Modified single-domain antibody

Abstract

This disclosure provides a single-domain antibody comprising, for example, a single-domain antibody at the carboxyl terminus (C-terminus) that reduces the binding of the single-domain antibody to the carboxyl terminus by an anti-drug antibody compared to an unmodified single-domain antibody. This application also provides fusion proteins and conjugates comprising the single-domain antibody, polynucleotides and recombinant vectors encoding the single-domain antibody, compositions and kits comprising the same, and host cells and methods for preparing the single-domain antibody.

Description

Cross-reference of related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 467,208, filed 17 May 2023, the disclosures of the said Provisional Patent Application are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing, which has been electronically filed in XML format and is incorporated herein by reference in its entirety. This XML copy, created on 30 April 2024, has the filename 260525_000044_SL.xml and a size of 257,846 bytes. [Technical Field]

[0003] This disclosure relates to a single-domain antibody comprising, for example, a carboxyl terminus (C-terminus) that reduces the binding of an anti-drug antibody to the carboxyl terminus of the single-domain antibody compared to an unmodified single-domain antibody, a method for preparing the same, and the use thereof. [Background technology]

[0004] Drug-induced immunogenicity is a major challenge in the development of biopharmaceuticals, as it can lead to reduced efficacy and adverse events in clinical settings. During clinical development, it is crucial to evaluate the immunogenicity potential of candidate biopharmaceuticals, particularly the extent to which they induce immune responses leading to the formation of anti-drug antibodies (ADAs). From a mechanistic perspective, ADAs can lead to suboptimal exposure and loss of efficacy by inactivating drugs and promoting enhanced clearance and / or loss of targeting of ADA-drug conjugates. For example, pre-existing ADAs against antibody fragments, including fragment antigen-binding (Fab) fragments, human heavy chain variable domains (VH), and single-domain antibodies (e.g., VHH), were first reported in drug-naive individuals more than 10 years ago. While up to 50% of healthy humans have been reported to possess pre-existing anti-VHH ADAs, the origin of these ADAs remains unclear. However, it has been confirmed that the carboxyl terminus (C-terminus) of VHH outside the IgG context can be a dominant epitope for these ADAs. Therefore, in this field of technology, there is a need to develop non-immunogenic VHH or functional fragments thereof that have been engineered to prevent or inhibit existing ADA from binding to VHH via its C-terminus or other means. [Overview of the project] [Problems that the invention aims to solve]

[0005] As described in the "Background Technology" section above, there is an unmet need in the field of technology for developing non-immunogenic VHHs or functional fragments thereof that have been engineered to prevent or inhibit existing ADAs from binding to VHHs, for example, via their C-terminus. This application provides compositions and methods to address this need and other related needs. [Means for solving the problem]

[0006] In one embodiment, the disclosure relates to a carboxyl terminus starting at position 111 according to Chothia, (1) V, (2) VX1, (3) VX1X2, (4) VX1X2G, or (5) VX1X2P [X1 is selected from the amino acids Ala(A), Asp(D), Glu(E), Gly(G), Ile(I), Lys(K), Leu(L), Asn(N), Pro(P), Arg(R), Ser(S), Thr(T), and Val(V), X2 is selected from the amino acids Ala(A), Asp(D), Glu(E), Gly(G), Ile(I), Lys(K), Leu(L), Asn(N), Pro(P), Gln(Q), Arg(R), Ser(S), Thr(T), and Val(V). We provide a single-domain antibody modified to contain an amino acid sequence selected from the following.

[0007] In some embodiments, X1 is selected from Ala(A), Gly(G), Pro(P), Asp(D), and Leu(L); and / or X2 is selected from the amino acids Ala(A), Gly(G), Pro(P), Asp(D), Gln(Q), and Leu(L).

[0008] In some embodiments, X1 is selected from Ala(A), Gly(G), and Pro(P); and / or X2 is selected from the amino acids Ala(A), Gly(G), Gln(Q), and Pro(P).

[0009] In some embodiments, the amino acid sequence is not 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 has a carboxy terminus starting at position 111 according to Chothia, (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, VVE, VVG, VVI, VVK, VVL, VVN, VVP, VVQ, VVR, VVS, VVT, or VVV; (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: 49), 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: 75), VLGG (SEQ ID NO: 75), VLGG (SEQ ID NO: 49), VGKG (SEQ ID NO: 51), VGLG (SEQ ID NO: 53), VGNG (SEQ ID NO: 55), Number 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), VP RG (sequence number 114), VPSG (sequence number 116), VPTG (sequence number 118), VPVG (sequence number 120), VRAG (sequence number 122), VRGG (sequence number 124), VRPG (sequence number 126), VRSG (sequence number 128), VRTG (sequence number 130), VSAG (sequence number 284), VSDG (sequence number 133), VSEG (sequence number 135), VSGG (sequence number 137), VSIG (sequence number 139), VSKG (sequence number 141), VSLG (sequence number 143), VSNG (sequence number 145), VSPG (sequence number 147),VSQG (sequence number 149), VSRG (sequence number 151), VSTG (sequence number 153), VSVG (sequence number 285), VTAG (sequence number 156), VTDG (sequence number 158), VTEG (sequence number 160), VTGG (sequence number 162), VTIG (sequence number 164), VTKG (sequence number 166), VTLG (sequence number 168), VTNG (sequence number 170), VTPG (sequence number 172), VTQG (sequence number 174), VTRG (sequence number 176), VTSG (sequence number 178), VTTG (sequence number 180), VTVG (sequence number 182), VVGG (sequence number 184), VVPG (sequence number 186), VVSG (sequence number 188), or VVTG (sequence number 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), VET P (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 (Distribution) Column number 78), VLPP (sequence number 80), VLSP (sequence number 81), VLTP (sequence number 83), VNAP (sequence number 85), VNGP (sequence number 87), VNPP (sequence number 89), VNSP (sequence number 91), VNTP (sequence number 93), VPAP (sequence number 95), VPDP (sequence number 97), VPEP (sequence number 99), VPGP (sequence number 101), VPIP (sequence number 103), VPKP (sequence number 105), VPLP (sequence number 107), VPNP (sequence number 109), VPPP (sequence number 111), VPQP (sequence number 113), VP RP (sequence code 115), VPSP (sequence code 117), VPTP (sequence code 119), VPVP (sequence code 121), VRAP (sequence code 123), VRGP (sequence code 125), VRPP (sequence code 127), VRSP (sequence code 129), VRTP (sequence code 131), VSAP (sequence code 132), VSDP (sequence code 134), VSEP (sequence code 136), VSGP (sequence code 138), VSIP (sequence code 140), VSKP (sequence code 142), VSLP (sequence code 144), VSNP (sequence code 146), VSPP (sequence code 148),VSQP (sequence number 150), VSRP (sequence number 152), VSTP (sequence number 154), VSVP (sequence number 155), VTAP (sequence number 157), VTDP (sequence number 159), VTEP (sequence number 161), VTGP (sequence number 163), VTIP (sequence number 165), VTKP (sequence number 167), VTLP (sequence number 169), VTNP (sequence number 171), VTPP (sequence number 173), VTQP (sequence number 175), VTRP (sequence number 177), VTSP (sequence number 179), VTTP (sequence number 181), VTVP (sequence number 183), VVGP (sequence number 185), VVPP (sequence number 187), VVSP (sequence number 189), or VVTP (sequence number 191), It contains an amino acid sequence selected from the following.

[0011] In some embodiments, the single-domain antibody contains 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) at the carboxy terminus starting at position 111 according to Chothia.

[0012] In some embodiments, the single-domain antibody contains 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) at the carboxy terminus starting at position 111 according to Chothia.

[0013] In some embodiments, the single-domain antibody comprises, at the carboxy terminus starting at 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, Leu (L) at position 11 is mutated to Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Asn (N), Gln (Q), Arg (R), Ser (S), Thr (T), Val (V), or Tyr (Y); Gln (Q) at position 13 is mutated to Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Leu (L), Asn (N), Arg (R), Ser (S), Thr (T), Val (V), or Tyr (Y); Thr (T) at position 87 is mutated to Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Leu (L), Asn (N), Gln (Q), Arg (R), Ser (S), Val (V), or Tyr (Y); Gly (G) at position 88 is mutated to Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Leu (L), Asn (N), Gln (Q), Arg (R), Ser (S), Thr (T), Val (V), or Tyr (Y); Val (V) or Ile (I) at position 89 is mutated to Ala (A), Asp (D), Glu (E), Lys (K), Leu (L), Asn (N), Gln (Q), Arg (R), Ser (S), Thr (T), or Tyr (Y); and / or Leu (L) or Gln (Q) at position 108 is mutated to Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Asn (N), Arg (R), Ser (S), Thr (T), Val (V), or Tyr (Y).

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

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

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

[0019] In another aspect, the Disclosure provides a single-domain antibody modified to include one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108. Leu (L), ranked 11th, is mutated to Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Asn (N), Gln (Q), Arg (R), Ser (S), Thr (T), Val (V), or Tyr (Y); The 13th most important gene, Gln(Q), is mutated into Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Leu(L), Asn(N), Arg(R), Ser(S), Thr(T), Val(V), or Tyr(Y); The 87th thr (T) is mutated to Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Leu (L), Asn (N), Gln (Q), Arg (R), Ser (S), Val (V), or Tyr (Y); Gly (G), ranked 88th, has mutated to Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Leu (L), Asn (N), Gln (Q), Arg (R), Ser (S), Thr (T), Val (V), or Tyr (Y); Val(V) or Ile(I) at position 89 is mutated to Ala(A), Asp(D), Glu(E), Lys(K), Leu(L), Asn(N), Gln(Q), Arg(R), Ser(S), Thr(T), or Tyr(Y); and / or Leu(L) or Gln(Q), ranked 108th, has mutated into Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Asn(N), Arg(R), Ser(S), Thr(T), Val(V), or Tyr(Y).

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

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

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

[0023] In some embodiments, single-domain antibodies exhibit reduced binding to their C-terminus by anti-drug antibodies (ADAs) compared to unmodified single-domain antibodies.

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

[0025] In some embodiments, single-domain antibodies exhibit at least approximately 80% less ADA binding to their C-terminus compared to unmodified single-domain antibodies.

[0026] In some embodiments, single-domain antibodies exhibit at least approximately 85% less ADA binding to their C-terminus compared to unmodified single-domain antibodies.

[0027] In some embodiments, single-domain antibodies exhibit at least approximately 90% less ADA binding to their C-terminus compared to unmodified single-domain antibodies.

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

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

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

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

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

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

[0034] In another embodiment, the Disclosure provides a conjugate comprising a single-domain antibody or a fusion protein as described herein, wherein the single-domain antibody or fusion protein is conjugated to a second portion.

[0035] In another aspect, the Disclosure provides polynucleotide molecules encoding a single-domain antibody or a fusion protein as described herein.

[0036] In another aspect, the present disclosure provides recombinant vectors comprising polynucleotide molecules described herein.

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

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

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

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

[0041] In another aspect, the present disclosure provides a method for modifying a single-domain antibody, wherein the amino acid sequence of the carboxy-terminus of a single-domain antibody starting at position 111 according to Chothia, (1) V, (2) VX1, (3) VX1X2, (4) VX1X2G, or (5) VX1X2P [X1 is selected from the amino acids Ala(A), Asp(D), Glu(E), Gly(G), Ile(I), Lys(K), Leu(L), Asn(N), Pro(P), Arg(R), Ser(S), Thr(T), and Val(V), X2 is selected from the amino acids Ala(A), Asp(D), Glu(E), Gly(G), Ile(I), Lys(K), Leu(L), Asn(N), Pro(P), Gln(Q), Arg(R), Ser(S), Thr(T), and Val(V). This includes mutating to an amino acid sequence selected from the available options.

