235V), Leu235Gln (L235Q), Gly236Arg (G236R), Met252Tyr (M252Y), Ser254Thr (S254T), Thr256Glu (T256E), Asp265Asn (D265N), Asp265Ala (D265A), Asp270Asn(D270N), Ser298Asn (S298N), Asn297Ala (N297A), Pro329Ala (P329A), Pro239Gly (P329G), Asn325Glu (N325E), and / or Ala327Ser (A327S). In some embodiments, modifications within the Fc region reduce binding to Fc-receptor-gamma receptors (Fey Rs) while have minimal impact on binding to the neonatal Fc receptor (FcRn).

[0229] In some embodiments, the human IgGl Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro239Ala (L234A / L235A / P329A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Asp265 (Kabat Numbering) to alter Fc receptor interactions, e.g Asp265Ala (D265A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat Numbering) to alter Fc receptor interactions, e.g., Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 265 and 329, e.g., Asp265Ala and Pro329Ala (D265A / P329A) or Asp265Ala and Pro329Gly (D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 265, e.g., Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, 265 and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding. For example, wherein Gly235 is deleted from the fusion protein.In some embodiments, the human IgGl Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A). In some embodiments, the human IgGl Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0230] In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Gly, Leu235Ser, Gly236Arg (L234G / L235S / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Thr, Gly236Arg (L234S / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Val, Gly236Arg (L234S / L235V / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Gln, Gly236Arg (L234T / L235Q / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Thr, Gly236Arg (L234T / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Thr, Leu235Thr, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 252, 254, and 256, e.g., Met252Tyr, Ser254Thr, Thr256Glu (M252Y / S254T / T256E).

[0231] In some embodiments, the Fc region of the fusion protein is lacking an amino acid at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In some embodiments, the Fc region of the fusion protein is lacking an amino acid at one or more of the following positions Glu233 (E233), Leu234 (L234), or Leu235 (L235) and is modified at one or more of the Asp265 (D265), Asn297 (N297), or Pro329 (P329) to reduce Fc receptor binding. For example, an Fc region included in a antigen binding polypeptide is derived from a human Fc domain, and comprises a three amino acid deletion in the lower hinge corresponding to IgGl E233, L234, and L235. In some embodiments, such Fc polypeptides do not engage FcyRs and thus are referred to as "effector silent" or "effector null." For example, Fc deletion of these three amino acids reduces the complement protein Clq binding. In some embodiments, a polypeptide with an Fc region with Fc deletion of these three amino acids retains binding to FcRn and therefore has extended half-life and transcytosis associated with FcRn mediated recycling.

[0232] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgGl Fc region, having an amino acid sequence:IgGl L234A, L235A (also known as "LALA" variant) (mutations bolded in the sequence below)DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 269)

[0233] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgGl Fc region, having an amino acid sequence:IgGl L234A, L235A, and P329A (also known as "LALAPA" variant) (mutations bolded in the sequence below)DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:270)

[0234] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgGl Fc region, having an amino acid sequence:IgGl D265A, N297A and P329A (also known as "DANAPA" variant) (mutations bolded in the sequence below)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAST YRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:271)

[0235] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgGl Fc region, having an amino acid sequence:IgGl L234A, L235A, and G237A (also known as "LALAGA" variant) (mutations bolded in the sequence below)DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTC VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:272)

[0236] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgGl Fc region, having an amino acid sequence:IgGl L234G / L235S / G236R (mutations bolded in the sequence below)DKTHTCPPCPAPEGSRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:287)

[0237] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgGl Fc region, having an amino acid sequence:IgGl L234S / L235T / G236R (mutations bolded in the sequence below)DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:288)

[0238] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgGl Fc region, having an amino acid sequence:IgGl L234S / L235V / G236R (mutations bolded in the sequence below)DKTHTCPPCPAPESVRGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:289)

[0239] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgGl Fc region, having an amino acid sequence:IgGl L234T / L235Q / G236R (mutations bolded in the sequence below)DKTHTCPPCPAPETQRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:290)

[0240] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgGl Fc region, having an amino acid sequence:IgGl L234T / L235T / G236R (mutations bolded in the sequence below)DKTHTCPPCPAPETTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:291)

[0241] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgGl Fc region, having an amino acid sequence:IgGl L234A / L235A / P329G (mutations bolded in the sequence below)DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:292)

[0242] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of humanIgGl Fc region, having an amino acid sequence:IgGl M252Y / S254T / T256E (mutations bolded in the sequence below)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ. ID NO: 293)

[0243] In some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (Kabat numbering, Dall'Acqua et al 2006, J. Biol Chem Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159), or Met252lle, Thr256Asp, Met428Leu (M252I, T256D, M428L, respectively) (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0244] In some embodiments, the Fc region lacks or has reduced fucose attached to the N-linked glycan-chain at N297. There are numerous ways to prevent fucosylation, including but not limited to production in a FUT8 deficient cell line; addition inhibitors to the mammalian cell culture media, for example Castanospermine; and metabolic engineering of the production cell line.

[0245] In some embodiments, the Fc domain included in a fusion protein or conjugate of the present disclosure is derived from a human Fc domain and comprises mutations M252Y and M428V. In some embodiments, the mutated or modified Fc polypeptide includes the following mutations: M252Y and M428L using the Kabat numbering system. In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5), while losing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn mediated recycling and extended half-life.

[0246] In some embodiments, the Fc domain included in a fusion protein or conjugate is derived from a human Fc domain and comprises mutations to induce heterodimerization. In some embodiments, such mutations include those referred to as "knob" and "hole" mutations. For example, having an amino acid modification within the CH3 domain at Thr366, which when replaced with a bulkier amino acid, e.g., Try (T366W), is able to preferentially pair with a second CH3 domain having amino acid modifications to less bulky amino acids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala and Vai, respectively (T366S / L368A / Y407V). In some embodiments, the "knob" Fc domain comprises the mutation T366W. In some embodiments, the "hole" Fc domain comprises mutations T366S, L368A, and Y407V.Heterodimerization via CH3 modifications can be further stabilized by the introduction of a disulfide bond, for example by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on opposite CH3 domains (Reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, Fc domains used for heterodimerization comprise additional mutations, such as the mutation S354C on afirst member of a heterodimeric Fc pair that forms an asymmetric disulfide with a corresponding mutation Y349C on the second member of a heterodimeric Fc pair. In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A binding while maintaining FcRn binding. In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc domain comprises the modification H435R or H435K (referred to as "hole-R" in some instances when the modification is H435R), while the knob Fc domain does not. In some instances, the hole-R mutation improves purification of the heterodimer over homodimeric hole Fc domains that may be present.

[0247] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the fusion proteins of the present disclosure are monomeric. For example, modification at residue Thr366 to a charged residue, e.g. Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0248] In some embodiments, the immunoglobulin Fc region of the fusion protein is of human lgG3 isotype, or a variant thereof. In one embodiment, the lgG3 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human lgG3 Fc region is modified at amino acid 435 to extend the half-life, e.g., Arg435His (R435H). In some embodiments, the human lgG3 Fc region lacks Lys447 (EU index of Kabat et al 1991).

[0249] In some embodiments, the immunoglobulin Fc region of the fusion protein is of human lgG4 isotype, or a variant thereof. In one embodiment, the human lgG4 Fc region is modified at amino acid 235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the human lgG4 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human lgG4 Fc region is lacks Lys447 (EU index of Kabat et al 1991).

[0250] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, L235E (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD lAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 273)

[0251] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, L235A (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 274)

[0252] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, F234A, L235E (mutations bolded in the sequence below) ESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 275)

[0253] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 S228P, F234A, L235A (mutations bolded in the sequence below) ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 276)

[0254] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human lgG4 Fc region, having an amino acid sequence: lgG4 P329G, S228P, L235E (mutations bolded in the sequence below)ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM HEALHNHYTQKSLSLSLGK (SEQ ID NO: 277)

[0255] Additional lgG4 heavy chain modifications suitable for use in the fusion proteins or conjugates of the present disclosure include those described in Tables 1 and 2 of Dumet et aL, mAbs, 11:8, 1341-1350, which is incorporated herein by reference in its entirety.

[0256] In some embodiments, the fusion protein or conjugate contains an immunoglobulin hinge region. In some embodiments, the hinge region serves as a linker to connect one or more antigen binding units (e.g., VHHs) to the Fc region. In other embodiments, the fusion protein can comprise a linker in addition to the hinge region to connect the one or more antigen binding units (e.g., VHHs) to the Fc region. The hinge region can be selected from any of the human IgG subclasses. For example, thefusion protein may contain a modified IgGl hinge having the sequence of EPKSSDKTHTCPPC (SEQ ID NO: 278), wherein the Cys220 that typically forms a disulfide bond with the C-terminal cysteine of the light chain is mutated to serine, e.g., Cys220Ser (C220S). In other embodiments, the fusion protein contains a truncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 279).

[0257] In some embodiments, the fusion protein or conjugate has a modified hinge from lgG4, which is modified to prevent or reduce strand exchange, e.g., Ser228Pro (S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 280).

[0258] In alternative embodiments, a fusion protein or conjugate of the present disclosure may comprise sequences other than an Fc region to achieve multimerization (e.g., dimerization). For example, an amino acid sequence containing at least one cysteine residue may be included to facilitate dimerization of two polypeptides by formation of a disulfide bond between the two polypeptides. In some embodiments, such multimerizing domain may comprise one or more cysteine residues, or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0259] Fc mutations suitable for use in the fusion proteins or conjugates disclosed herein are also discussed in, e.g., Wilkinson et aL, Fc-engineered antibodies with immune effector functions completely abolished. PLoS One. 2021; WO2021234402A2; US 8,969,526; EP3692065B1; and US 7,083,784, each of which is incorporated herein by reference.Fusion or Conjugation to Half-Life Extension Moieties

[0260] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise one or more other moieties which provide the fusion protein or conjugate with increased (in vivo) halflife. In vivo half-life extension means, that the fusion protein or conjugate has an increased half-life in a mammal, such as a human subject, after administration.