[0042] In some embodiments, X1 is selected from Ala(A), Gly(G), Pro(P), Asp(D), and Leu(L); and / or X2 is selected from the amino acids Ala(A), Gly(G), Pro(P), Asp(D), Gln(Q), and Leu(L).

[0043] In some embodiments, X1 is selected from Ala(A), Gly(G), and Pro(P); and / or X2 is selected from the amino acids Ala(A), Gly(G), Gln(Q), and Pro(P).

[0044] In some embodiments, the amino acid sequence is not 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: (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, VVE, VVG, VVI, VVK, VVL, VVN, VVP, VVQ, VVR, VVS, VVT, or VVV; (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: 49), 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: 75), VLGG (SEQ ID NO: 75), VLGG (SEQ ID NO: 49), VGKG (SEQ ID NO: 51), VGLG (SEQ ID NO: 53), VGNG (SEQ ID NO: 55), Number 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), VP RG (sequence number 114), VPSG (sequence number 116), VPTG (sequence number 118), VPVG (sequence number 120), VRAG (sequence number 122), VRGG (sequence number 124), VRPG (sequence number 126), VRSG (sequence number 128), VRTG (sequence number 130), VSAG (sequence number 284), VSDG (sequence number 133), VSEG (sequence number 135), VSGG (sequence number 137), VSIG (sequence number 139), VSKG (sequence number 141), VSLG (sequence number 143), VSNG (sequence number 145), VSPG (sequence number 147),VSQG (sequence number 149), VSRG (sequence number 151), VSTG (sequence number 153), VSVG (sequence number 285), VTAG (sequence number 156), VTDG (sequence number 158), VTEG (sequence number 160), VTGG (sequence number 162), VTIG (sequence number 164), VTKG (sequence number 166), VTLG (sequence number 168), VTNG (sequence number 170), VTPG (sequence number 172), VTQG (sequence number 174), VTRG (sequence number 176), VTSG (sequence number 178), VTTG (sequence number 180), VTVG (sequence number 182), VVGG (sequence number 184), VVPG (sequence number 186), VVSG (sequence number 188), or VVTG (sequence number 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), VET P (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 (Distribution) Column number 78), VLPP (sequence number 80), VLSP (sequence number 81), VLTP (sequence number 83), VNAP (sequence number 85), VNGP (sequence number 87), VNPP (sequence number 89), VNSP (sequence number 91), VNTP (sequence number 93), VPAP (sequence number 95), VPDP (sequence number 97), VPEP (sequence number 99), VPGP (sequence number 101), VPIP (sequence number 103), VPKP (sequence number 105), VPLP (sequence number 107), VPNP (sequence number 109), VPPP (sequence number 111), VPQP (sequence number 113), VP RP (sequence code 115), VPSP (sequence code 117), VPTP (sequence code 119), VPVP (sequence code 121), VRAP (sequence code 123), VRGP (sequence code 125), VRPP (sequence code 127), VRSP (sequence code 129), VRTP (sequence code 131), VSAP (sequence code 132), VSDP (sequence code 134), VSEP (sequence code 136), VSGP (sequence code 138), VSIP (sequence code 140), VSKP (sequence code 142), VSLP (sequence code 144), VSNP (sequence code 146), VSPP (sequence code 148),VSQP (sequence number 150), VSRP (sequence number 152), VSTP (sequence number 154), VSVP (sequence number 155), VTAP (sequence number 157), VTDP (sequence number 159), VTEP (sequence number 161), VTGP (sequence number 163), VTIP (sequence number 165), VTKP (sequence number 167), VTLP (sequence number 169), VTNP (sequence number 171), VTPP (sequence number 173), VTQP (sequence number 175), VTRP (sequence number 177), VTSP (sequence number 179), VTTP (sequence number 181), VTVP (sequence number 183), VVGP (sequence number 185), VVPP (sequence number 187), VVSP (sequence number 189), or VVTP (sequence number 191), Selected from.

[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 contains 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) at the carboxy terminus starting at position 111 according to Chothia.

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

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

[0050] In some embodiments, Leu (L), ranked 11th, can be mutated into Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Asn (N), Gln (Q), Arg (R), Ser (S), Thr (T), Val (V), or Tyr (Y); The 13th position Gln(Q) can be mutated into Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Leu(L), Asn(N), Arg(R), Ser(S), Thr(T), Val(V), or Tyr(Y); The 87th position Thr(T) can be mutated into Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Leu(L), Asn(N), Gln(Q), Arg(R), Ser(S), Val(V), or Tyr(Y); Gly (G), at position 88, can be mutated into Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Leu (L), Asn (N), Gln (Q), Arg (R), Ser (S), Thr (T), Val (V), or Tyr (Y); Val(V) or Ile(I) at position 89 is mutated to Ala(A), Asp(D), Glu(E), Lys(K), Leu(L), Asn(N), Gln(Q), Arg(R), Ser(S), Thr(T), or Tyr(Y); and / or Leu(L) or Gln(Q) at position 108 can be mutated into Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Asn(N), Arg(R), Ser(S), Thr(T), Val(V), or Tyr(Y).

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

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

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

[0054] In another aspect, the Disclosure provides a method for modifying a single-domain antibody, the method comprising introducing one or more amino acid substitutions in the single-domain antibody at positions 11, 13, 87, 88, 89, and / or 108. Leu (L), ranked 11th, can be mutated into Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Asn (N), Gln (Q), Arg (R), Ser (S), Thr (T), Val (V), or Tyr (Y); The 13th position Gln(Q) can be mutated into Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Leu(L), Asn(N), Arg(R), Ser(S), Thr(T), Val(V), or Tyr(Y); The 87th position Thr(T) can be mutated into Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Leu(L), Asn(N), Gln(Q), Arg(R), Ser(S), Val(V), or Tyr(Y); Gly (G), at position 88, can be mutated into Ala (A), Asp (D), Glu (E), Ile (I), Lys (K), Leu (L), Asn (N), Gln (Q), Arg (R), Ser (S), Thr (T), Val (V), or Tyr (Y); Val(V) or Ile(I) at position 89 is mutated to Ala(A), Asp(D), Glu(E), Lys(K), Leu(L), Asn(N), Gln(Q), Arg(R), Ser(S), Thr(T), or Tyr(Y); and / or Leu(L) or Gln(Q) at position 108 can be mutated into Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Asn(N), Arg(R), Ser(S), Thr(T), Val(V), or Tyr(Y).

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

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

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

[0058] In some embodiments, single-domain antibodies exhibit reduced binding of anti-drug antibodies (ADAs) to their C-terminus compared to unmodified single-domain antibodies.

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

[0060] In some embodiments, single-domain antibodies exhibit at least approximately 80% less ADA binding to their C-terminus compared to unmodified single-domain antibodies.

[0061] In some embodiments, single-domain antibodies exhibit at least approximately 85% less ADA binding to their C-terminus compared to unmodified single-domain antibodies.

[0062] In some embodiments, single-domain antibodies have at least approximately 90% less ADA binding to their C-terminus compared to unmodified single-domain antibodies.

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

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

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

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

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

[0068] In some embodiments, the single-domain antibody is located within the fusion protein, or at the carboxyl terminus of the fusion protein. [Brief explanation of the drawing]

[0069] [Figure 1] Figure 1 shows the locations of engineering activity at the C-terminus and within the framework of VHH. Engineering at the C-terminus was concentrated on residues H112-113. Engineering within the framework was performed on residues H11, H13, H87-89, and H108. Each residue is indicated by a stick (PDB: 7LVU). [Figure 2A]Figures 2A-2E show annotations of VHH sequences based on Kabat and Chothia. Annotations of human DP47 germline VH sequences and exemplary camelid VHH sequences using Kabat and Chothia with H-numbering are shown. Framework regions 1-4 and CDR regions 1-3 are labeled. The positions used in the manipulation are highlighted in black. Sequence IDs 206 and 207 are disclosed in order of appearance in the figures. [Figure 2B] Continuation of Figure 2A. [Figure 2C] Continuation of Figure 2B. [Figure 2D] Continuation of Figure 2C. [Figure 2E] Continuation of Figure 2D. [Figure 3] Figure 3 shows a bar graph of the binding of pre-anti-drug antibodies (pre-ADA) (human IgG) to purified V bodies. 452 manipulated V bodies were incubated with human intravenous IgG (IVIg), and the binding of human IgG was detected. The original V body sequences, which were not manipulated in terms of framework or C-terminal sequence, were labeled as WT (wild-type) (black bars, dotted lines). Based on the absorbance values ​​of WT, a 90% reduction in signal was calculated and shown as a 90% reduction in the binding of pre-ADA (dashed line). Phosphate-buffered saline (PBS) was used as a background control. All values ​​shown are after subtracting blank values. [Figure 4A] Figures 4A–4W show bar graphs of the binding of pre-existing anti-drug antibodies (pre-ADA) (human IgG) to purified V bodies, normalized against the control VHH ODY-349. 452 purified and manipulated V bodies were incubated with IVIg, and human IgG binding was detected. PBS was used as a background control, and all values ​​shown have been subtracted from the blank value. The absorbance of the unmodified V body (ODY-349) was set to 1, and all values ​​were normalized accordingly. [Figure 4B] Continuation of Figure 4A. [Figure 4C] Continuation of Figure 4B. [Figure 4D] Continuation of Figure 4C. [Figure 4E] Continuation of Figure 4D. [Figure 4F] Continuation of Figure 4E. [Figure 4G] Continuation of Figure 4F. [Figure 4H] Continuation of Figure 4G. [Figure 4I] Continuation of Figure 4H. [Figure 4J] Continuation of Figure 4I. [Figure 4K] Continuation of Figure 4J. [Figure 4L] Continuation of Figure 4K. [Figure 4M] Continuation of Figure 4L. [Figure 4N] Continuation of Figure 4M. [Figure 4O] Continuation of Figure 4N. [Figure 4P] Continuation of Figure 4O. [Figure 4Q] Continuation of Figure 4P. [Figure 4R] Continuation of Figure 4Q. [Figure 4S] Continuation of Figure 4R. [Figure 4T] Continuation of Figure 4S. [Figure 4U] Continuation of Figure 4T. [Figure 4V] Continuation of Figure 4U. [Figure 4W] Continuation of Figure 4V. [Figure 5A]Figures 5A–5D show an overview of the binding of existing ADA (human IgG) to purified V bodies. These panels show normalized enzyme-linked immunosorbent assay (ELISA) values ​​(WT sequence = 1.000; PBS control subtracted) and their corresponding C-terminal sequences. Values ​​exceeding the WT value were capped at 100% (1.000). Along the vertical and horizontal axes, the amino acids at positions H112 and H113 are highlighted, respectively. The C-terminal 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 indicates V bodies below the concentration threshold for this experiment. Strikethrough V bodies were not tested. The grayscale ranges from white (0.000) to medium gray (0.500) and then to dark gray (1.000). [Figure 5B] Continuation of Figure 5A. [Figure 5C] Continuation of Figure 5B. [Figure 5D] Continuation of Figure 5C. [Figure 6] Figure 6 shows a heatmap of optical density (OD) values ​​for pre-existing ADA binding analysis for 24 V-body variants screened in individual serum samples from 50 healthy human donors. The grayscale codes are based on the average OD value per donor on a logarithmic scale. The grayscale shows labels on a logarithmic scale (values ​​in parentheses represent the original scale). The intensity of the grayscale represents the detected level of binding of pre-existing ADA to the purified V-bodies. Sequence numbers 5, 11, 23, 41, 47, 59, 94, 100, 110, 112, 14, 22, 95, 101, and 107 are disclosed in order of appearance in the figure. [Figure 7A]Figure 7A shows that C-terminal modification of a tetravalent V-body agonist targeting hepatocyte membrane proteins significantly reduced hepatotoxicity. The relative viability of primary hepatocytes, measured by the CellTiter-Glo assay (CTG), is plotted on the y-axis against the indicated concentrations (x-axis) of the wild-type V-body agonist (wt-Vtetra3) and modified Vtetra3-VPAG in the presence and absence of human intravenous IgG (IVIg) after 24-hour (hr) incubation (Figure 7A). The horizontal dashed line highlights, for comparison, the hepatocyte viability for the highest agonist concentration tested in the absence of IVIg. Structural models of the V-body agonist and modification (the C-terminal end) are also included at the bottom of the figure panel. [Figure 7B] Figure 7B shows that C-terminal modification of a tetravalent V-body agonist targeting hepatocyte membrane proteins significantly reduced hepatotoxicity. The relative viability of primary hepatocytes, measured by the CellTiter-Glo assay (CTG), is plotted on the y-axis against the indicated concentrations (x-axis) of the wild-type V-body agonist (wt-Vtetra3) and modified Vtetra3-VAGG in the presence and absence of IVIg after 24-hour incubation (Figure 7B). The horizontal dashed line highlights, for comparison, the hepatocyte viability for the highest agonist concentration tested in the absence of IVIg. Structural models of the V-body agonist and modification locations (C-terminal end) are also included at the bottom of the figure panel. [Modes for carrying out the invention]

[0070] definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. For the interpretation of this specification, the following definitions of terms shall apply, and where appropriate, a singular term shall also include its plural form, and vice versa. All patents, applications, published applications, and other publications are incorporated herein by reference in their entirety. If any definition of a term contained herein conflicts with any document incorporated herein by reference, the definition of the term contained below shall prevail.

[0071] Where used herein, the term "about" means, when used in relation to a particular number, that number may vary by no more than 5% from the stated number. For example, where used herein, the expression "about 100" includes 95 and 105, as well as any value in between (e.g., 96, 97, 98, 99, etc.).

[0072] The term “antigen” encompasses any factor (e.g., proteins, peptides, polysaccharides, glycoproteins, glycolipids, nucleotides, parts thereof, or combinations thereof) to which a specific humoral or cellular immune product, such as an antibody molecule or a T cell receptor, may specifically bind. In various embodiments of this disclosure, the antigens described herein may be human, cynomolgus monkey, and / or mouse antigens.

[0073] The term "epitope" refers 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. Therefore, multiple different antibodies may bind to different regions of a single antigen, resulting in different biological effects (e.g., agonist or antagonist effects). Epitopes can be structural or linear. Structural epitopes are formed by spatially juxtaposed amino acids derived from multiple different segments of a linear polypeptide chain. Linear epitopes are formed by adjacent amino acid residues within a single polypeptide chain. In some cases, they may include non-peptide moieties on the antigen, such as sugars, phosphoryl groups, or sulfonyl groups.

[0074] The terms "antibody" and "immunoglobulin" or "Ig" are used interchangeably and in their broadest sense, encompassing, for example, individual monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or complete monoclonal antibodies), antibody compositions having polyepitope or monoepitope specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies (e.g., VHH), single-chain antibodies, intrabodies, anti-idiotype (anti-Id) antibodies, and antigen-binding fragments of antibodies. Antibodies may be human antibodies, humanized antibodies, camelid antibodies, recombinant antibodies, chimeric antibodies, synthetic antibodies, affinity-demature antibodies, and / or affinity-mature antibodies, as well as antibodies derived from other species such as mice, camels, llamas, and rabbits. An "antigen-binding fragment" generally refers to a portion of an antibody heavy chain and / or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment originates. Non-limiting examples of antigen-binding fragments include single-domain antibodies (e.g., VHH), single-chain Fv(scFv), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-bonded Fv(sdFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies, or chemically modified derivatives thereof.Such antibody fragments are described, for example, in 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); Plueckthun and Skerra, Meth. Enzymol., 178:497-515 (1989); and Day, ED, Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, NY (1990). The antibodies provided herein may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a, and IgG2b) of immunoglobulin molecules.

[0075] Where used herein, the term “single-domain antibody” or “sdAb” refers to an antibody or antibody fragment that contains a single antibody variable domain capable of binding to a specific antigen and does not require another antibody variable domain. The complementarity-determining region (CDR) of a single-domain antibody is part of the single antibody variable domain. Examples of single-domain antibodies include, but are not limited to, heavy-chain antibodies, naturally occurring light-chain-free antibodies, single-domain antibodies derived from conventional four-chain antibodies, modified 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 mice, humans, camels, llamas, sharks, goats, rabbits, and / or cattle. In some embodiments, the single-domain antibodies used herein are naturally occurring single-domain antibodies known as light-chain-free heavy-chain antibodies. For clarity, variable domains derived from naturally occurring light-chain-free heavy-chain antibodies are referred to herein as VHH to distinguish them from VH of conventional four-chain immunoglobulins. Such VHH molecules may be derived from antibodies produced in the bodies of camelid species, such as camels, llamas, dromedaries, alpacas, and guanacos. Other species may also naturally produce heavy chain antibodies that do not contain light chains, and these are also within the scope of the present invention. For example, cartilaginous fish such as sharks can produce immunoglobulin-like structures known as VNARs. In some embodiments, single-domain antibodies can be obtained from camelid VH domains. In some embodiments, single-domain antibodies can be obtained from human VH by camelidization. For a review of single-domain antibodies, see Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608 (disclosures in this document are incorporated herein by reference).

[0076] When used in the context of single-domain antibodies, polypeptides, polynucleotides, and vectors, the term “isolated” means that the antibody, polypeptide, polynucleotide, and vector are, at least partially, free from other biological molecules derived from the cells or cell cultures in which they were produced. Such biological molecules include nucleic acids, proteins, other antibody or antigen-binding fragments, lipids, carbohydrates, or other substances such as cell residues and growth media. An isolated single-domain antibody may further be, at least partially, free from components of the expression system, such as host cell-derived biological molecules, or its growth medium. Generally, the term “isolated” is not intended to mean the complete absence of such biological molecules (for example, trace or insignificant amounts of impurities may remain), nor is it intended to mean the absence of water, buffers, or salts, or components of a pharmaceutical formulation containing a single-domain antibody.

[0077] As used herein, the term “operably linked” may refer to a functional relationship between two or more regions of a polypeptide chain such that these two or more regions are linked together to produce a functional polypeptide.

[0078] Where used herein, the terms “variant,” “derivative,” or “derived from” in the context of a protein or polypeptide (e.g., a single-domain antibody or its domain) mean: (a) a polypeptide having at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with respect to the polypeptide that is the variant or derivative; (b) a polypeptide having at least 40%, 45%, 50% sequence identity with respect to the nucleotide sequence encoding the polypeptide that is the variant or derivative. (c) a polypeptide encoded by a nucleotide sequence having sequence identity of %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%; (c) a polypeptide having 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) to a polypeptide that is a variant or derivative of that polypeptide. (d) a polypeptide, which is encoded by a nucleic acid that can hybridize to a nucleic acid encoding a polypeptide in which the polypeptide is a variant or derivative under high, moderate, or typical stringency hybridization conditions; (e) a polypeptide, which is encoded by a nucleotide sequence that can hybridize to a nucleotide sequence encoding a fragment of the polypeptide in which the polypeptide is a variant or derivative, consisting of at least 20 consecutive amino acids, at least 30 consecutive amino acids, at least 40 consecutive amino acids, at least 50 consecutive amino acids, at least 75 consecutive amino acids, at least 100 consecutive amino acids, at least 125 consecutive amino acids, or at least 150 consecutive amino acids under high, moderate, or typical stringency hybridization conditions;Alternatively, (f) a fragment, which is a fragment of a polypeptide in which the fragment is a variant or derivative. These terms also encompass fusion proteins or polypeptides, which include a polypeptide in which the fusion protein or polypeptide is a variant or derivative.

[0079] When referring to nucleic acids or fragments thereof, the terms “substantial identity” or “substantially identical” indicate that, when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, nucleotide sequence identity exists in at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any known sequence identity algorithm such as FASTA, BLAST, or Gap, as described below. A nucleic acid molecule having substantial identity with a reference nucleic acid molecule may, in certain examples, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0080] When applied to polypeptides, the term “substantial similarity” or “substantially similar” means that when two peptide sequences are optimally aligned using a program such as GAP or BESTFIT with default gap weighting, they share at least 95% sequence identity, more preferably at least 98% or 99%. Preferably, the positions of non-identical residues are different due to conservative amino acid substitutions. A “conservative amino acid substitution” is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences are different from each other due to conservative substitutions, the conservative nature of the substitutions can be corrected by adjusting the degree of % sequence identity or similarity upwards. Means for making such adjustments are known to those skilled in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24: 307-331 (the disclosures of this document are incorporated herein by reference). Examples of groups of amino acids having 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: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256: 1443-1445 (the disclosures of which are incorporated herein by reference). A "moderately conservative" permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0081] Polypeptide sequence similarity, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conserved amino acid substitutions. For example, GCG software includes programs such as Gap and Bestfit, and by using these programs with default parameters, sequence homology or sequence identity can be determined between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their mutaines. See, for example, GCG Version 6.1. Polypeptide sequences can also be compared using FASTA, a program within GCG Version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and % sequence identity for the region with the most overlap between the query sequence and the search sequence (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of this disclosure with a database containing numerous sequences from multiple different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. For example, see Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402 (each referenced herein).

[0082] The terms “enhance,” “promote,” “increase,” “expand,” or “improve” generally refer to the ability of a composition considered herein to produce, induce, or cause a higher physiological response (i.e., downstream effect) compared to the response caused by the vehicle or control molecule / composition. Measurable physiological responses, as is evident from the understanding in the art and the description herein, include, in particular, increased proliferation, activation, effector function, and persistence of immune cells, and / or increased tumor cell killing ability. In certain embodiments, the "increased" or "enhanced" amount may be a "statistically significant" amount and may include increases of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more (e.g., 500, 1000) of the response produced by the vehicle or control composition (e.g., 1.5, 1.6, 1.7, 1.8, etc., including any integers and decimals in between and greater than 1).

[0083] The terms “decrease,” “lower,” “lessen,” “reduce,” “abate,” or “impede” generally refer to the ability of a composition considered herein to produce, induce, or cause a lower physiological response (i.e., downstream effect) compared to the response caused by the vehicle or control molecule / composition. In certain embodiments, “decrease” refers to the ability to produce a lower physiological response (i.e., downstream effect). The amount "reduced" or "amount reduced" may be a "statistically significant" amount and may include reductions of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times, or more (e.g., 500 times, 1000 times) of the response produced by the vehicle or control composition (reference response) (e.g., 1.5, 1.6, 1.7, 1.8, etc., including any integers and decimals in between and greater than 1).

[0084] The terms “treat” a state, disorder, or condition, or “treatment” thereof, include: (1) preventing, delaying, or reducing the occurrence and / or likelihood of the appearance of at least one clinical or subclinical symptom of the state, disorder, or condition in a subject who is suffering from or predisposed to the state, disorder, or condition but has not yet experienced or presented any clinical or subclinical symptom of the state, disorder, or condition; or (2) inhibiting the state, disorder, or condition, i.e., preventing, reducing, or delaying the progression or recurrence of the disease, or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, i.e., causing regression of at least one of the state, disorder, or condition, or its clinical or subclinical symptom thereof. The benefit to the subject being treated must be statistically significant or at least perceptible to the patient or physician.