[0261] Non-limiting examples of half-life extension moieties suitable for use in the present disclosure include polyethylene glycol (PEG) molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

[0262] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG. In one embodiment, a fusion protein or conjugate of the present disclosure may comprise a binding moiety that can bind to human serum albumin. In one embodiment, the binding moiety is a single-domain antibody (e.g., VHH).

[0263] For example and without limitation, albumin binders that are described in, e.g., WO 04 / 041865, WO 06 / 122787, W02012 / 175400, WO 2012 / 175741, WO2015 / 173325, W02017 / 080850, WO2017 / 085172, WO2018 / 104444, W02018 / 134235, WO2018 / 134234, each of which is incorporated herein by reference in its entirety, can be used in the fusion protein or conjugate of the present disclosure.Fusion or Conjugation to Other Moieties

[0264] Single-domain antibodies provided herein may be operably linked, directly or indirectly, to a second moiety, such as but not limited to, a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a small molecule drug, a chemotherapeutic agent, a therapeutic agent, a diagnostic agent, or a combination thereof.

[0265] In some embodiments, a conjugate of the present disclosure comprises a label, which can generate a detectable signal. Such conjugates can be used for research or diagnostic purposes, such as for the in vivo detection of cancer. Preferably, the label is capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope (such as 3H, 14C, 32P, 35S, 1231, 1251, 1311); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound (such as fluorescein isothiocyanate, rhodamine or luciferin); an enzyme (such as fJ- galactosidase, alkaline phosphatase, or horseradish peroxidase); an imaging agent; or a metal ion. In some embodiments, the label is a radioactive atom for scintigraphic studies, for example 99Tc or 1231, or a spin label for nuclear magnetic resonance (NMR) imaging, such as zirconium-89, iodine-123, iodine- 131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Zirconium-89 may also be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011 / 056983).

[0266] Single-domain antibodies of the present disclosure may be conjugated to another moiety, such as an epitope tag, e.g., for the purpose of purification or detection. Examples of such molecules that are useful in protein purification include those that present structural epitopes capable of being recognized by a second molecule. This is commonly employed in protein purification by affinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding the immobilized compound. Non-limiting examples of epitope tag molecules that can be conjugated to single-domain antibodies of the present disclosure, e.g., for the purposes of molecular recognition include a poly-histidine tag (His-tag), a myc-tag, human influenza hemagglutinin (HA) tag, a FLAG-tag, maltose-binding protein, glutathione-S- transferase, biotin, and streptavidin. Conjugates containing the epitopes presented by these molecules are capable of being recognized by complementary molecules such as maltose, glutathione, a nickel- containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti-HA antibody, streptavidin, or biotin, respectively. For example, one can purify a single-domain antibody of the present disclosure that has been conjugated to an epitope tag from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc) by treating the mixture with a solid phase resin containing a complementary molecule that can selectively recognize and bind the epitope tag of the antigen antibody or fragment thereof. Examples of solid phase resins include agarose beads, which are compatible with purifications in aqueous solution.

[0267] In some embodiments, a conjugate of the present disclosure may comprise one or more VHH domains described herein conjugated to a therapeutic agent, which can be cytotoxic, cytostatic or otherwise provides some therapeutic benefit. In some embodiments, the cytotoxic agent is a drug, a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate). Such conjugates may be applicable to, e.g., the treatment or prevention of a disease associated with autoreactive cytotoxic T-cell activity. In some embodiments, antibody drug conjugates described herein may allow targeted delivery of a drug moiety to a target tissue (e.g., tumors).

[0268] In some embodiments, a conjugate of the present disclosure comprises a toxin. In some embodiments, the toxin includes, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et aL, J. Nat. Cancer Inst.92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et aL, Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et aL, Cancer Res. 53:3336-3342 (1993)). The toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Examples of other therapeutic agents that can be conjugated to a single-domain antibody of the present disclosure are described herein.

[0269] In some embodiments, single-domain antibodies of the present disclosure may be fused or conjugated to one or more moieties that facilitate delivery to the central nervous system (CNS) / brain. The moiety that can facilitate delivery of a single-domain antibody to the central nervous system (CNS) / brain can be for example, a peptide, a polypeptide, small molecule, a lipid, or a synthetic polymer.Various approaches to deliver single-domain antibodies into the brain are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.

[0270] As a non-limiting example, a single-domain antibody of the present disclosure may be fused or conjugated to a moiety (e.g., an antibody) that binds to the transferrin receptor (TfR) or insulin receptor. The transferrin receptor (TfR) is highly expressed by brain capillary endothelial cells (BCECs) forming the blood-brain barrier (BBB) and has been utilized as a target for brain drug delivery. Monoclonal antibodies binding to the TfR, such as clone Ri7, have been shown to internalize into BCECs in vivo. As another example, a single-domain antibody of the present disclosure may be conjugated to hydrophobic fatty acid moieties, such as C18 fatty acid (stearic acid), C16 fatty acid (palmitic acid) or C8 fatty acid (octanoic acid) moieties; or amphiphilic block copolymer moieties, such as polyethylene oxide)- poly(propylene oxide)-poly(ethylene oxide) (pluronics or poloxamers) or poly(2-oxasolines). Various fatty acid moieties and block copolymer moieties that can be utilized for brain delivery of proteins are described in, e.g., Yi and Kabanov, J Drug Target. 2013; 21(10): 940-955, which is incorporated herein by reference in its entirety.

[0271] Example methods for attaching a moiety, such as a label, to a binding protein include those described in Hunter, et al., Nature 144:945 (1962); David, et aL, Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel and Meares, Elsevier, N.Y. (1983); and Colcher et aL, Meth. Enzymol., 121 :802-16 (1986). Additional suitable methods for preparing the conjugates of the present disclosure include those described in, e.g., WO 2009 / 067800, WO 2011 / 133886, and US2014322129, incorporated by reference herein in their entirety.

[0272] In some embodiments, the attachment between a single-domain antibody and a second moiety can be covalent or non-covalent, e.g., via a biotin-streptavidin non-covalent interaction. In some embodiments, a second moiety can be attached to a single-domain antibody using any of various molecular biological or chemical conjugation and linkage methods known in the art and described below. In some embodiments, linkers such as peptide linkers, cleavable linkers, non-cleavable linkers or linkers that aid in the conjugation reaction, can be used to link or conjugate a second moiety to a singledomain antibody described herein.

[0273] In some embodiments, a single-domain antibody is conjugated to one or more second moieties, e.g., about 1 to about 20 moieties per molecule, optionally via a linker. In some embodiments, the one or more second moieties can be the same or different. The linker may be composed of one or more linker components. For covalent attachment of an antibody and the second moiety, the linker typically has two reactive functional groups, i.e., bivalency in a reactive sense. Bivalent linker reagents which areuseful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups have been described in, e.g., Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242.

[0274] In some embodiments, a linker used in a conjugate of the present disclosure may include 6- maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), a alaninephenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-Succinimidyl 4-(2- pyridylthio)pentanoate ("SPP"), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-l carboxylate ("SMCC"), or N-Succinimidyl(4-iodo-acetyl)aminobenzoate ("STAB"), or a combination thereof.

[0275] In some embodiments, a linker used in a conjugate of the present disclosure may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include valine-citrulline (vc or val-cit), alaninephenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues used in an amino acid linker component may include naturally occurring amino acids, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.

[0276] Conjugates of a single-domain antibody and second moiety (e.g., cytotoxic agent) can be made using a variety of bifunctional protein-coupling agents such as N-succinim idyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bisactive fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).

[0277] Conjugates of the present disclosure can be prepared by a variety of methods. For example, the conjugation method may include: (1) reaction of a nucleophilic group of a VHH domain with a bivalent linker reagent, to form VHH-Linker, via a covalent bond, followed by reaction with a drug moiety; or (2) reaction of a nucleophilic group of a drug moiety with a bivalent linker reagent, to form drug-linker, via a covalent bond, followed by reaction with the nucleophilic group of a VHH domain.

[0278] Nucleophilic groups on proteins including antibodies (e.g., VHH domains), include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups (e.g., lysine), (iii) side chain thiol groups (e.g., cysteine), and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Additional nucleophilic groups can be introduced into proteins (e.g., antibodies such as VHH domains) through the reaction of lysines with 2- iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into a protein (e.g., antibody such as a VHH domain) by introducing one, two, three, four, or more cysteine residues.

[0279] Conjugates, such as antibody drug conjugates, may also be produced by modification of an antibody, such as a VHH domain, to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may lead with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium metaperiodate, resulting in production of an aldehyde in place of the first amino acid. Such aldehyde can be reacted with a drug moiety or linker nucleophile.

[0280] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBi esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[0281] Alternatively, a fusion protein containing a VHH domain and cytotoxic agent may be made, e.g., by recombinant DNA techniques or peptide synthesis. A DNA sequence may be engineered to comprise respective regions encoding the two portions of the fusion protein either adjacent to one another or separated by a region encoding a linker peptide which does not impair the desired properties of the fusion protein. The DNA sequence can be then transfected into a host cell that expresses the fusionprotein. The fusion protein can be recovered from the cell culture and purified using techniques known in the art.Linkers

[0282] In some embodiments, the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via peptide linkers. A peptide linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects a peptide linker ranges from 3 amino acids to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length.

[0283] In some embodiments, a peptide linker, e.g., a peptide linker separating two VHH domains or an VHH domain and a heavy chain constant region, is at least 5 amino acids, at least 6 amino acids or at least 7 amino acids in length and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids or up to 60 amino acids in length.

[0284] In some embodiments, the linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids in length. In other embodiments of the foregoing, the linker ranges from 6 amino acids to 50 amino acids in length, e.g., ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids in length. In yet other embodiments of the foregoing, the linker ranges from 7 amino acids to 50 amino acids in length, e.g., ranges from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino acids in length.

[0285] In some embodiments, charged (e.g., charged hydrophilic linkers) and / or flexible linkers are used. Examples of flexible linkers that can be used in the fusion proteins of the disclosure include those disclosed by Chen et ai, 2013, Adv Drug Deliv Rev. 65(10): 1357-1369 and Klein et a / ., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are or comprise repeats of glycines and serines (termed "GS-linker" herein), e.g., a monomer or multimer of GnS (SEQ ID NO: 254) or SGn(SEQ ID NO: 255), where n is an integer from 1 to 10, e.g., 1 2, 3, 4, 5, 6, or 7, 8, 9 or 10. In one embodiment, the linker is or comprises a monomer or multimer of repeat of G4S (SEQ ID NO: 210), e.g., (GGGGS)n (SEQ ID NO: 256).

[0286] Polyglycine linkers can suitably be used in the fusion proteins of the disclosure. In some embodiments, a peptide linker used herein comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 257), five consecutive glycines (5Gly) (SEQID NO: 258), six consecutive glycines (6Gly) (SEQ ID NO: 259), seven consecutive glycines (7Gly) (SEQ ID NO: 260), eight consecutive glycines (8Gly) (SEQ ID NO: 261), or nine consecutive glycines (9Gly) (SEQ ID NO: 262).

[0287] In some embodiments, a GS-linker used herein comprises an amino acid sequence selected from GGSGGS, i.e., (GGS)2(SEQ ID NO: 263); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 264); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 265); and GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 266). In some embodiments, the fusion proteins can include a combination of a GS-linker and a glycine linker.

[0288] In one embodiment, two or more VHHs are linked via a GGGGSGGGGSGGGGS (SEQ ID NO: 211) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGS (SEQ ID NO: 267) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGSESKYGPPCPSCP (SEQ ID NO: 249) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGS (SEQ ID NO: 210) linker.

[0289] In some embodiments, the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via a "rigid" peptide linker. Such peptidic linker may comprise a prolinerich peptide. In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAP (SEQ ID NO: 250). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 253). In one embodiment, a rigid peptide linker comprises A(EAAAK)nA (SEQ ID NO: 268), where n is any integer, e.g., 1 2, 3, 4, 5, 6, or 7, 8, 9 or 10.

[0290] Other exemplary peptide linkers that can be used in the fusion proteins described herein are shown in Table 2.Table 2. Exemplary Peptide Linker SequencesPolynucleotide Molecules

[0291] In another aspect, provided herein are polynucleotide molecules encoding the single-domain antibodies or fusion proteins described herein. Polynucleotide molecules encoding polypeptide portion(s) of a conjugate of the present disclosure are also encompassed within the present disclosure.

[0292] A polynucleotide molecule may be used to transform / transfect a host cell or host organism, e.g., for expression and / or production of a polypeptide. Suitable hosts or host cells for production of an polypeptides described herein include any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. A host or host cell comprising a polynucleotidemolecule encoding a single-domain antibody or fusion protein described herein is also encompassed by the present disclosure.

[0293] A polynucleotide molecule may be for example DNA, RNA, or a hybrid thereof, and may also comprise (e.g., chemically) modified nucleotides, like locked nucleic acids (LNA) or peptide nucleic acids (PNA). In some embodiments, the polynucleotide is single-stranded. In some embodiments, the polynucleotide is double-stranded. In one embodiment, the polynucleotide is in the form of doublestranded DNA (e.g., plasmid). In some embodiments, the polynucleotide is in the form of a singlestranded RNA (e.g., mRNA).

[0294] Techniques for generating polynucleotides may include, for example but not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and / or regions that may easily be digested and / or ligated using suitable restriction enzymes), and / or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers. Alternatively, polynucleotides of the present disclosure may be isolated from a suitable natural source. Polynucleotide sequences encoding naturally occurring (poly)peptides can for example be subjected to site-directed mutagenesis, to generate a polynucleotide molecule encoding polypeptide with sequence variation.Vectors

[0295] Also provided herein are vectors comprising the polynucleotide molecules encoding the singledomain antibodies, fusion proteins, or other relevant polypeptides of the present disclosure. A "vector" as used herein is a vehicle suitable for carrying genetic material into a host cell. A vector can include a nucleic acid vector, such as a plasmid or mRNA, or nucleic acids embedded into a bigger structure, such as a liposome or viral vector.

[0296] A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters, enhancers, terminators) that regulate the expression of a polypeptide of interest, and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, 0-galactosidase). For DNA-based vectors, this usually includes the presence of elements for transcription (e.g., a promoter and a polyA signal) and translation (e.g., Kozak sequence). In some embodiments, the vector is anexpression vector, i.e. a vector suitable for expressing an encoded polypeptide or construct under suitable conditions in a host cell.

[0297] To express a single-domain antibody or fusion protein (or fragments thereof) of the present disclosure, polynucleotides encoding partial or full-length polypeptide chains, e.g., obtained as described above (e.g., VHH, VHH-Fc-VHH), can be inserted into expression vectors such that the genes are operatively linked to one or more transcriptional and translational control sequences. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Polynucleotides encoding the two or more polypeptide chains (when present and differ from one another) of a single-domain antibody or fusion protein of the present disclosure can be inserted into separate vectors, or, optionally, incorporated into the same expression vector.

[0298] In addition to polynucleotides encoding the polypeptide chain(s) of a single-domain antibody or fusion protein, the recombinant expression vectors of the invention may include regulatory sequences that control the expression of genes encoding the polypeptide chain(s) in a host cell. The design of the expression vector, including the selection of regulatory sequences, may depend on the choice of the host cell to be transformed and / or the desired level of protein expression. For example, suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Additional examples of viral regulatory elements, and sequences thereof, include those described in, e.g., U.S. Pat. Nos. 5, 168,062; 4,510,245; and 4,968,615; the disclosures of each of which are incorporated herein by reference.

[0299] Recombinant expression vectors of the present disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., US4,399,216; US 4,634,665; and US 5,179,017; the disclosure of each of which is incorporated herein by reference in its entirety). For example, typically the selectable marker gene confers resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin, or cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cell into which the vector has been introduced. Suitable selectable marker genes can include the dihydrofolate reductase (DHFR) gene (for use in DHFR deficient host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0300] Vectors of the present disclosure may further include sequence elements that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5' and 3' untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.

[0301] Viral vectors can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral vectors are particularly useful for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents to induce gene integration. Examples of suitable viral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses (AAV) such as AAV2, AAV8, AAV9), negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpes virus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), baculovirus, coronavirus, and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding polypeptides of the present disclosure include, for example Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include, but are not limited to, avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M.1996. Fundamental Virology, DMKDN Fields, PM Howley, ed. (Philadelphia, Lippincott-Raven Publishers): 763-843., the disclosure of which is incorporated herein by reference). Other examples of viral genomes useful in the compositions and methods of the present disclosure include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline sarcoma virus, feline leukemia virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses.Host Cells

[0302] In one aspect, the present disclosure also provides host cells or host organisms that comprise the polynucleotides or vectors encoding the single-domain antibodies, fusion proteins, or other relevantpolypeptides described herein. Suitable host cells or host organisms can be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells can also include cells transfected in vivo with a polynucleotide(s) or vector provided herein.

[0303] Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris); plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), COS cells, SP2 / 0 cells, and 293 and CHO cells, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Exemplary prokaryotic cells include bacterial cells such as Escherichia coli.Preparation Methods

[0304] The present disclosure also provides methods of producing the single-domain antibodies, fusion proteins, or conjugates described herein.

[0305] In one aspect, the present disclosure provides methods of modifying a single-domain antibody (e.g., VHH) described herein to have reduced binding by anti-drug antibodies to the C-terminus, the method comprising modification of the amino acid sequence of the single-domain antibody (e.g., VHH) at its carboxy-terminus. In some embodiments, the method may comprise mutation of single-domain antibody (e.g., VHH) within FR1, FR2, FR3, or FR4, or a combination thereof. In some embodiments, modification(s) of the amino acid sequence of the single-domain antibody (e.g., VHH) at its carboxyterminus and mutation(s) of the amino acid sequence of the single-domain antibody (e.g., VHH) within FR1, FR2, FR3 or FR4, or a combination thereof, may be combined (in any combination) in the practice of the methods described herein. In some embodiments, the methods of modifying the single-domain antibody (e.g., VHH) described herein may comprise, e.g., modification and / or mutation of the singledomain antibody (e.g., VHH) by any of the modifications and / or mutations set forth in Tables 1-1, 1-2, or 1-4, or a combination thereof, described herein.

[0306] In some embodiments, a method of producing the single-domain antibodies, fusion proteins, or conjugates described herein may comprise transforming / transfecting a host cell or host organism with a polynucleotide encoding a single-domain antibody, fusion protein, or other relevant polypeptide(s)described herein, expressing the single-domain antibody, fusion protein, or other relevant polypeptide(s) in the host, optionally followed by one or more isolation and / or purification steps.

[0307] When recombinant expression vectors encoding one or more polypeptide(s) of a single-domain antibody, fusion protein, or conjugate of the present disclosure are introduced into mammalian host cells, the host cells are cultured for a period of time sufficient to allow for expression of the protein(s) or polypeptide(s) in the host cells or secretion of the protein(s) or polypeptide(s) into the culture medium in which the host cells are grown. Protein(s) or polypeptide(s) can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as VHH domains.