[0085] The term “effective amount” or “therapeutic effective amount” refers to the amount and / or concentration of a composition containing an active ingredient (e.g., a single-domain antibody, or its fusion protein or conjugate) that, when administered to a patient alone (i.e., as monotherapy) or in combination with other therapeutic agents, results in a significant reduction in disease progression, for example, by relieving or eliminating the symptoms and / or etiology of the disease. The effective amount may be the amount that alleviates, reduces, or mitigates at least one symptom, physiological response, or effect associated with the disease or disorder, prevents the progression of the disease or disorder, or improves the patient’s physical function. The therapeutic effective amount of a composition containing an activator may vary depending on factors such as the individual’s disease state, age, sex, and weight, as well as the ability of the activator to induce a desired response in the individual’s body. The therapeutic effective amount is also the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the activator. The therapeutic effective amount may be delivered in one or more doses. The therapeutically effective dose refers to the amount of medication that is effective in achieving the desired therapeutic and / or preventive outcome at the required dosage and duration.

[0086] The terms “individual,” “subject,” and “patient” are used interchangeably herein and refer to animals, such as mammals. These terms include humans and veterinary subjects. In some embodiments, methods are provided for treating mammals, including but not limited to humans, rodents, apes, cats, dogs, horses, cattle, pigs, sheep, goats, mammalian laboratory animals, mammalian domestic animals, mammalian sports animals, and mammalian pets. Subjects may be male or female and may be of any appropriate age, including infants, boys, adolescents, adults, and elderly subjects. In some embodiments, subjects may be subjects requiring treatment for a disease or disorder. In certain embodiments, the subject is human.

[0087] Single-domain antibody In some embodiments, the Disclosure provides single-domain antibodies (also called “sdAbs”) that have been modified, for example, to reduce ADA binding at the carboxyl terminus. The single-domain antibodies of the Disclosure may originate from a number of sources including, but not limited to, VHH, VNAR, or VH domains (naturally occurring or modified VH domains). VHH can be generated from antibodies and libraries of camelid heavy chains only. VNAR can be generated from antibodies and libraries of cartilaginous fish heavy chains only. Various methods have been implemented, including interface manipulation and selection of specific germline families, to generate monomeric sdAbs from conventional heterodimer VH and VL domains. In some embodiments, the sdAbs of the Disclosure are human sdAbs or humanized sdAbs.

[0088] In some embodiments, the single-domain antibody described herein is a VHH fragment (also known as a nanobody). The VHH fragment is also referred to herein as a "V-body". In some embodiments, VHH is camelid VHH, humanized VHH, or camelid VH. In some embodiments, the single-domain antibody described herein is a VH domain. In some embodiments, the single-domain antibody described herein is a naturally occurring VH domain or a modified VH domain.

[0089] The variable domain of the single-domain antibody of this disclosure comprises at least three complementarity-determining regions (CDRs) that determine its binding specificity. Preferably, within the variable domain, the CDRs are distributed between framework regions. The variable domain typically comprises four framework FR regions separated by three CDR regions, thereby yielding the following typical antibody variable domain structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs and / or FRs of the single-domain antibody of the present invention may be fragments or derivatives derived from naturally occurring antibody variable domains, or they may be synthetically produced.

[0090] This disclosure provides, in particular, single-domain antibodies (e.g., VHH) comprising one or more modifications. In certain embodiments, the single-domain antibodies (e.g., VHH) of this disclosure are modified by one or more modifications to the carboxyl terminus (C-terminus), i.e., the single-domain antibodies may be modified to include one or more C-terminal modifications. While we do not wish to be bound by theory, it has been explained that the exposed C-terminus of an immunoglobulin variable domain may form an epitope, such as a B-cell epitope, which may induce a novel antibody, such as an anti-drug antibody (ADA), and / or interact with an existing antibody. In some embodiments, a single-domain antibody modified at the C-terminus may have reduced ADA binding to the C-terminus compared to an unmodified single-domain antibody. Single-domain antibodies can be modified in particular to reduce the binding of single-domain antibodies described herein (e.g., VHH) to ADA (or other nonspecific proteins) in human blood, including whole blood, serum, and / or plasma (or other bodily fluids, but not limited to bronchoalveolar fluid, ocular fluid, cerebrospinal fluid, and mucus) compared to unmodified (e.g., "wild-type") single-domain antibodies.

[0091] In some embodiments, the C-terminal modification described herein may include, for example, a modification to framework 4 (FR4) of a single-domain antibody (e.g., VHH) described herein. In some embodiments, the C-terminal modification may include a modification to one or more amino acids at positions 111, 112, 113, or 114 of the C-terminus (numbered by Chothia) or any combination thereof (see, for example, Figures 1-2).

[0092] In some embodiments, the single-domain antibodies (e.g., VHH) described herein may contain one or more amino acid mutations. Non-limiting examples of amino acid mutations include amino acid substitutions, insertions, additions, and / or deletions. Amino acid substitution means that an amino acid residue is replaced by a substitute amino acid residue at the same position. The inserted amino acid residue may be inserted at any position, and some or all of the inserted amino acid residues may be inserted adjacent to each other, or none of the inserted amino acid residues may be inserted adjacent to any other inserted amino acid residue. In some embodiments, the single-domain antibodies (e.g., VHH) of this disclosure may be modified to contain one or more amino acid mutations (e.g., one or more substitution mutations) at any of the various amino acid positions described herein. In some embodiments, the single-domain antibodies (e.g., VHH) described herein may be modified to contain at least one substitution mutation. In some embodiments, the single-domain antibodies (e.g., VHH) described herein may be modified to contain one substitution mutation.

[0093] In some embodiments, the single-domain antibodies (e.g., VHH) described herein may contain one or more amino acid mutations in the C-terminus of the single-domain antibody (e.g., in any or a combination of residues between positions 111 and 114 (numbered by Chothia)) (see, for example, Figures 1 and 2).

[0094] In some embodiments, the single-domain antibodies described herein (e.g., VHH) may contain one or more amino acid mutations within the framework region of the single-domain antibody (e.g., FR1-4, or a combination thereof) (see, for example, Figures 1-2).

[0095] In some embodiments, the single-domain antibody described herein (e.g., VHH) may contain one or more amino acid mutations within FR1. In some embodiments, FR1 of the single-domain antibody described herein may be modified to include, for example, an amino acid mutation at position 11 and / or position 13 (Chothia numbering), but is not limited to these. In some embodiments, FR2 of the single-domain antibody described herein may be modified to include, for example, an amino acid mutation at position 87, 88, or 89 (Chothia numbering), or a combination thereof. In some embodiments, FR4 of the single-domain antibody described herein may be modified to include, for example, an amino acid mutation at position 108, 111, 112, 113, or 114 (Chothia numbering), or a combination thereof, but is not limited to these. In some embodiments, the FR regions (e.g., FR1-4, or combinations thereof) may be modified to include amino acid mutations at any of the following positions: 11, 13, 87, 88, 89, 108, 111, 112, 113, or 114 (Chothia numbering), or combinations thereof. In some embodiments, the mutations within any of FR1-4, or combinations thereof, may include one or more substitution mutations.

[0096] In some embodiments, a single-domain antibody may include one or more C-terminal modifications described herein, or one or more of the various other mutations described herein (e.g., framework mutations), or a combination thereof. In some embodiments, a single-domain antibody described herein may include one or more C-terminal modifications and one or more framework mutations described herein (e.g., substitution mutations). In some embodiments, a single-domain antibody described herein may include one or more C-terminal modifications, or one or more framework mutations described herein (e.g., substitution mutations).

[0097] In some embodiments, the single-domain antibodies described herein may include one of the various modifications disclosed herein at the C-terminus starting at position 111 (Chothia numbering). In some embodiments, the single-domain antibodies may include one of the various modifications at, for example, position 111, 112, 113, or 114 (Chothia numbering), or a combination thereof.

[0098] In some embodiments, the single-domain antibodies of this disclosure (e.g., VHH) may be modified to include one or more mutations (i.e., one or more framework mutations) in, for example, the framework regions FR1, FR3, and / or FR4, or combinations thereof. In some embodiments, such mutations (e.g., one or more substitution mutations) may include one or more mutations at any of the amino acid positions 11, 13, 87, 88, 89, or 108 (Chothia numbering), or combinations thereof.

[0099] In some embodiments, modified single-domain antibodies exhibit ADA binding reduced by at least approximately 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 approximately 60% to 70%, approximately 70% to 80%, approximately 80% to 85%, approximately 85% to 90%, approximately 90% to 95%, or approximately 90% to 100% compared to unmodified single-domain antibodies.

[0100] In some embodiments, single-domain antibodies exhibit approximately 80% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 81% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 82% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 83% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 84% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit 85% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 86% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 87% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 88% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 89% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 90% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 91% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 92% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 93% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 94% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 95% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 96% reduced ADA binding compared to unmodified single-domain antibodies.In some embodiments, single-domain antibodies exhibit approximately 97% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 98% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 99% reduced ADA binding compared to unmodified single-domain antibodies. In some embodiments, single-domain antibodies exhibit approximately 100% reduced ADA binding compared to unmodified single-domain antibodies.

[0101] In some embodiments, ADA binding to the single-domain antibodies described herein is measured using an enzyme-linked immunosorbent assay (ELISA) assay. A variety of assays, methods, and techniques exist for performing ADA assays, including, but not limited to, ligand-binding assays in different formats such as (i) bridging format; (ii) direct format; (iii) indirect format; (iv) radio immunoprecipitation assay (RIP); or (v) surface plasmon resonance (e.g., ELISA, electrochemiluminescence immunoassay (ECLIA)). For example, see Table 1 of the review by Mire-Sluis et al., J. Immunol. Meth. 289 (2004), 1-16; Table 1 of the 2010 review by Wadwha and Thorpe, Bioanalysis (2010), 2(6), 1073-1084; and Table 2 of the 2006 paper by Wadwha and Thorpe, Journal of Immunotoxicology, 3:115-121, 2006 (each reference is incorporated in its entirety by reference). Non-limiting examples of anti-drug antibody (ADA) assays are described in Example 2 below.

[0102] In some embodiments, the ADA assays of this disclosure may be carried out in accordance with any of the methods and / or techniques described, for example, International Publication No. 2016 / 118733, Accaert 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.

[0103] C-terminus modification In various embodiments, the single-domain antibodies (VHHs) described herein include one or more modifications at the C-terminus, i.e., one or more C-terminal modifications. In some embodiments, the single-domain antibodies of this disclosure have a C-terminus (starting at position 111 according to Chothia) (1) V, (2) VX1, (3) VX1X2, (4) VX1X2G, or (5) VX1X2P It may be modified to include an amino acid sequence selected from the above.

[0104] In various embodiments, X1 is selected from the amino acids Ala(A), Asp(D), Glu(E), Gly(G), Ile(I), Lys(K), Leu(L), Asn(N), Pro(P), Arg(R), Ser(S), Thr(T), and Val(V).

[0105] In various embodiments, X2 is selected from the amino acids Ala(A), Asp(D), Glu(E), Gly(G), Ile(I), Lys(K), Leu(L), Asn(N), Pro(P), Gln(Q), Arg(R), Ser(S), Thr(T), and Val(V).

[0106] In various embodiments, single-domain antibodies exhibit reduced ADA binding to the C-terminus compared to unmodified (e.g., wild-type) single-domain antibodies.

[0107] In some embodiments, an unmodified (e.g., wild-type) single-domain antibody contains a VSS at its carboxyl terminus.

[0108] In some embodiments, X1 is selected from Ala(A), Gly(G), Pro(P), Asp(D), and Leu(L).

[0109] In some embodiments, X2 is selected from the amino acids Ala(A), Gly(G), Pro(P), Asp(D), Gln(Q), and Leu(L).

[0110] In some embodiments, X1 is selected from Ala(A), Gly(G), Pro(P), Asp(D), and Leu(L); X2 is selected from the amino acids Ala(A), Gly(G), Pro(P), Asp(D), Gln(Q), and Leu(L).