[0308] Once a protein or polypeptide of the present disclosure has been produced by recombinant expression, it can be purified by any method known in the art for purification of a protein or polypeptide, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for antigen after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the proteins or polypeptides of the present disclosure can be fused to heterologous polypeptide sequences described herein (e.g., His-tag) or otherwise known in the art to facilitate purification or to produce therapeutic conjugates below). Once isolated, a protein or polypeptide of the present disclosure can, if desired, be further purified, e.g., by high performance liquid chromatography, or by gel filtration chromatography, such as on a Superdex™ column.Compositions and Formulations

[0309] The present disclosure also provides a composition comprising a single-domain antibody, fusion protein, or conjugate of the present technology, at least one polynucleotide molecule encoding the same, at least one vector comprising such a polynucleotide molecule, or at least one host cell comprising the polynucleotide molecule or vector. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprise one or more further pharmaceutically active polypeptides and / or compounds.

[0310] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, which is incorporatedherein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions.

[0311] Examples of suitable formulations include, but are not limited to, solutions, suspensions, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Phdomain Sci Technol 52:238- 311.

[0312] A pharmaceutical composition of the present disclosure may be formulated according to its intended route of administration. Examples of suitable routes of administration include, e.g., intravenous, subcutaneous, intratumoral, oral (e.g., buccal, sublingual), intranasal, inhalation, intraocular, intramuscular, intradermal, transdermal (i.e., topical), intraperitoneal, transmucosal, vaginal, and rectal administration, or injection to the CNS / brain (e.g., intraspinal, intracerebral, or intrathecal administration). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; fixed oils; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as phosphates, acetates, or citrates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of plastic or glass.

[0313] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include, for example, physiological saline, bacteriostatic water, Cremophor EL®, or phosphate buffered saline (PBS). The composition is preferably sterile and has a proper fluidity. In most embodiments, the composition is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, e.g., water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the contamination by microorganisms can be achieved by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0314] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and / or freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously steri I e-f i Itered solution thereof.

[0315] Oral compositions may include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, capsules, or liquid forms. Formulation in tablet and liquid forms may be used for protease insensitive VHHs. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0316] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0317] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0318] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0319] For brain delivery, compounds of the present disclosure may be formulated to facilitate crossing of the blood-brain barrier. For example, single-domain antibodies, fusion proteins, or conjugates of the present disclosure may be encapsulated into brain targeted liposomes, lipid nanoparticles, lipid microparticles, or lipid microcapsules for brain delivery. Example liposomes delivery systems are described in Pothin et aL, Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.

[0320] In some embodiments, the active compounds are prepared with carriers that can protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US 4,522,811, which is incorporated herein by reference in its entirety.

[0321] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure is dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0322] The pharmaceutical compositions (or components thereof) can be included in a kit, container, pack, or dispenser together with instructions for administration. These pharmaceutical compositions can be included in diagnostic kits with instructions for use.

[0323] Pharmaceutical compositions are administered in an amount effective for treatment or prophylaxis of the specific indication. The therapeutically effective amount is typically dependent on the weight of the subject being treated, the physical or health condition of the subject, the extensiveness of the condition to be treated, or the age of the subject being treated. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 50 pg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 100 pg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 100 pg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering a pharmaceutical composition of the present disclosure may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well- known methods in the art (e.g., Mordenti et al., 1991, Phdomainaceut. Res. 8:1351).

[0324] In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 200 mg per dose.

[0325] In some embodiments wherein the single-domain antibodies of the present disclosure are administered as a viral vector (e.g., an AAV), dose ranges and frequency of administration of the viral vector described herein can vary depending on the nature of the viral vector, and the medical condition, as well as parameters of a specific patient and the route of administration used. In some embodiments, viral vector compositions can be administered to a subject at a dose ranging from about Ixio5plaque forming units (pfu) to about lxlO15pfu, depending on mode of administration, the route of administration, the nature of the disease and condition of the subject. In some cases, the viral vectorcompositions can be administered at a dose ranging from about lxlO8pfu to about lxlO15pfu, or from about lxlO10pfu to about lxlO15pfu, or from about lxlO8pfu to about lxlO12pfu. A more accurate dose can also depend on the subject in which it is being administered. For example, a lower dose may be required if the subject is juvenile, and a higher dose may be required if the subject is an adult human subject. In certain embodiments, a more accurate dose can depend on the weight of the subject. In certain embodiments, for example, a juvenile human subject can receive from about lxlO8pfu to about lxlO10pfu, while an adult human subject can receive a dose from about lxlO10pfu to about lxlO12pfu.

[0326] Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, intraspinal, intracerebral, intrathecal and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

[0327] A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0328] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Grit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target,thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0329] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, intramuscular, intratumoral, intraperitoneal, intraspinal, intracerebral, and intrathecal injections, drip infusions, etc. In one embodiment, the injectable preparations may be prepared, e.g., by dissolving, suspending or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0001] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the single-domain antibodies described herein may be about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, the single-domain antibodies described herein may be contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

[0330] The pharmaceutical composition may be administered as needed to a subject. In some embodiments, an effective dose of the pharmaceutical composition is administered to a subject one or more times. In various embodiments, an effective dose of the pharmaceutical composition is administered to the subject once a month, less than once a month, such as, for example, every two months, every three months, or every six months. In other embodiments, an effective dose of the pharmaceutical composition is administered more than once a month, such as, for example, every two weeks, every week, twice per week, three times per week, daily, or multiple times per day. An effective dose of the pharmaceutical composition is administered to the subject at least once. In some embodiments, the effective dose of the pharmaceutical composition may be administered multiple times, including for periods of at least a month, at least six months, or at least a year. In someembodiments, the pharmaceutical composition is administered to a subject as needed to alleviate one or more symptoms of a condition.

[0331] In some embodiments, a pharmaceutical composition of the present disclosure may be administered to a subject at levels lower than that required to achieve the desired therapeutic effect and the dosage may be gradually increased until the desired effect is achieved. Alternatively, a pharmaceutical composition of the present disclosure may be administered at a high dose and subsequently administered progressively lower doses until a therapeutic effect is achieved. In general, a suitable daily dose of a single-domain antibody of the invention is an amount of the antibody which is the lowest dose effective to produce a therapeutic effect.

[0332] Pharmaceutical compositions of the present disclosure may optionally include more than one active agent.Kits

[0333] The present disclosure further comprises a kit comprising any of various compositions of the present disclosure, including the single-domain antibodies, fusion proteins, or conjugates comprising the single-domain antibodies, polynucleotide molecules, vectors, or cells, of the disclosure. In some embodiments, the kits comprising any of the compositions described herein may, optionally, comprise instructions and / or packaging for same.

[0334] In various embodiments, kits of the present disclosure may comprise a lyophilized formulation of the composition(s) disclosed herein in a suitable container and / or instructions for its reconstitution and / or use. Examples of suitable containers include vials, syringes, bottles, flasks, and test tubes. The container may be produced from a variety of materials such as plastic or glass.

[0335] The kit and / or container may comprise instructions associated with or on the container which may include, for example, instructions for reconstitution of the lyophilized formulation and / or use of the kit. In some embodiments, the label may specify that the lyophilized formulation can be reconstituted to an appropriate composition concentration. The label may specify that the formulation is useful or intended for any route of administration, e.g., parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation, or intranasal routes.

[0336] In some embodiments, a kit may contain an apparatus (e.g., one or more needles, syringes, eye droppers, pipettes, etc.), which may allow for administration of the agents of the disclosure that are components of the present kit.

[0337] The container holding the formulation may be a multi-use vial. In some embodiments, the muti- use vial may allow for repeat administrations (e.g., from 2-20 administrations) of the reconstituted formulation. The kit may further comprise a second container which comprises a suitable diluent (e.g., normal saline, sterile water for inject, or dextrose 5% in water).

[0338] Upon mixing of the diluent and the lyophilized formulation, the final concentration of the composition within the reconstituted formulation can be reached. The kit may further include additional materials which may be desirable from a user and / or commercial angle. Such additional materials may include syringes, needles, diluents, buffers, filters, and / or package inserts which may comprise, for example, instructions for use.

[0339] Kits of described herein may have a single container that contains the formulation of the compositions according to the present disclosure with or without other components (e.g., other compounds or pharmaceutical compositions of such compounds) or may have a separate container for each component. In some embodiments, when there is more than one component, the kit may comprise a second vial or other container, which may allow for separate dosing. The kit may also comprise another container for a pharmaceutically acceptable liquid.

[0340] In some embodiments, kits of described herein may include a formulation of the disclosure packaged for use in combination with the co-administration of a second compound (such as adjuvants, e.g., a chemotherapeutic agent, a hormone, an antagonist, a natural product, or a chelator) or a pharmaceutical composition thereof. The components of the kit may be pre-complexed or each component may be in a separate individual container.

[0341] The components of the kit may be provided in one or more liquid solutions. A liquid solution described herein may be an aqueous solution which may be, e.g., a sterile aqueous solution. The components of the kit may be, in some cases, provided as solids, which may be transformed into liquids such as by addition of suitable solvents, which may be provided in another distinct container.EXAMPLES

[0342] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.Example 1. Expression and purification of VHHs

[0343] C-terminal-engineered constructs (Table 1-1) and framework modified constructs (Table 1-2) were designed. Bacterial expression plasmids coding for all designed VHHs were transformed into a chemically competent E. coli T7Shuffle strain. Transformed bacteria were used for overnight proteinexpression in autoinduction Terrific Broth (TB)-medium at 30°C, in 96-well plate format and / or 1-liter shake flask format. E. coli cells were harvested the next morning by centrifugation and treated with a combination of high- and low-sucrose-containing buffer to release VHHs from the bacterial periplasm by osmotic shock. The periplasmic extract was then cleared by centrifugation and purified via Ni-NTA affinity chromatography, employing standard protocols and buffers. Following elution from the Ni-NTA resin, the VHHs were rebuffered into 1 x phosphate buffered saline (PBS) for further storage and flash- frozen in liquid nitrogen.Example 2. Anti-drug antibody (ADA) assay

[0344] To measure binding of pre-existing anti-drug antibodies (ADAs) to the engineered VHHs, affinity- purified VHHs were coated onto MaxiSorp plates at 4°C overnight (per well, 10 pil VHH solution and 40 pil PBS were added). Following, the samples were washed twice with 0.05% (v / v) PBS-Tween20 and blocked with Casein solution (Thermo Fisher, Cat. #: 37528) on a plate shaker at room temperature (RT) for 1 hour. After two washes with 0.05% (v / v) PBS-Tween20, the samples were incubated with human intravenous IgG ( I Vlg), Creative Biomart, Cat. #: THP-0108) diluted 1:3,150 in 0.01% (v / v) Casein-PBS on a plate shaker at RT for 1 hour. Subsequently, the samples were washed five times with 0.05% (v / v) PBS- Tween20 and incubated with anti-human-IgG (Fc-specific)-HRP (Sigma-Aldrich, Cat. #: A0170) diluted 1:60,000 in 0.01% (v / v) Casein-PBS on a plate shaker at RT for 1 hour protected from light. After five washes with 0.05% (v / v) PBS-Tween20 and two washes with PBS, 50 pL TMB One Component HRP Microwell Substrate (Tebubio) was added per well. The plate was incubated in the dark at RT for 10 min and the reaction was stopped by adding H2SO4. Absorbance was measured at 450 nm and at 620 nm for reference. A bar graph of binding of pre-anti-drug antibodies (pre-ADAs) (human IgG) to purified V- bodies described herein is shown in Figure 3 and a corresponding summary of the data normalized to control VHH ODY-349 is shown in Figure 4. For generation of these data, a total of 452 engineered V- bodies were incubated with human intravenous IgG (IVIg) and binding of human IgG was detected. The results are also summarized in Figures 5A-5D.Example 3. Pre-existing ADA binding from human sera to modified V-bodies

[0345] Pre-existing anti-drug antibody (ADA) binding the C-terminally modified V-bodies was evaluated using individual sera of 50 healthy human donors in an ADA assay. WT VHH (ODY-349) with the C- terminal amino acid sequence VSS was used as a control. To measure binding of pre-existing ADAs to the engineered VHHs, affinity-purified VHHs were coated onto MaxiSorp plates (per well, 1 pg VHH in PBS,0.1% BSA, 0.05% Tween20 was added). The samples were then washed and blocked on a plate shaker at room temperature (RT) for 2 hours. Serum samples of healthy, naive donors were added (dilution 1:20 in PBS, 0.1% BSA, 0.05% Tween20) and incubated for 2 hours at room temperature, while shaking. Subsequently, the samples were washed and incubated with anti-human-IgG (Fc-specific)-HRP (Sigma- Aldrich, Cat. #: A0170) diluted 1:100,000 in PBS, 0.1% BSA, 0.05% Tween20 on a plate shaker at RT. After washing, (3,3',5,5'-Tetramethylbenzidine) (TMB) horseradish peroxidase (HRP) Substrate was added, and the plate was incubated in the dark at RT. The reaction was stopped by adding H2SO4and absorbance was measured at 450 nm.

[0346] Figure 6 shows a heat map presenting the optical density (OD) values of pre-existing ADA binding analysis for 24 V-body variants screened against 50 individual sera from human healthy donors. Intensity of color represents pre-existing ADA binding to purified V-bodies.Example 4. C-terminal modification of tetravalent V-body agonists targeting a hepatocyte membrane protein significantly reduced hepatotoxicity

[0347] A tetravalent unmodified V-body agonist (wt-Vtetra) as well as two C-terminally modified versions of the same molecule (Vtetra3-VPAG and Vtetra3-VAGG) that target a hepatocyte membrane protein were incubated on primary hepatocytes at concentrations between 0.1-1000 pM in the presence and absence of pre-existing anti-drug antibodies (ADAs) containing human intravenous IgG (IVIg). A summary of data generated in the present Example are depicted in Figures 7A-7B. In Figure 7A, the relative viability of the primary hepatocytes as measured by CellTiter-Glo assay (CTG) on the y-axis is plotted against the indicated concentration of wild-type V-body agonist (wt-Vtetra3) and modified Vtetra3-VPAG in the presence and absence of human IVIg after 24 hours of incubation (x-axis). In Figure 7B, the relative viability of the primary hepatocytes as measured by CellTiter-Glo assay (CTG) on the y- axis is plotted against the indicated concentration of wild-type V-body agonist (wt-Vtetra3) and modified Vtetra3-VAGG in the presence and absence of human IVIg after 24 hours of incubation (x-axis). Dashed horizontal lines in Figures 7A-7B highlight the viability of the hepatocytes for the highest concentration of agonist tested in the absence of IVIg for comparison. Structural models of the V-body agonists and position of the modifications (very C-terminus) are depicted at the bottom of the figure panels. The results showed that while the unmodified tetravalent V-body agonist elicited pre-ADA induced hepatotoxicity, the C-terminally modified versions (VPAG (SEQ ID NO: 94) and VAGG (SEQ ID NO: 5)) of the same molecule significantly reduced hepatotoxicity.* * *

[0348] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0349] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.

Claims

Claims1. A single-domain antibody which is modified to comprise, at the carboxy-terminus starting from position 111 according to Chothia, an amino acid sequence selected from(1) V,(2) VXb(3) VXxX2,(4) VXIX2G, or(5) VXiX2P; wherein Xi is selected from amino acids Ala (A), Asp (D), Glu (E), Gly (G), lie (I), Lys (K), Leu (L), Asn (N), Pro (P), Arg (R), Ser (S), Thr (T), and Vai (V), andX2is selected from amino acids Ala (A), Asp (D), Glu (E), Gly (G), He (I), Lys (K), Leu (L), Asn (N), Pro (P), Gin (Q), Arg (R), Ser (S), Thr (T), and Vai (V).

2. The single-domain antibody of claim 1, wherein Xi is selected from Ala (A), Gly (G), Pro (P), Asp(D), and Leu (L); and / or X2is selected from amino acids Ala (A), Gly (G), Pro (P), Asp (D), Gin (Q), and Leu (L).

3. The single-domain antibody of claim 2, wherein X2is selected from Ala (A), Gly (G), and Pro (P); and / or X2is selected from amino acids Ala (A), Gly (G), Gin (Q), and Pro (P).

4. The single-domain antibody of any one of claims 1-3, wherein the amino acid sequence is not one selected from VSS, VE, VEG, VEP, VEPG (SEQ ID NO: 35), VK, VKS, VKG, VKP, VKPG (SEQ ID NO: 294), VQS, VS, VSE, VSEG (SEQ ID NO: 135), VSK, VSKG (SEQ ID NO: 141), VRP, VRPG (SEQ ID NO: 126), VDP, VDPG (SEQ ID NO: 25), VSSP (SEQ ID NO: 295), and VSSG (SEQ ID NO: 286).