[0111] In some embodiments, X1 is selected from Ala(A), Gly(G), and Pro(P).

[0112] In some embodiments, X2 is selected from the amino acids Ala(A), Gly(G), Gln(Q), and Pro(P).

[0113] In some embodiments, X1 is selected from Ala(A), Gly(G), and Pro(P); X2 is selected from the amino acids Ala(A), Gly(G), Gln(Q), and Pro(P).

[0114] In some embodiments, the single-domain antibody has a carboxyl terminus (starting at position 111 according to Chothia), (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, VVE, VVG, VVI, VVK, VVL, VVN, VVP, VVQ, VVR, VVS, VVT, or VVV; (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: 49), 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: 75), VLGG (SEQ ID NO: 75), VLGG (SEQ ID NO: 49), VGKG (SEQ ID NO: 51), VGLG (SEQ ID NO: 53), VGNG (SEQ ID NO: 55), Number 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), VP RG (sequence number 114), VPSG (sequence number 116), VPTG (sequence number 118), VPVG (sequence number 120), VRAG (sequence number 122), VRGG (sequence number 124), VRPG (sequence number 126), VRSG (sequence number 128), VRTG (sequence number 130), VSAG (sequence number 284), VSDG (sequence number 133), VSEG (sequence number 135), VSGG (sequence number 137), VSIG (sequence number 139), VSKG (sequence number 141), VSLG (sequence number 143), VSNG (sequence number 145), VSPG (sequence number 147),VSQG (sequence number 149), VSRG (sequence number 151), VSTG (sequence number 153), VSVG (sequence number 285), VTAG (sequence number 156), VTDG (sequence number 158), VTEG (sequence number 160), VTGG (sequence number 162), VTIG (sequence number 164), VTKG (sequence number 166), VTLG (sequence number 168), VTNG (sequence number 170), VTPG (sequence number 172), VTQG (sequence number 174), VTRG (sequence number 176), VTSG (sequence number 178), VTTG (sequence number 180), VTVG (sequence number 182), VVGG (sequence number 184), VVPG (sequence number 186), VVSG (sequence number 188), or VVTG (sequence number 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), VET P (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 (Distribution) Column number 78), VLPP (sequence number 80), VLSP (sequence number 81), VLTP (sequence number 83), VNAP (sequence number 85), VNGP (sequence number 87), VNPP (sequence number 89), VNSP (sequence number 91), VNTP (sequence number 93), VPAP (sequence number 95), VPDP (sequence number 97), VPEP (sequence number 99), VPGP (sequence number 101), VPIP (sequence number 103), VPKP (sequence number 105), VPLP (sequence number 107), VPNP (sequence number 109), VPPP (sequence number 111), VPQP (sequence number 113), VP RP (sequence code 115), VPSP (sequence code 117), VPTP (sequence code 119), VPVP (sequence code 121), VRAP (sequence code 123), VRGP (sequence code 125), VRPP (sequence code 127), VRSP (sequence code 129), VRTP (sequence code 131), VSAP (sequence code 132), VSDP (sequence code 134), VSEP (sequence code 136), VSGP (sequence code 138), VSIP (sequence code 140), VSKP (sequence code 142), VSLP (sequence code 144), VSNP (sequence code 146), VSPP (sequence code 148),VSQP (sequence number 150), VSRP (sequence number 152), VSTP (sequence number 154), VSVP (sequence number 155), VTAP (sequence number 157), VTDP (sequence number 159), VTEP (sequence number 161), VTGP (sequence number 163), VTIP (sequence number 165), VTKP (sequence number 167), VTLP (sequence number 169), VTNP (sequence number 171), VTPP (sequence number 173), VTQP (sequence number 175), VTRP (sequence number 177), VTSP (sequence number 179), VTTP (sequence number 181), VTVP (sequence number 183), VVGP (sequence number 185), VVPP (sequence number 187), VVSP (sequence number 189), or VVTP (sequence number 191), It contains an amino acid sequence selected from the following.

[0115] In some embodiments, the single-domain antibody contains an amino acid sequence at the carboxyl terminus (starting at position 111 according to Chothia) 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).

[0116] In some embodiments, the single-domain antibody contains an amino acid sequence at its carboxyl terminus (starting at position 111 according to Chothia) 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).

[0117] In some embodiments, the single-domain antibody does not contain 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) at its carboxyl terminus (starting at position 111 according to Chothia).

[0118] In some embodiments, the single-domain antibody contains the amino acid sequence VAGG (SEQ ID NO: 5) or VPAG (SEQ ID NO: 94) at its carboxyl terminus (starting at position 111 according to Chothia).

[0119] In various embodiments, the single-domain antibodies described herein may contain Val(V), Ser(S), and another Ser(S) (i.e., VSS) at the C-terminus starting at amino acid position 111 (according to Chothia), so that in the VSS sequence, Val(V) is at position 111, Ser(S) is at position 112, and another Ser(S) is at position 113. A VSS sequence containing Val(V) at position 111, Ser(S) at position 112, and another Ser(S) at position 113 may be referred to herein as an "unmodified" VSS sequence (or "wild-type" VSS sequence). In various embodiments, the wild-type VSS sequence may be modified according to any of the various modifications described herein.

[0120] In some embodiments, any one of positions 111, 112, 113, or 114, or any combination thereof, can be modified according to any of the various modifications described herein.

[0121] In some embodiments, the single-domain antibody of the Disclosure contains Val(V) at position 111. In some embodiments, the single-domain antibody of the Disclosure contains Pro(P) at position 111.

[0122] In some embodiments, the single-domain antibodies described herein may, but are not limited to, include Ser(S), Ala(A), Asp(D), Glu(E), Gly(G), Ile(I), Leu(L), Asn(N), Pro(P), Arg(R), Thr(T), Val(V), or Lys(K) at position 112. In some embodiments, the amino acid at position 112 may be absent (e.g., deleted), and therefore position 112 is not occupied by an amino acid. The deletion of one or more amino acids from the amino acid positions described herein may be represented by a dash, i.e., "-", in place of the one or more deleted amino acids. For example, a C-terminal sequence with amino acid deletions from positions 112 and 113 may contain the sequence "V--", where V is at position 111.

[0123] In some embodiments, the single-domain antibodies described herein may, but are not limited to, contain Ser(S), Asp(D), Glu(E), Gly(G), Lys(K), Asn(N), Pro(P), Gln(Q), Arg(R), Thr(T), Ala(A), Ile(I), Leu(L), or Val(V) at position 113. In some embodiments, the amino acid at position 113 may be absent (e.g., deleted), and therefore position 113 is not occupied by an amino acid.

[0124] In some embodiments, the single-domain antibodies described herein may include a C-terminal sequence longer than the unmodified VSS sequence (i.e., 3 amino acid length). In some embodiments, but not limited to, the length of the C-terminal sequence including the 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 longer. In some embodiments, the single-domain antibodies disclosed herein may include one or more amino acids added or inserted to the C-terminus of a sequence containing any of various amino acids at positions 111-113, and thus positions 114 (and above) may be occupied by amino acids such as Gly(G) and / or Pro(P), but not limited to. In some embodiments, the single-domain antibodies described herein may include Gly(G) or Pro(P) at position 114, but not limited to. In some embodiments, the above sequence, which includes any of various amino acids at positions 111-113, may include one or more amino acid additions (or insertions) at the C-terminus. In some embodiments, the amino acid at position 114 may be absent (e.g., deleted), and therefore position 114 is not occupied by an amino acid. Non-limiting examples of C-terminal addition modifications to single-domain antibodies are shown in Table 1-1.

[0125] In some embodiments, the single-domain antibodies described herein may include a C-terminal sequence that is shortened in length compared to the wild-type VSS sequence (i.e., 3 amino acid length). As an unrestricted example, when the C-terminal domain of a single-domain antibody described herein is shortened, amino acids may be absent at positions 112 and / or 113. Unrestricted examples of shortened C-terminal amino acid sequences (starting from position 111 according to Chothia) include, for example, PP-, V--, VA-, VD-, VE-, VG-, VI-, VK-, VL-, VN-, VP-, VR-, VS-, VT-, and VV-. Unrestricted examples of C-terminal shortening modifications for single-domain antibodies are listed in Table 1-1.

[0126] In some embodiments, the single-domain antibodies described herein may include a C-terminal sequence that is the same length as the wild-type VSS sequence (i.e., 3 amino acid length) 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 containing a C-terminal mutation may include Val(V) at position 111, along with any amino acids at positions 112 and 113, except for the combination of Ser(S) at position 112 and Ser(S) at position 113. In some embodiments, a single-domain antibody containing a C-terminal variant sequence (i.e., a C-terminal sequence containing a C-terminal mutation) may include Ala(A), Asp(D), Glu(E), Gly(G), Ile(I), Leu(L), Asn(N), Pro(P), Arg(R), Ser(S), Thr(T), or Val(V) at position 112. In some embodiments, single-domain antibodies containing a C-terminal variant sequence (i.e., a C-terminal sequence containing a C-terminal mutation) include Ala(A), Asp(D), Glu(E), Gly(G), Ile(I), Lys(K), Leu(L), Asn(N), Pro(P), Gln(Q), Arg(R), Ser(S), Thr(T), or Val(V) at position 113. Non-limiting examples of C-terminal variant modifications for single-domain antibodies are listed in Table 1-1.

[0127] [Table 1-1] TIFF2026521677000002.tif255159TIFF2026521677000003.tif255158TIFF2026521677000004.tif255158TIFF20265216770 00005.tif255158TIFF2026521677000006.tif255159TIFF2026521677000007.tif255159TIFF2026521677000008.tif217162

[0128] Framework mutation In various embodiments, the single-domain antibodies (e.g., VHH) described herein include one or more modifications, each containing one or more amino acid mutations at one or more positions in any of the framework regions FR1-FR4 or any combination thereof. Such one or more amino acid mutations may include any of the various mutations described herein. In some embodiments, the one or more amino acid mutations include, but are not limited to, amino acid substitutions, insertions, deletions, or any combination thereof. In some embodiments, the single-domain antibodies (e.g., VHH) described herein may contain one or more amino acid substitutions. Non-limiting examples of exemplary amino acid substitutions are listed in Table 1-2. In some embodiments, the single-domain antibodies described herein may contain one or more amino acid mutations paired (i.e., combined) with one or more C-terminal modifications described herein, such as the framework mutations described herein.

[0129] Exemplary framework variants considered herein are listed in Table 1-2.

[0130] [Table 1-2] TIFF2026521677000010.tif162148

[0131] In some embodiments, one or more amino acid substitutions may be any of the various conservative amino acid substitutions described herein. For example, but not limited to, one or more amino acid substitutions may be one or more conservative amino acid substitutions among at least one of FR1, FR2, FR3, and FR4. In some embodiments, FR1, FR2, FR3, and / or FR4 may comprise at least one, two, three, four, five, six, seven, eight, nine, or ten, or even more, conservative amino acid substitutions.

[0132] In some embodiments, the single-domain antibodies described herein (e.g., VHH) may contain one or more amino acid mutations within framework region 1 (FR1). The one or more mutations within FR1 may include any one or more mutations described herein at any of the amino acid positions 1-25 (Chothia numbering) or in combination thereof (see, for example, Figure 2). In some embodiments, the FR1 of the single-domain antibodies described herein may be modified to include, for example, an amino acid mutation at position 11 and / or position 13 (Chothia numbering).

[0133] In some embodiments, the single-domain antibodies described herein (e.g., VHH) may contain one or more amino acid mutations within framework region 2 (FR2). The one or more mutations within FR2 may include any one or more mutations described herein at any of the amino acid positions 33-51 (Chothia numbering) or any combination thereof (see, for example, Figure 2).