5. The single-domain antibody of claim 1, wherein the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, an amino acid sequence selected from(1) V;(2) VA, VD, VE, VG, VI, VK, VL, VN, VP, VR, VS, VT, or VV;(3) VAA, VAD, VAE, VAG, VAI, VAK, VAL, VAN, VAP, VAQ, VAR, VAS, VAT, VAV, VDA, VDD, VDE, VDG, VDI, VDK, VDL, VDN, VDP, VDQ, VDR, VDS, VDT, VDV, VEA, VED, VEE, VEG, VEI, VEK, VEL, VEN, VEP, VEQ, VER, VES, VET, VEV, VGA, VGD, VGE, VGG, VGI, VGK, VGL, VGN, VGP, VGQ, VGR, VGS, VGT, VGV, VIA, VID, VIE, VIG, VII, VIK, VIL, VIN, VIP, VIQ, VIR, VIS, VIT, VIV, VLA, VLD, VLE, VLG, VLI, VLK, VLL, VLN, VLP, VLQ, VLR, VLS, VLT, VLV, VNA, VND, VNE, VNG, VNI, VNK, VNL, VNN, VNP, VNQ, VNR, VNS, VNT, VNV, VPA, VPD, VPE, VPG, VPI, VPK, VPL, VPN, VPP, VPQ, VPR, VPS, VPT, VPV, VRA, VRD, VRE, VRG, VRI, VRK, VRL, VRN, VRP, VRQ, VRR, VRS, VRT, VRV, VSA, VSD, VSE, VSG, VSI, VSK, VSL, VSN, VSP, VSQ, VSR, VST, VSV, VTA, VTD, VTE, VTG, VTI, VTK, VTL, VTN, VTP, VTQ, VTR, VTS, VTT, VTV, VVA, VVD, WE, VVG, VVI, WK, WL, WN, WP, VVQ, WR, WS, WT, or VW;(4) VADG (SEQ ID NO: 1), VAEG (SEQ ID NO: 3), VAGG (SEQ ID NO: 5), VAKG (SEQ ID NO: 7), VANG (SEQ ID NO: 9), VAPG (SEQ ID NO: 11), VAQG (SEQ ID NO: 13), VARG (SEQ ID NO: 15), VASG (SEQ ID NO: 17), VATG (SEQ ID NO: 19), VDAG (SEQ ID NO: 21), VDGG (SEQ ID NO: 23), VDPG (SEQ ID NO: 25), VDSG (SEQ ID NO: 27), VDTG (SEQ ID NO: 29), VEAG (SEQ ID NO: 31), VEGG (SEQ ID NO: 33), VEPG (SEQ ID NO: 35), VESG (SEQ ID NO: 37), VETG (SEQ ID NO: 39), VGAG (SEQ ID NO: 41), VGDG (SEQ ID NO: 43), VGEG (SEQ ID NO: 45), VGGG (SEQ ID NO: 47), VGIG (SEQ ID NO: 49), VGKG (SEQ ID NO: 51), VGLG (SEQ ID NO: 53), VGNG (SEQ ID NO: 55), VGPG (SEQ ID NO: 57), VGQG (SEQ ID NO: 59), VGRG (SEQ ID NO: 61), VGSG (SEQ ID NO: 63), VGTG (SEQ ID NO: 65), VGVG (SEQ ID NO: 67), VIGG (SEQ ID NO: 69), VIPG (SEQ ID NO: 71), VISG (SEQ ID NO: 73), VITG (SEQ ID NO: 75), VLGG (SEQ ID NO: 77), VLPG (SEQ ID NO: 79), VLSG (SEQ ID NO: 283), VLTG (SEQ ID NO: 82), VNAG (SEQ ID NO: 84), VNGG (SEQ ID NO: 86), VNPG (SEQ ID NO: 88), VNSG (SEQ ID NO: 90), VNTG (SEQ ID NO: 92), VPAG (SEQ ID NO: 94), VPDG (SEQ ID NO: 96), VPEG (SEQ ID NO: 98), VPGG (SEQ ID NO: 100), VPIG (SEQ ID NO: 102), VPKG (SEQ ID NO: 104), VPLG (SEQ ID NO: 106), VPNG (SEQ ID NO: 108), VPPG (SEQ ID NO: 110), VPQG (SEQ ID NO: 112), VPRG (SEQ ID NO: 114), VPSG (SEQ ID NO: 116), VPTG (SEQ ID NO: 118), VPVG (SEQ ID NO: 120), VRAG (SEQ ID NO: 122), VRGG (SEQ ID NO: 124), VRPG (SEQ ID NO: 126), VRSG (SEQ ID NO: 128), VRTG (SEQ ID NO: 130), VSAG (SEQ ID NO: 284), VSDG (SEQ ID NO: 133), VSEG (SEQ ID NO: 135), VSGG (SEQ ID NO: 137), VSIG (SEQ ID NO: 139), VSKG (SEQ ID NO: 141), VSLG (SEQ ID NO: 143), VSNG (SEQ ID NO: 145), VSPG (SEQ ID NO: 147), VSQG (SEQ ID NO: 149), VSRG (SEQ ID NO: 151), VSTG (SEQ ID NO: 153), VSVG (SEQ ID NO: 285), VTAG (SEQ ID NO: 156), VTDG (SEQ ID NO: 158), VTEG (SEQ ID NO: 160), VTGG (SEQ ID NO: 162), VTIG (SEQ ID NO: 164), VTKG (SEQ ID NO: 166), VTLG (SEQ ID NO: 168), VTNG (SEQ ID NO: 170), VTPG (SEQ ID NO: 172), VTQG (SEQ ID NO: 174), VTRG (SEQ ID NO: 176), VTSG (SEQ ID NO: 178), VTTG (SEQ ID NO: 180), VTVG (SEQ ID NO: 182), WGG (SEQ ID NO: 184), WPG (SEQ ID NO: 186), WSG (SEQ ID NO: 188), or WTG (SEQ ID NO: 190); or(5) VADP (SEQ ID NO: 2), VAEP (SEQ ID NO: 4), VAGP (SEQ ID NO: 6), VAKP (SEQ ID NO: 8), VANP (SEQ ID NO: 10), VAPP (SEQ ID NO: 12), VAQP (SEQ ID NO: 14), VARP (SEQ ID NO: 16), VASP (SEQ ID NO: 18), VATP (SEQ ID NO: 20), VDAP (SEQ ID NO: 22), VDGP (SEQ ID NO: 24), VDPP (SEQ ID NO: 26), VDSP (SEQ ID NO: 28), VDTP (SEQ ID NO: 30), VEAP (SEQ ID NO: 32), VEGP (SEQ ID NO: 34), VEPP (SEQ ID NO: 36), VESP (SEQ ID NO: 38), VETP (SEQ ID NO: 40), VGAP (SEQ ID NO: 42), VGDP (SEQ ID NO: 44), VGEP (SEQ ID NO: 46), VGGP (SEQ ID NO: 48), VGIP (SEQ ID NO: 50), VGKP (SEQ ID NO: 52), VGLP (SEQ ID NO: 54), VGNP (SEQ ID NO: 56), VGPP (SEQ ID NO: 58), VGQP (SEQ ID NO: 60), VGRP (SEQ ID NO: 62), VGSP (SEQ ID NO: 64), VGTP (SEQ ID NO: 66), VGVP (SEQ ID NO: 68), VIGP (SEQ ID NO: 70), VIPP (SEQ ID NO: 72), VISP (SEQ ID NO: 74), VITP (SEQ ID NO: 76), VLGP (SEQ ID NO: 78), VLPP (SEQ ID NO: 80), VLSP (SEQ ID NO: 81), VLTP (SEQ ID NO: 83), VNAP (SEQ ID NO: 85), VNGP (SEQ ID NO: 87), VNPP (SEQ ID NO: 89), VNSP (SEQ ID NO: 91), VNTP (SEQ ID NO: 93), VPAP (SEQ ID NO: 95), VPDP (SEQ ID NO: 97), VPEP (SEQ ID NO: 99), VPGP (SEQ ID NO: 101), VPIP (SEQ ID NO: 103), VPKP (SEQ ID NO: 105), VPLP (SEQ ID NO: 107), VPNP (SEQ ID NO: 109), VPPP (SEQ ID NO: 111), VPQP (SEQ ID NO: 113), VPRP (SEQ ID NO: 115), VPSP (SEQ ID NO: 117), VPTP (SEQ ID NO: 119), VPVP (SEQ ID NO: 121), VRAP (SEQ ID NO: 123), VRGP (SEQ ID NO: 125), VRPP (SEQ ID NO: 127), VRSP (SEQ ID NO: 129), VRTP (SEQ ID NO: 131), VSAP (SEQ ID NO: 132), VSDP (SEQ ID NO: 134), VSEP (SEQ ID NO: 136), VSGP (SEQ ID NO: 138), VSIP (SEQ ID NO: 140), VSKP (SEQ ID NO: 142), VSLP (SEQ ID NO: 144), VSNP (SEQ ID NO: 146), VSPP (SEQ ID NO: 148), VSQP (SEQ ID NO: 150), VSRP (SEQ ID NO: 152), VSTP (SEQ ID NO: 154), VSVP (SEQ ID NO: 155), VTAP (SEQ ID NO: 157), VTDP (SEQ ID NO: 159), VTEP (SEQ ID NO: 161), VTGP (SEQ ID NO: 163), VTIP (SEQ ID NO: 165), VTKP (SEQ ID NO: 167), VTLP (SEQ ID NO: 169), VTNP (SEQ ID NO: 171), VTPP (SEQ ID NO: 173), VTQP (SEQ ID NO: 175), VTRP (SEQ ID NO: 177), VTSP (SEQ ID NO: 179), VTTP (SEQ ID NO: 181), VTVP (SEQ ID NO: 183), VVGP (SEQ ID NO: 185), WPP (SEQ ID NO: 187), VVSP (SEQ ID NO: 189), or VVTP (SEQ ID NO: 191).

6. The single-domain antibody of claim 5, wherein the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, an amino acid sequence selected from VR, VG, VP, VA, VPG, VDG, VPQ, VPA, VAGG (SEQ ID NO: 5), VAPG (SEQ ID NO: 11), VAQP (SEQ ID NO: 14), VPAG (SEQ ID NO: 94), VPGG (SEQ ID NO: 100), VPPG (SEQ ID NO: 110), VPQG (SEQ ID NO: 112), VPAP (SEQ ID NO: 95), VPGP (SEQ ID NO: 101), VPLP (SEQ ID NO: 107), VGP, VGAG (SEQ ID NO: 41), VGGG (SEQ ID NO: 47), VGQG (SEQ ID NO: 59), VDGG (SEQ ID NO: 23), or VDAP (SEQ ID NO: 22).

7. The single-domain antibody of claim 5 or 6, wherein the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, an amino acid sequence selected from VAGG (SEQ ID NO: 5), VPA, VAPG (SEQ ID NO: 11), VAQP (SEQ ID NO: 14), VPAG (SEQ ID NO: 94), VPGG (SEQ ID NO: 100), VPPG (SEQ ID NO: 110), VPQ, VPQG (SEQ ID NO: 112), VGAG (SEQ ID NO: 41), VGGG (SEQ ID NO: 47), VGQG (SEQ ID NO: 59), VDGG (SEQ ID NO: 23), or VDAP (SEQ ID NO: 22).

8. The single-domain antibody of any one of claims 5-7, wherein the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, the amino acid sequence VAGG (SEQ ID NO: 5) or VPAG (SEQ ID NO: 94).

9. The single-domain antibody of any one of claims 1-8, wherein the single-domain antibody further comprises one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108.

10. The single-domain antibody of claim 9, wherein(a) the Leu (L) at position 11 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(b) the Gin (Q) at position 13 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(c) the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y);(d) the Gly (G) at position 88 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(e) the Vai (V) or lie (I) at position 89 is mutated to Ala (A), Asp (D), Glu (E), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), or Tyr (Y); and / or(f) the Leu (L) or Gin (Q) at position 108 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

11. The single-domain antibody of claim 9, wherein the single-domain antibody comprises at least an amino acid substitution at position 87.

12. The single-domain antibody of claim 11, wherein the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y).

13. The single-domain antibody of claim 12, wherein the Thr (T) at position 87 is mutated to Ala (A), Ser (S), or Vai (V).

14. A single-domain antibody which is modified to comprise one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108, wherein(a) the Leu (L) at position 11 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(b) the Gin (Q) at position 13 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(c) the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y);(d) the Gly (G) at position 88 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(e) the Vai (V) or lie (I) at position 89 is mutated to Ala (A), Asp (D), Glu (E), Lys (K), Leu (L), Asn ( N ), Gin (Q), Arg (R), Ser (S), Thr (T), or Tyr (Y); and / or(f) the Leu (L) or Gin (Q) at position 108 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

15. The single-domain antibody of claim 14, wherein the single-domain antibody comprises at least an amino acid substitution at position 87.

16. The single-domain antibody of claim 15, wherein the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y).

17. The single-domain antibody of claim 16, wherein the Thr (T) at position 87 is mutated to Ala (A), Ser (S), or Vai (V).

18. The single-domain antibody of any one of claims 1-17, wherein the single-domain antibody has reduced binding by anti-drug antibodies (ADAs) to its C-terminus as compared to an unmodified single-domain antibody.

19. The single-domain antibody of claim 18, wherein the unmodified single-domain antibody comprises VSS at the C-terminus.

20. The single-domain antibody of claim 18 or 19, wherein the single-domain antibody has at least about 80% reduced ADA binding to its C-terminus as compared to an unmodified single-domain antibody.

21. The single-domain antibody of claim 20, wherein the single-domain antibody has at least about 85% reduced ADA binding to its C-terminus as compared to an unmodified single-domain antibody.

22. The single-domain antibody of claim 21, wherein the single-domain antibody has at least about 90% ADA binding to its C-terminus as compared to an unmodified single-domain antibody.

23. The single-domain antibody of any one of claims 18-22, wherein the ADA binding is measured using an enzyme-linked immunosorbent assay (ELISA).

24. The single-domain antibody of any one of claims 1-23, wherein the single-domain antibody is a VHH, or a VH domain.

25. The single-domain antibody of claim 24, wherein the single-domain antibody is a camelid VHH.

26. The single-domain antibody of claim 24, wherein the single-domain antibody is a humanized VHH.

27. The single-domain antibody of claim 24, wherein the single-domain antibody is a camelized VH.

28. A fusion protein comprising one or more of the single-domain antibody of any one of claims 1- 27, wherein at least one single-domain antibody is at the carboxy-terminus of the fusion protein.

29. A conjugate comprising the single-domain antibody of any one of claims 1-27 or the fusion protein of claim 28, wherein the single-domain antibody or fusion protein is conjugated to a second moiety.

30. A polynucleotide molecule encoding the single-domain antibody of any one of claims 1-27 or the fusion protein of claim 28.

31. A recombinant vector comprising the polynucleotide molecule of claim 30.

32. A host cell comprising the polynucleotide molecule of claims 30, or the recombinant vector of claim 31.

33. A kit comprising the single-domain antibody of any one of claims 1-27, the fusion protein of claim 28, the conjugate of claim 29, the polynucleotide molecule of claim 30, or the recombinant vector of claim 31, and optionally, instructions and / or packaging for the same.

34. A method for producing a single-domain antibody or a fusion protein comprising expressing the polynucleotide sequence of claim 30 or the recombinant vector of claim 31 in a host cell.

35. A composition comprising the single-domain antibody of any one of claims 1-27, the fusion protein of claim 28, the conjugate of claim 29, the polynucleotide molecule of claim 30, or the recombinant vector of claim 31, and at least one carrier, diluent or excipient.

36. A method of modifying a single-domain antibody, the method comprising mutating the amino acid sequence, at the carboxy-terminus of the single-domain antibody starting from position 111 according to Chothia, to an amino acid sequence selected from(1) V,(2) VX1;(3) VXiX2,(4) VX1X2G, or(5) VX1X2P;wherein Xi is selected from amino acids Ala (A), Asp (D), Glu (E), Gly (G), lie (I), Lys (K), Leu (L),Asn (N), Pro (P), Arg (R), Ser (S), Thr (T), and Vai (V), andX2 is selected from amino acids Ala (A), Asp (D), Glu (E), Gly (G), He (I), Lys (K), Leu (L), Asn (N), Pro (P), Gin (Q), Arg (R), Ser (S), Thr (T), and Vai (V).

37. The method of claim 36, wherein Xi is selected from Ala (A), Gly (G), Pro (P), Asp (D), and Leu (L); and / or X2 is selected from amino acids Ala (A), Gly (G), Pro ( P), Asp (D), Gin (Q).. and Leu (L).

38. The method of claim 37, wherein Xi is selected from Ala (A), Gly (G), and Pro (P); and / or X2is selected from amino acids Ala (A), Gly (G), Gin (Q.), and Pro (P).

39. The method of any one of claims 36-38, wherein the amino acid sequence is not one selected from VSS, VE, VEG, VEP, VEPG (SEQ ID NO: 35), VK, VKS, VKG, VKP, VKPG (SEQ ID NO: 294), VQS, VS, VSE, VSEG (SEQ ID NO: 135), VSK, VSKG (SEQ ID NO: 141), VRP, VRPG (SEQ ID NO: 126), VDP, VDPG (SEQ ID NO: 25), VSSP (SEQ ID NO: 295), and VSSG (SEQ ID NO: 286).