[0134] In some embodiments, the single-domain antibodies described herein (e.g., VHH) may contain one or more amino acid mutations within framework region 3 (FR3). The one or more mutations within FR3 may include any one or more mutations described herein at any of the amino acid positions 57-81, 82, 82A, 82B, 82C, or 83-94 (Chothia numbering), or any combination thereof (see, for example, Figure 2). In some embodiments, the FR3 of the single-domain antibodies described herein may be modified to include, but are not limited to, an amino acid mutation at any of the positions 87, 88, or 89 (Chothia numbering), or any combination thereof.

[0135] In some embodiments, the single-domain antibodies described herein (e.g., VHH) may contain one or more amino acid mutations within framework region 4 (FR4). The one or more mutations within FR4 may include any one or more mutations described herein at any of the amino acid positions 103-113 (Chothia numbering) or any combination thereof (see, for example, Figure 2). In some embodiments, the FR4 of the single-domain antibodies described herein may be modified to include, but are not limited to, an amino acid mutation at any of the positions 108, 111, 112, or 113 (Chothia numbering) or any combination thereof.

[0136] In some embodiments, the single-domain antibodies described herein (e.g., VHH) may contain an amino acid mutation at position 114 (Chothia numbering). In some embodiments, the single-domain antibodies described herein may be modified to contain one or more amino acid mutations at any of the following positions, or combinations thereof: positions 11, 13, 87, 88, 89, 108, 111, 112, 113, or 114.

[0137] In some embodiments, the FRs (e.g., FR1-4, or combinations thereof) of the single-domain antibodies (e.g., VHH) described herein may be modified to include mutations in one or more amino acids at any of the following positions: 11, 13, 87, 88, 89, 108, 111, 112, 113, or 114, or combinations thereof.

[0138] In some embodiments, the single-domain antibody of this disclosure (e.g., VHH) may be modified by one or more framework mutations (e.g., substitution mutations) at any of the amino acid positions 11, 13, 87, 88, 89, or 108 (Chothia numbering), or a combination thereof.

[0139] In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may include, for example, Leu(L) (Leu11) at amino acid position 11 in FR1. In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may include, for example, Gln(Q) (Gln13) at amino acid position 13 in FR1. In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may include, for example, Thr(T) (Thr87) at amino acid position 87 in FR3. In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may include, for example, Gly(G) (Gly88) at amino acid position 88 in FR3. In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may include, for example, Val(V) or Ile(I) (Val89 or Ile89, respectively) at amino acid position 89 in FR3. In some embodiments, the unmodified (i.e., wild-type) single-domain antibody may include, for example, Gln(Q) or Leu(L) at amino acid position 108 within FR4 (Gln108 or Leu108, respectively).

[0140] In some embodiments, the single-domain antibody described herein may be modified at position Leu11 within framework region 1 (FR1). In some embodiments, the single-domain antibody described herein may be modified at position Gln13 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 Gln108 or Leu108 within framework region 4 (FR4).

[0141] In some embodiments, the single-domain antibody (e.g., VHH) described herein may be modified at position Leu11 in framework region 1 (FR1), position Gln13 in framework region 1 (FR1), Thr87 in framework region 3 (FR3), Gly88 in framework region 3 (FR3), Val89 and / or Ile89 in framework region 3 (FR3), or Gln108 and / or Leu108 in framework region 4 (FR4), or any combination thereof.

[0142] In some embodiments, the single-domain antibody contains one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108.

[0143] In some embodiments, the Leu(L) at position 11 is mutated to Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Asn(N), Gln(Q), Arg(R), Ser(S), Thr(T), Val(V), or Tyr(Y).

[0144] In some embodiments, the 13th position Gln(Q) is mutated to Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Leu(L), Asn(N), Arg(R), Ser(S), Thr(T), Val(V), or Tyr(Y).

[0145] In some embodiments, Thr(T) at position 87 is mutated to Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Leu(L), Asn(N), Gln(Q), Arg(R), Ser(S), Val(V), or Tyr(Y). In some embodiments, Thr(T) at position 87 is mutated to Ala(A), Ser(S), or Val(V).

[0146] In some embodiments, the Gly(G) at position 88 is mutated to Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Leu(L), Asn(N), Gln(Q), Arg(R), Ser(S), Thr(T), Val(V), or Tyr(Y).

[0147] In some embodiments, the 89th position Val(V) or Ile(I) is mutated to Ala(A), Asp(D), Glu(E), Lys(K), Leu(L), Asn(N), Gln(Q), Arg(R), Ser(S), Thr(T), or Tyr(Y).

[0148] In some embodiments, the Leu(L) or Gln(Q) at position 108 is mutated to Ala(A), Asp(D), Glu(E), Ile(I), Lys(K), Asn(N), Arg(R), Ser(S), Thr(T), Val(V), or Tyr(Y).

[0149] In some embodiments, the single-domain antibodies of this disclosure may contain one or more substitution mutations. Non-limiting examples of substitution mutations include (a) Leu11Asp (L11D), Leu11Glu (L11E), Leu11Lys (L11K), Leu11Asn (L11N), Leu11Arg (L11R), Leu11Ser (L11S), Leu11Thr (L11T), Leu11Val (L11V), Leu11Ala (L11A), Leu11Ile (L11I), Leu11Gln (L11Q), or Leu11T yr(L11Y); (b) Gln13Asn(Q13N), Gln13Ser(Q13S), Gln13Thr(Q13T), Gln13Ala(Q13A), Gln13Asp(Q13D), Gln13Glu(Q13E), Gln13Ile(Q13I), Gln13Lys(Q13K), Gln13Leu(Q13L), Gln13Arg(Q13R), Gln13Val(Q13V), or Gln13Tyr(Q13Y) ;(c)Thr87Asn(T87N), Thr87Ser(T87S), Thr87Ala(T87A), Thr87Asp(T87D), Thr87Glu(T87E), Thr87Ile(T87I), Thr 87Lys(T87K), Thr87Leu(T87L), Thr87Gln(T87Q), Thr87Arg(T87R), Thr87Val(T87V), or Thr87Tyr(T87Y);(d)Gly8 8Asp(G88D), Gly88Glu(G88E), Gly88Lys(G88K), Gly88Arg(G88R), Gly88Ala(G88A), Gly88Ile(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), Ile89Leu(I89L), Il e89Asn(I89N), Ile89Ser(I89S), Ile89Thr(I89T), Ile89Ala(I89A), Ile89Asp(I89D), Ile89Glu(I89E), Ile89Lys(I89K), Ile89Gln(I89Q), Ile89Arg(I89R), or Ile89Tyr(I89Y); and / or (f)Leu108Asn(L108N), Leu108S er(L108S), Leu108Thr(L108T), Leu108Ala(L108A), Leu108Asp(L108D), Leu108Glu(L108E), Leu108Ile(L108 I), Leu108Lys(L108K), Leu108Arg(L108R), Leu108Val(L108V), Leu108Tyr(L108Y), Gln108Asn(Q108N), Gln10 Examples include 8Ser(Q108S), Gln108Thr(Q108T), Gln108Ala(Q108A), Gln108Asp(Q108D), Gln108Glu(Q108E), Gln108Ile(Q108I), Gln108Lys(Q108K), Gln108Arg(Q108R), Gln108Val(Q108V), Gln108Tyr(Q108Y), or any combination thereof.

[0150] In some embodiments, the single-domain antibodies of this disclosure may contain substitution mutations selected from Leu11Lys(L11K), Leu11Arg(L11R), Leu11Asp(L11D), or Leu11Glu(L11E).

[0151] In some embodiments, the single-domain antibodies of this disclosure may contain substitutional mutations selected from Ala88Glu (A88E), Ala88Asp (A88D), Ala88Arg (A88R), or Ala88Lys (A88K).

[0152] In some embodiments, the single-domain antibodies described herein may be modified to include one or more substitutional mutations within FR1. In some embodiments, the substitutional mutations may include mutations at amino acid position 11 or amino acid position 13 within FR1, or a combination thereof. In some embodiments, the one or more substitutional mutations at amino acid position 11 may include, but are not limited to, Leu11Asp(L11D), Leu11Glu(L11E), Leu11Lys(L11K), Leu11Asn(L11N), Leu11Arg(L11R), Leu11Ser(L11S), Leu11Thr(L11T), Leu11Val(L11V), Leu11Ala(L11A), Leu11Ile(L11I), Leu11Gln(L11Q), or Leu11Tyr(L11Y). In some embodiments, one or more substitutional mutations at amino acid position 13 in FR1 may include, but are not limited to, Gln13Asn(Q13N), Gln13Ser(Q13S), Gln13Thr(Q13T), Gln13Ala(Q13A), Gln13Asp(Q13D), Gln13Glu(Q13E), Gln13Ile(Q13I), Gln13Lys(Q13K), Gln13Leu(Q13L), Gln13Arg(Q13R), Gln13Val(Q13V), or Gln13Tyr(Q13Y).

[0153] In some embodiments, the single-domain antibody may contain an amino acid at position 11 (numbered by Chothia) that has been mutated to Ser(S) or Val(V).

[0154] In some embodiments, the single-domain antibody may contain an amino acid at position 11 (numbered by Chothia) that has been mutated to Asp(D), Glu(E), Lys(K), Asn(N), Arg(R), Ser(S), Thr(T), or Val(V). In some embodiments, the mutation at amino acid position 11 that may be present in the single-domain antibody of this disclosure is as described in U.S. Patent No. 10,526,397, U.S. Patent No. 11,306,139, U.S. Patent Publication No. 2022 / 0324951, U.S. Patent Publication No. 2022 / 0119497, U.S. Patent No. 11,220,539, U.S. Patent No. 11,312,765, and U.S. Patent No. 11,485 This may include any of the following: U.S. Patent No. 777, U.S. Patent No. 10,858,418, U.S. Patent No. 11,192,937, European Patent No. 3143042, U.S. Patent Publication No. 2017 / 0121399, U.S. Patent Publication No. 2022 / 0332807, and European Patent No. 3693386, each of which is incorporated herein by reference in its entirety.

[0155] In some embodiments, the single-domain antibody may contain an amino acid at position 13 (numbered by Chothia) that is mutated to Asn(N), Ser(S), or Thr(T). In some embodiments, the mutations at amino acid position 13 that may be present in the single-domain antibody of this disclosure may include any of those described in U.S. Patent No. 11,306,139 and U.S. Patent Application Publication 2022 / 0324951, each of which is incorporated herein by reference in its entirety.

[0156] In some embodiments, the single-domain antibodies described herein may be modified to include Val(V) at position 11 and Leu(L) at position 89. In some embodiments, if the single-domain antibody may include Val(V) at position 11 and Leu(L) at position 89, the amino acid at position 110 may be, for example, Thr(T), Ile(I), Ala(A), Lys(K), or Gln(Q); and / or (ii) the amino acid residue at position 112 may be, for example, Ser(S), Phe(F), Lys(K), or Gln(Q).