40. The method of claim 36, wherein the amino acid sequence is selected from(1) V;(2) VA, VD, VE, VG, VI, VK, VL, VN, VP, VR, VS, VT, or VV;(3) VAA, VAD, VAE, VAG, VAI, VAK, VAL, VAN, VAP, VAQ, VAR, VAS, VAT, VAV, VDA, VDD, VDE, VDG, VDI, VDK, VDL, VDN, VDP, VDQ, VDR, VDS, VDT, VDV, VEA, VED, VEE, VEG, VEI, VEK, VEL, VEN, VEP, VEQ, VER, VES, VET, VEV, VGA, VGD, VGE, VGG, VGI, VGK, VGL, VGN, VGP, VGQ, VGR, VGS, VGT, VGV, VIA, VID, VIE, VIG, VII, VIK, VIL, VIN, VIP, VIQ, VIR, VIS, VIT, VIV, VLA, VLD, VLE, VLG, VLI, VLK, VLL, VLN, VLP, VLQ, VLR, VLS, VLT, VLV, VNA, VND, VNE, VNG, VNI, VNK, VNL, VNN, VNP, VNQ, VNR, VNS, VNT, VNV, VPA, VPD, VPE, VPG, VPI, VPK, VPL, VPN, VPP, VPQ, VPR, VPS, VPT, VPV, VRA, VRD, VRE, VRG, VRI, VRK, VRL, VRN, VRP, VRQ, VRR, VRS, VRT, VRV, VSA, VSD, VSE, VSG, VSI, VSK, VSL, VSN, VSP, VSQ, VSR, VST, VSV, VTA, VTD, VTE, VTG, VTI, VTK, VTL, VTN, VTP, VTQ, VTR, VTS, VTT, VTV, VVA, VVD, WE, VVG, VVI, WK, WL, WN, WP, WQ, WR, WS, WT, or VW;(4) VADG (SEQ ID NO: 1), VAEG (SEQ ID NO: 3), VAGG (SEQ ID NO: 5), VAKG (SEQ ID NO: 7), VANG (SEQ ID NO: 9), VAPG (SEQ ID NO: 11), VAQG (SEQ ID NO: 13), VARG (SEQ ID NO: 15), VASG (SEQ ID NO: 17), VATG (SEQ ID NO: 19), VDAG (SEQ ID NO: 21), VDGG (SEQ ID NO: 23), VDPG (SEQ ID NO: 25), VDSG (SEQ ID NO: 27), VDTG (SEQ ID NO: 29), VEAG (SEQ ID NO: 31), VEGG (SEQ ID NO: 33), VEPG (SEQID NO: 35), VESG (SEQ ID NO: 37), VETG (SEQ ID NO: 39), VGAG (SEQ ID NO: 41), VGDG (SEQ ID NO: 43), VGEG (SEQ ID NO: 45), VGGG (SEQ ID NO: 47), VGIG (SEQ ID NO: 49), VGKG (SEQ ID NO: 51), VGLG (SEQ ID NO: 53), VGNG (SEQ ID NO: 55), VGPG (SEQ ID NO: 57), VGQG (SEQ ID NO: 59), VGRG (SEQ ID NO: 61), VGSG (SEQ ID NO: 63), VGTG (SEQ ID NO: 65), VGVG (SEQ ID NO: 67), VIGG (SEQ ID NO: 69), VIPG (SEQ ID NO: 71), VISG (SEQ ID NO: 73), VITG (SEQ ID NO: 75), VLGG (SEQ ID NO: 77), VLPG (SEQ ID NO: 79), VLSG (SEQ ID NO: 283), VLTG (SEQ ID NO: 82), VNAG (SEQ ID NO: 84), VNGG (SEQ ID NO: 86), VNPG (SEQ ID NO: 88), VNSG (SEQ ID NO: 90), VNTG (SEQ ID NO: 92), VPAG (SEQ ID NO: 94), VPDG (SEQ ID NO: 96), VPEG (SEQ ID NO: 98), VPGG (SEQ ID NO: 100), VPIG (SEQ ID NO: 102), VPKG (SEQ ID NO: 104), VPLG (SEQ ID NO: 106), VPNG (SEQ ID NO: 108), VPPG (SEQ ID NO: 110), VPQG (SEQ ID NO: 112), VPRG (SEQ ID NO: 114), VPSG (SEQ ID NO: 116), VPTG (SEQ ID NO: 118), VPVG (SEQ ID NO: 120), VRAG (SEQ ID NO: 122), VRGG (SEQ ID NO: 124), VRPG (SEQ ID NO: 126), VRSG (SEQ ID NO: 128), VRTG (SEQ ID NO: 130), VSAG (SEQ ID NO: 284), VSDG (SEQ ID NO: 133), VSEG (SEQ ID NO: 135), VSGG (SEQ ID NO: 137), VSIG (SEQ ID NO: 139), VSKG (SEQ ID NO: 141), VSLG (SEQ ID NO: 143), VSNG (SEQ ID NO: 145), VSPG (SEQ ID NO: 147), VSQG (SEQ ID NO: 149), VSRG (SEQ ID NO: 151), VSTG (SEQ ID NO: 153), VSVG (SEQ ID NO: 285), VTAG (SEQ ID NO: 156), VTDG (SEQ ID NO: 158), VTEG (SEQ ID NO: 160), VTGG (SEQ ID NO: 162), VTIG (SEQ ID NO: 164), VTKG (SEQ ID NO: 166), VTLG (SEQ ID NO: 168), VTNG (SEQ ID NO: 170), VTPG (SEQ ID NO: 172), VTQG (SEQ ID NO: 174), VTRG (SEQ ID NO: 176), VTSG (SEQ ID NO: 178), VTTG (SEQ ID NO: 180), VTVG (SEQ ID NO: 182), WGG (SEQ ID NO: 184), WPG (SEQ ID NO: 186), WSG (SEQ ID NO: 188), or VVTG (SEQ ID NO: 190); or(5) VADP (SEQ ID NO: 2), VAEP (SEQ ID NO: 4), VAGP (SEQ ID NO: 6), VAKP (SEQ ID NO: 8), VANP (SEQ ID NO: 10), VAPP (SEQ ID NO: 12), VAQP (SEQ ID NO: 14), VARP (SEQ ID NO: 16), VASP (SEQ ID NO: 18), VATP (SEQ ID NO: 20), VDAP (SEQ ID NO: 22), VDGP (SEQ ID NO: 24), VDPP (SEQ ID NO: 26), VDSP (SEQ ID NO: 28), VDTP (SEQ ID NO: 30), VEAP (SEQ ID NO: 32), VEGP (SEQ ID NO: 34), VEPP (SEQ ID NO: 36), VESP (SEQ ID NO: 38), VETP (SEQ ID NO: 40), VGAP (SEQ ID NO: 42), VGDP (SEQ ID NO: 44), VGEP (SEQ ID NO: 46), VGGP (SEQ ID NO: 48), VGIP (SEQ ID NO: 50), VGKP (SEQ ID NO: 52), VGLP (SEQ ID NO: 54), VGNP (SEQ ID NO: 56), VGPP (SEQ ID NO: 58), VGQP (SEQ ID NO: 60), VGRP (SEQ ID NO: 62), VGSP (SEQ ID NO: 64), VGTP (SEQ ID NO: 66), VGVP (SEQ ID NO: 68), VIGP (SEQ ID NO: 70), VIPP (SEQ ID NO: 72), VISP (SEQ ID NO: 74), VITP (SEQ ID NO: 76), VLGP (SEQ ID NO: 78), VLPP (SEQ ID NO: 80), VLSP (SEQ ID NO: 81), VLTP (SEQ ID NO: 83), VNAP (SEQ ID NO: 85), VNGP (SEQ ID NO: 87), VNPP (SEQ ID NO: 89), VNSP (SEQ ID NO: 91), VNTP (SEQ ID NO: 93), VPAP (SEQ ID NO: 95), VPDP (SEQ ID NO: 97), VPEP (SEQ ID NO: 99), VPGP (SEQ ID NO: 101), VPIP (SEQ ID NO: 103), VPKP (SEQ ID NO: 105), VPLP (SEQ ID NO: 107), VPNP (SEQ ID NO: 109), VPPP (SEQ ID NO: 111), VPQP (SEQ ID NO: 113), VPRP (SEQ ID NO: 115), VPSP(SEQ ID NO: 117), VPTP (SEQ ID NO: 119), VPVP (SEQ ID NO: 121), VRAP (SEQ ID NO: 123), VRGP (SEQ ID NO: 125), VRPP (SEQ ID NO: 127), VRSP (SEQ ID NO: 129), VRTP (SEQ ID NO: 131), VSAP (SEQ ID NO: 132), VSDP (SEQ ID NO: 134), VSEP (SEQ ID NO: 136), VSGP (SEQ ID NO: 138), VSIP (SEQ ID NO: 140), VSKP (SEQ ID NO: 142), VSLP (SEQ ID NO: 144), VSNP (SEQ ID NO: 146), VSPP (SEQ ID NO: 148), VSQP (SEQ ID NO: 150), VSRP (SEQ ID NO: 152), VSTP (SEQ ID NO: 154), VSVP (SEQ ID NO: 155), VTAP (SEQ ID NO: 157), VTDP (SEQ ID NO: 159), VTEP (SEQ ID NO: 161), VTGP (SEQ ID NO: 163), VTIP (SEQ ID NO: 165), VTKP (SEQ ID NO: 167), VTLP (SEQ ID NO: 169), VTNP (SEQ ID NO: 171), VTPP (SEQ ID NO: 173), VTQP (SEQ ID NO: 175), VTRP (SEQ ID NO: 177), VTSP (SEQ ID NO: 179), VTTP (SEQ ID NO: 181), VTVP (SEQ ID NO: 183), VVGP (SEQ ID NO: 185), WPP (SEQ ID NO: 187), VVSP (SEQ ID NO: 189), or VVTP (SEQ ID NO: 191).

41. The method of claim 40, wherein the amino acid sequence is selected from VR, VG, VP, VA, VPG, VDG, VPQ, VPA, VAGG (SEQ ID NO: 5), VAPG (SEQ ID NO: 11), VAQP (SEQ ID NO: 14), VPAG (SEQ ID NO: 94), VPGG (SEQ ID NO: 100), VPPG (SEQ ID NO: 110), VPQG (SEQ ID NO: 112), VPAP (SEQ ID NO: 95), VPGP (SEQ ID NO: 101), VPLP (SEQ ID NO: 107), VGP, VGAG (SEQ ID NO: 41), VGGG (SEQ ID NO: 47), VGQG (SEQ ID NO: 59), VDGG (SEQ ID NO: 23), or VDAP (SEQ ID NO: 22).

42. The single-domain antibody of claim 40 or 41, wherein the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, an amino acid sequence selected from VAGG (SEQ ID NO: 5), VAPG (SEQ ID NO: 11), VAQP (SEQ ID NO: 14), VPA, VPAG (SEQ ID NO: 94), VPGG (SEQ ID NO: 100), VPPG (SEQ ID NO: 110), VPQ, VPQG (SEQ ID NO: 112), VGAG (SEQ ID NO: 41), VGGG (SEQ ID NO: 47), VGQG (SEQ ID NO: 59), VDGG (SEQ ID NO: 23), or VDAP (SEQ ID NO: 22).

43. The single-domain antibody of any one of claims 40-42, wherein the single-domain antibody comprises, at the carboxy-terminus starting from position 111 according to Chothia, the amino acid sequence VAGG (SEQ ID NO: 5) or VPAG (SEQ ID NO: 94).

44. The method of any one of claims 36-42, wherein the method comprises introducing into the single-domain antibody one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108.

45. The method of claim 44, wherein(a) the Leu (L) at position 11 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(b) the Gin (Q.) at position 13 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(c) the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y);(d) the Gly (G) at position 88 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(e) the Vai (V) or lie (I) at position 89 is mutated to Ala (A), Asp (D), Glu (E), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), or Tyr (Y); and / or(f) the Leu (L) or Gin (Q) at position 108 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

46. The method of claim 44, wherein the single-domain antibody comprises at least an amino acid substitution at position 87.

47. The method of claim 46, wherein the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y).

48. The method of claim 47, wherein the Thr (T) at position 87 is mutated to Ala (A), Ser (S), or Vai (V).

49. A method of modifying a single-domain antibody, the method comprising introducing into the single-domain antibody one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108, wherein(a) the Leu (L) at position 11 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(b) the Gin (Q) at position 13 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(c) the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y);(d) the Gly (G) at position 88 is mutated to Ala (A), Asp ( D), Glu (E), He (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y);(e) the Vai (V) or He (I) at position 89 is mutated to Ala (A), Asp (D), Glu (E), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Thr (T), or Tyr (Y); and / or(f) the Leu (L) or Gin (Q) at position 108 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Asn (N), Arg (R), Ser (S), Thr (T), Vai (V), or Tyr (Y).

50. The method of claim 49, wherein the single-domain antibody comprises at least an amino acid substitution at position 87.

51. The method of claim 50, wherein the Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), lie (I), Lys (K), Leu (L), Asn (N), Gin (Q), Arg (R), Ser (S), Vai (V), or Tyr (Y).

52. The method of claim 51, wherein the Thr (T) at position 87 is mutated to Ala (A), Ser (S), or Vai (V).

53. The method of any one of claims 36-52, wherein the single-domain antibody has reduced binding by anti-drug antibodies (ADAs) to its C-terminus as compared to an unmodified singledomain antibody.

54. The method of claim 53, wherein the unmodified single-domain antibody comprises VSS at the C-terminus.

55. The method of claim 53 or 54, wherein the single-domain antibody has at least about 80% reduced ADA binding to its C-terminus as compared to an unmodified single-domain antibody.

56. The method of claim 55, wherein the single-domain antibody has at least about 85% reduced ADA binding to its C-terminus as compared to an unmodified single-domain antibody.

57. The method of claim 56, wherein the single-domain antibody has at least about 90% ADA binding to its C-terminus as compared to an unmodified single-domain antibody.

58. The method of any one of claims 55-57, wherein the ADA binding is measured using an enzyme- linked immunosorbent assay (ELISA).

59. The method of any one of claims 36-58, wherein the single-domain antibody is a VHH, or a VH domain.

60. The method of claim 59, wherein the single-domain antibody is a camelid VHH.

61. The method of claim 59, wherein the single-domain antibody is a humanized VHH.

62. The method of claim 59, wherein the single-domain antibody is a camelized VH.

63. The method of any one of claims 36-62, wherein the single-domain antibody is present in a fusion protein, and the single-domain antibody is at the carboxy-terminus of the fusion protein.