[0157] In some embodiments, the single-domain antibodies described herein may be modified to include one or more substitutional mutations within FR3. In some embodiments, the substitutional mutations may include mutations at amino acid positions 87, 88, or 89, or combinations thereof, within FR3. In some embodiments, the substitutional mutation at amino acid position 87 may include, but is not limited to, any of Thr87Asn (T87N), Thr87Ser (T87S), Thr87Ala (T87A), Thr87Asp (T87D), Thr87Glu (T87E), Thr87Ile (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 include Thr87Ala (T87A), Thr87Ser (T87S), or Thr87Val (T87V). In some embodiments, the substitution mutation at amino acid position 88 may include, but are not limited to, Gly88Asp (G88D), Gly88Glu (G88E), Gly88Lys (G88K), Gly88Arg (G88R), Gly88Ala (G88A), Gly88Ile (G88I), Gly88Leu (G88L), Gly88Asn (G88N), Gly88Gln (G88Q), Gly88Ser (G88S), Gly88Thr (G88T), Gly88Val (G88V), or Gly88Tyr (G88Y).In some embodiments, substitution mutations at amino acid position 89 are, but are not limited to, Val89Leu (V89L), Val89Asn (V89N), Val89Ser (V89S), Val89Thr (V89T), Val89Ala (V89A), Val89Asp (V89D), Val89Glu (V89E), Val89Lys (V89K), Val89Gln (V89Q), Val89Arg (V89R), Va It may contain any of the following: l89Tyr(V89Y), Ile89Leu(I89L), Ile89Asn(I89N), Ile89Ser(I89S), Ile89Thr(I89T), Ile89Ala(I89A), Ile89Asp(I89D), Ile89Glu(I89E), Ile89Lys(I89K), Ile89Gln(I89Q), Ile89Arg(I89R), or Ile89Tyr(I89Y).

[0158] In some embodiments, the single-domain antibody may contain an amino acid at position 87 (numbered by Chothia) that is mutated to Asn(N) or Ser(S). In some embodiments, the mutations at amino acid position 87 that may be present in the single-domain antibody of this disclosure may include any of those described in U.S. Patent No. 11,306,139 and U.S. Patent Application Publication No. 2022 / 0324951, which are each incorporated herein by reference in their entirety.

[0159] In some embodiments, the single-domain antibody may contain an amino acid at position 88 (numbered by Chothia) that is mutated to Asp(D), Glu(E), Lys(K), or Arg(R). In some embodiments, the mutations at amino acid position 88 that may be present in the single-domain antibody of this disclosure may include any of those described in U.S. Patent Application Publication No. 2020 / 0140525, which is incorporated herein by reference in its entirety.

[0160] In some embodiments, the single-domain antibody may contain an amino acid at position 88 (numbered by Chothia) that is mutated to Lys(L), Asn(N), Ser(S), or Thr(T). In some embodiments, the mutations at amino acid position 88 that may be present in the single-domain antibody of this disclosure are as described in U.S. Patent Nos. 11,220,539, 11,306,139, 11,485,777, U.S. Patent Publication No. 2022 / 0324951, 11,312,765, U.S. Patent Publication No. 2017 / 0121399, and 10,030,068. This application may include any of the following: the Book, U.S. Patent Application Publication No. 2022 / 0332807, U.S. Patent No. 11,014,977, European Patent No. 3143042, European Patent No. 3066120, European Patent No. 3693386, U.S. Patent Application Publication No. 2021 / 0347855, and European Patent No. 3447068, each of which is incorporated herein by reference in its entirety.

[0161] In some embodiments, the single-domain antibodies described herein may be modified to include one or more substitutional mutations within FR4. In some embodiments, the substitutional mutations may include, for example, a mutation at amino acid position 108 within FR4. In some embodiments, the substitutional mutations at amino acid position 108 may include, but are not limited to, Leu108Asn (L108N), Leu108Ser (L108S), Leu108Thr (L108T), Leu108Ala (L108A), Leu108Asp (L108D), Leu108Glu (L108E), Leu108Ile (L108I), Leu108Lys (L108K), Leu108Arg (L108R), Leu108Val (L10 8V), Leu108Tyr(L108Y), Gln108Asn(Q108N), Gln108Ser(Q108S), Gln108Thr(Q108T), Gln108Ala(Q108A), Gln108Asp(Q108D), Gln108Glu(Q108E), Gln108Ile(Q108I), Gln108Lys(Q108K), Gln108Arg(Q108R), Gln108Val(Q108V), or Gln108Tyr(Q108Y) It may include any of the following.

[0162] In some embodiments, the modified single-domain antibody contains Gln(Q) or Leu(L) at position 108 (Chothia numbering).

[0163] In some embodiments, the single-domain antibody may contain an amino acid at position 108 (numbered by Chothia) that is mutated to Asn(N), Ser(S), or Thr(T). In some embodiments, the mutations at amino acid position 88 that may be present in the single-domain antibody of this disclosure may include any of those described in U.S. Patent No. 11,306,139, European Patent No. 3,271391, and U.S. Patent Application Publication 2022 / 0324951, each of which is incorporated herein by reference in its entirety.

[0164] In some embodiments, the single-domain antibodies described herein may include modifications at amino acid position 110 (Chothia numbering). In some embodiments, the amino acid at position 110 is Lys(K) or Gln(Q). In some embodiments, if the amino acid at position 110 is Lys(K) or Gln(Q), the amino acid at position 11 may be Val(V), or the amino acid at position 89 in Kabat may be Leu(L), or a combination thereof. In some embodiments of any of the above modifications, the amino acid at position 108 may be Gln(Q) or Leu(L). In some embodiments, if the amino acid at position 110 is Lys(K) or Gln(Q), the amino acid at position 11 may be Val(V) according to Chothia, and / or the amino acid at position 89 may be Leu(L).

[0165] In some embodiments, the single-domain antibodies described herein may contain an amino acid at position 112 (numbered by Chothia) which is Lys(K) or Gln(Q). In some embodiments, if the single-domain antibody contains an amino acid at position 112 which is Lys(K) or Gln(Q), the amino acid at position 11 may be, for example, Leu(L), Glu(E), Lys(K), Val(V), or Tyr(Y). In some embodiments, if the single-domain antibody contains an amino acid at position 112 which is Lys(K) or Gln(Q), the amino acid at position 13 may be, for example, Ser(S), Thr(T), Ala(A), Leu(L), Pro(P), Phe(F), Glu(E), or Val(V).

[0166] In some embodiments, the single-domain antibodies described herein are used in U.S. Patent Publication No. 2022 / 0332807, U.S. Patent Publication No. 2022 / 0332806, U.S. Patent Publication No. 2022 / 0324951, U.S. Patent Publication No. 2022 / 0119497, U.S. Patent Publication No. 2021 / 0403536, U.S. Patent Publication No. 2021 / 0388062, U.S. Patent Publication No. 2021 / 0347855, U.S. Patent Publication No. 2021 / 0 U.S. Patent No. 341490, U.S. Patent Application Publication No. 2020 / 0325221, U.S. Patent Application Publication No. 2020 / 0140525, U.S. Patent Application Publication No. 2018 / 0355031, U.S. Patent Application Publication No. 2018 / 0009888, U.S. Patent Application Publication No. 2017 / 0121399, U.S. Patent Application Publication No. 2015 / 0050266, U.S. Patent No. 9,150,640, U.S. Patent No. 11,485,777, U.S. Patent No. 11,426,468, U.S. Patent U.S. Patent No. 11,312,765, U.S. Patent No. 11,312,764, U.S. Patent No. 11,306,139, U.S. Patent No. 11,220,539, U.S. Patent No. 11,192,938, U.S. Patent No. 11,192,937, U.S. Patent No. 11,021,544, U.S. Patent No. 11,014,977, U.S. Patent No. 11,009,511, U.S. Patent No. 10,858,418, U.S. Patent No. 10,526,397, U.S. Patent No. 10,526, The patent application may be modified to include any number of variations, such as substitutional mutations, as described in U.S. Patent No. 397, U.S. Patent No. 10,030,068, European Patent No. 3693386, European Patent No. 3447068, European Patent No. 3339322, European Patent No. 3271391, European Patent No. 3143042, European Patent No. 3066120, and European Patent No. 2987806, each of these documents is incorporated into this application by reference in its entirety.

[0167] In some embodiments, the single-domain antibodies described herein may contain any mutation in any of FR1-4 described herein, together with any other mutation in any of FR1-4 described herein. In some embodiments, one or more amino acid mutations in FR1 may be combined with one or more other amino acid mutations in FR1. In some embodiments, one or more amino acid mutations in FR2 may be combined with one or more other amino acid mutations in FR2. In some embodiments, one or more amino acid mutations in FR3 may be combined with one or more other amino acid mutations in FR3. In some embodiments, one or more amino acid mutations in FR4 may be combined with one or more other amino acid mutations in FR4. In some embodiments, one or more amino acid mutations in FR1 described herein may be combined with one or more amino acid mutations in FR2, FR3, and / or FR4, or combinations thereof, described herein. In some embodiments, one or more arbitrary amino acid mutations within FR2 as described herein may be combined with one or more arbitrary amino acid mutations from FR1, FR3, and / or FR4, or combinations thereof, as described herein. In some embodiments, one or more arbitrary amino acid mutations within FR3 as described herein may be combined with one or more arbitrary amino acid mutations from FR1, FR2, and / or FR4, or combinations thereof, as described herein. In some embodiments, one or more arbitrary amino acid mutations within FR4 as described herein may be combined with one or more arbitrary amino acid mutations from FR1, FR2, and / or FR3, or combinations thereof, as described herein.

[0168] Within the scope of this disclosure, any combination of any one or more amino acid mutations from FR1, FR2, FR3, and / or FR4, or any combination thereof, as described herein, may be paired (i.e., combined) with any one or more or any combination thereof of any C-terminal modifications described herein. As a non-limiting example, any of the amino acid mutations listed in Table 1-2 may be combined with any of the C-terminal modifications listed in Table 1-1.

[0169] In some embodiments, (a) Leu11Asp(L11D), Leu11Glu(L11E), Leu11Lys(L11K), Leu11Asn(L11N), Leu11Arg(L11R), Leu11Ser(L11S), Leu11Thr(L11T), Leu11Val(L11V), Leu11Ala(L11A), Leu11Ile(L11I), Leu11Gln(L11Q), or Leu11Tyr(L11Y); (b) Gln13Asn(Q13N), Gln13Ser(Q13S), Gln13Thr(Q13T), G ln13Ala(Q13A), Gln13Asp(Q13D), Gln13Glu(Q13E), Gln13Ile(Q13I), Gln13Lys(Q13K), Gln13Leu(Q13L), Gln13Arg(Q13R), Gln13Val(Q13V), or Gln13Tyr(Q13Y); (c)Thr87Asn(T87N), Thr87Ser(T87S), Thr87Ala(T87A), Thr87Asp(T87D), Thr87Glu(T87E), Thr87Ile(T87I), Thr87Lys(T87K), Thr 87Leu(T87L), Thr87Gln(T87Q), Thr87Arg(T87R), Thr87Val(T87V), Thr87Tyr(T87Y);(d)Gly88Asp(G88D), Gly88Glu(G88E), Gly88Lys(G88K), Gly8 8Arg(G88R), Gly88Ala(G88A), Gly88Ile(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(V 89E), Val89Lys (V89K), Val89Gln (V89Q), Val89Arg (V89R), Val89Tyr (V89Y), Ile89Leu (I89L), Ile89Asn (I89N), Ile89Ser (I89S), Ile89Thr (I89T),Ile89Ala(I89A), Ile89Asp(I89D), Ile89Glu(I89E), Ile89Lys(I89K), Ile89Gln(I89Q), Ile89Arg(I89R), or Ile89Tyr(I89Y); or (f)Leu108Asn(L108N), Leu108Ser(L108S), Leu108Thr(L108T), Leu108Ala(L108A), Leu108Asp(L108D), Leu108Glu(L108E), Leu108Ile(L108I), Leu108Lys(L1 08K), Leu108Arg(L108R), Leu108Val(L108V), Leu108Tyr(L108Y), Gln108Asn(Q108N), Gln108Ser(Q108S), Gln108Thr(Q108T), Gln108Ala(Q108A), Gln108Asp(Q108D), Gln108Glu(Q108E), Gln108Ile(Q108I), Gln108Lys(Q108K), Gln108Arg(Q108R), Gln108Val(Q108V), or Gln108Tyr(Q108Y), or This selects any amino acid mutation from these combinations, resulting in 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), VGN P (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: 71), VIPP (SEQ ID NO: 72), VISG (SEQ ID NO: 73), VISP (SEQ ID NO: 74), VITG (SEQ ID NO: 71), VGPGVGQG (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 (S Column number 75), VITP (sequence number 76), VLGG (sequence number 77), VLGP (sequence number 78), VLPG (sequence number 79), VLPP (sequence number 80), VLSP (sequence number 81), VLTG (sequence number 82), VLTP (sequence number 83), VNAG (sequence number 84), VNAP (sequence number 85), VNGG (sequence number 86), VNGP (sequence number 87), VNPG (sequence number 88), VNPP (sequence number 89), VNSG (sequence number 90), VNSP (sequence number 91), VNTG (sequence number 92), VNTP (sequence number 93), VPAG (sequence number 94), VPAP (sequence number 95), VPDG (sequence number 96), VPDP (sequence number 97), VPEG (sequence number 98), VPEP (sequence number 99), VPGG (sequence number 100), VPGP (sequence number 101), VPIG (sequence number 102), VPIP (sequence number 103), VPKG (sequence number 104), VPKP (sequence number 105), VPLG (sequence number 106), VPLP (sequence number 107), VPNG (sequence number 108), VPNP (sequence number 109), VPPG (sequence number 110), VPPP (sequence number 111), VPQG (sequence number 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: 113) 30), VRTP (sequence number 131), VSAP (sequence number 132), VSDG (sequence number 133), VSDP (sequence number 134), VSEG (sequence number 135), VSEP (sequence number 136), VSGG (sequence number 137), VSGP (sequence number 138), VSIG (sequence number 139), VSIP (sequence number 140), VSKG (sequence number 141), VSKP (sequence number 142), VSLG (sequence number 143), VSLP (sequence number 144), VSNG (sequence number 145), VSNP (sequence number 146), VSPG (sequence number 147), VSPP (array Number 148), VSQG (sequence number 149), VSQP (sequence number 150), VSRG (sequence number 151), VSRP (sequence number 152), VSTG (sequence number 153), VSTP (sequence number 154), VSVP (sequence number 155), VTAG (sequence number 156), VTAP (sequence number 157), VTDG (sequence number 158), VTDP (sequence number 159), VTEG (sequence number 160), VTEP (sequence number 161), VTGG (sequence number 162), VTGP (sequence number 163), VTIG (sequence number 164), VTIP (sequence number 165), VTK G (sequence code 166), VTKP (sequence code 167), VTLG (sequence code 168), VTLP (sequence code 169), VTNG (sequence code 170), VTNP (sequence code 171), VTPG (sequence code 172), VTPP (sequence code 173), VTQG (sequence code 174), VTQP (sequence code 175), VTRG (sequence code 176), VTRP (sequence code 177), VTSG (sequence code 178), VTSP (sequence code 179), VTTG (sequence code 180), VTTP (sequence code 181), VTVG (sequence code 182), VTVP (sequence code 183),VVGG (SEQ ID NO: 184), VVGP (SEQ ID NO: 185), VVPG (SEQ ID NO: 186), VVPP (SEQ ID NO: 187), VVSG (SEQ ID NO: 188), VVSP (SEQ ID NO: 189), VVTG (SEQ ID NO: 190), VVTP (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-, VV-, 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, VVA, VVD, VVE, VVG, VVI, VVK, VVL, VVN, VVP, VVQ, VVR, VVS, VVT, VVV, VEA, VET, or any C-terminal sequence selected from VND, or a combination thereof, starting from position 111 according to Chothia, may be combined.

[0170] Within the scope of this disclosure, any combination of any one or more amino acid mutations from FR1, FR2, FR3, and / or FR4 as described herein, or any combination thereof, may be paired (i.e., combined) with any one or more C-terminal modifications or combination thereof as described herein, or, optionally, with any one or more modifications (for example, but not limited to, those listed in Table 1-4) to any amino acid position in any one or more of CDR1, CDR2, and / or CDR3 as described herein.

[0171] In some embodiments, the amino acids at one or more of the positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 112, 113, or 114 (Chothia numbering), or any combination thereof, of the single-domain antibodies described herein may be glycosylated or contain glycosylation sites capable of being glycosylated. For example, but not limited to, the amino acids at one or more of the 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 contain N-glycosylated or N-glycosylation sites. In some embodiments, position 11 may be Val(V) or Lys(K), and one or more amino acid residues at positions 10, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 110, 112, 113, or 114, or any combination thereof, may be glycosylated or contain glycosylated sites. In some embodiments, position 89 may be Thr(T) or Leu(L), and one or more amino acid residues at positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 108, 110, 112, 113, or 114, or any combination thereof, may be glycosylated or contain glycosylated sites.

[0172] Single-domain antibodies (e.g., VHH) can be obtained by immunizing dromedary camels, camels, llamas, alpacas, or sharks with a desired antigen, and then isolating the mRNA encoding the heavy-chain antibody. Antigens can be purified from natural sources or during recombinant production. Immunization and / or immunoglobulin sequence screening can be performed using peptide fragments of such antigens. Reverse transcription and polymerase chain reaction (PCR) can be used to create gene libraries of single-domain antibodies containing millions of clones. Screening techniques such as phage display, yeast display, and ribosome display are useful for identifying clones that bind to the antigen. Methods for generating heavy chain antibody fragments are described, for example, in International Publication No. 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 these documents is incorporated herein by reference in its entirety).

[0173] Alternatively, gene libraries derived from animals that have not been previously immunized may be used. Since such naive libraries typically contain only antibodies with low affinity for the desired antigen, an additional step is required: affinity maturation through random mutagenesis. See, for example, Saerens, D.; et al. (2008). “Single-domain antibodies as building blocks for novel therapeutics”. Current Opinion in Pharmacology 8 (5): 600-608.

[0174] Affinity maturation strategies can be categorized into targeted / rational approaches or non-targeted / random approaches. Targeted approaches require information about the VHH of interest, such as affinity maturation hotspots or structural information of the VHH:antigen complex, while non-targeted approaches do not require prior information. Targeted approaches applicable to VHH affinity maturation include site-directed in vitro mutagenesis and in silico / computational approaches. Common non-targeted approaches used for VHH affinity maturation include random in vitro mutagenesis, CDR swapping, and autonomous high-frequency mutant yeast surface display, the latter two being novel, emerging, and highly time-efficient techniques. Common to most of these strategies is that after applying a specific randomization strategy to generate a mutant library, the resulting library can be screened using standard display techniques such as yeast, phage, or ribosome display to select the best conjugate. The choice of display system often depends on the size of the library being displayed; yeast display is approximately 10 7 ~10 9 Phage displays are approximately 10 8 ~10 10 The ribosome display is approximately 10 12 ~10 13 The library size can be handled (Chan and Groves, 2021). In particular, during affinity maturation, the number of highly interacting residues such as aromatic amino acids increases within the CDR region. Selected affinity-matured clones can be further evaluated by feasibility assessments to test undesirable properties such as nonspecific binding to off-targets or VHH instability.

[0175] Targeted in vitro mutagenesis allows for mutations in selected sets of residues within the CDR of VHH (Tiller et al., 2017; Yau et al., 2005). These residues can be pre-selected by identifying mutation hotspot residues using alanine scanning or by identifying mutation sites using structural data of the antigen:VHH complex. These sites can then be subjected to saturation mutagenesis, substituting specific sites with any possible amino acids or specific amino acid substitutions to obtain several small libraries. After mutagenesis, the conjugates can be displayed to select the best mature candidate. Typically, targeted mutagenesis is performed multiple times using separate sublibraries to obtain individual combinations of mutations that synergistically increase binding affinity.

[0176] In many cases, computer-aided / in silico methods are used to induce targeted in vitro mutagenesis. Target: Homology modeling or docking of the VHH complex can be used to identify mutation hotspots, which are then subjected to in vitro mutagenesis (Bert Schepens et al., 2021; Cheng et al., 2019; Inoue et al., 2013; Mahajan et al., 2018). Furthermore, in silico methods utilize a virtual library (approximately 10 components). 40 By searching through all designed variants within a given set in a relatively short time, a suitable number of promising candidates for experimental testing can be identified. These techniques can be particularly useful when structural data for drug-target interactions are available.

[0177] Non-targeted / random affinity maturation strategies applicable to VHH affinity maturation include random in vitro mutagenesis, CDR shuffling / swapping, and in vivo affinity maturation by yeast display. Random in vitro mutagenesis randomly mutates either the entire VHH sequence or only the CDR sequence (Chen et al., 2021; Ye et al., 2021; Zupancic et al., 2021). The most commonly used technique is error-prone PCR, which uses a DNA polymerase lacking proofreading activity and PCR conditions that further increase the polymerase error rate. This technique is applicable without further structural knowledge or information regarding the importance of residues contributing to antigen:VHH interaction. The resulting mutant library can then be displayed to select the best-matured candidates. This technique can be combined with NGS sequencing of the display elutes to thoroughly read out all obtained candidates, thereby identifying sparse but promising clones (Chen et al., 2021).

[0178] In some embodiments, CDR shuffling or swapping is applied to VHH affinity maturation, as described in Zupancic et al., 2021. In CDR swapping, the enriched library can be used as input material for a PCR reaction, allowing for the individual amplification of the VHH's CDRs. The PCR products are then mixed and reconstituted using overlap PCR to generate a complete plasmid for further display to select the best mature conjugate. One limitation of this approach, as with synthetic libraries, is that it can only be used with VHHs containing the same framework.

[0179] In some embodiments, in vivo affinity maturation by yeast display is applied to VHH affinity maturation, as described in Wellner et al., 2021. This method is based on an autonomous hypermutation yeast surface display (AHEAD) that mimics somatic high-frequency mutations during VHH selection using a modified yeast strain. The yeast error-prone orthogonal DNA replication system can generate novel variants during plasmid replication by randomly introducing mutations. These novel variants can then be displayed and selected using the yeast surface display to identify the best conjugate. This allows for the generation of high-affinity clones in a significantly shorter time (approximately two weeks) than conventional affinity maturation procedures. This method can be applied using synthetic or immunolibraries, and can be used with unenriched libraries, enriched libraries, or subsets of pre-selected clones.

[0180] As in the case of V-bodies, when a moderately affinity conjugate is needed and the affinity of identified candidates needs to be reduced, very similar techniques can be applied. For example, mutations aimed at reducing affinity can be introduced using the same targeted or non-targeted approaches described for affinity maturation. Subsequent selection can be adapted as appropriate. When larger libraries are generated and screening by display techniques is required, the selection strategy can be adapted to enrich moderately affinity conjugates while eliminating high-affinity candidates. This could, for example, involve pre-panning with low-antigen concentration phage display to remove all high-affinity candidates, followed by selection at high-antigen concentration to obtain moderately affinity VHHs. For library sizes up to 1000 candidates, characterization of dynamic dissociation rates can be used to obtain immediate information about the dynamic behavior of candidates.

[0181] Once the most potent clone is identified, its stability against enzymes, for example, can be improved by optimizing its DNA sequence. Another goal is humanization to prevent the immune response of humans to the antibody. Humanization can be achieved based on homology between camelid VHH and human VH fragments, which will be described in more detail later. Finally, the optimized single-domain antibody can be translated and expressed in suitable organisms such as Escherichia coli (E. coli) or Saccharomyces cerevisiae.

[0182] Single-domain antibodies may be derived from conventional antibodies. In some embodiments, single-domain antibodies can be produced from conventional four-chain mouse or human IgG. The process is similar and includes a gene library from an immunized or naive donor and a display technique to identify the most specific antigen. However, the binding region of conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate due to their lipophilicity. Monomerization can be achieved by substituting lipophilic amino acids with hydrophilic amino acids (see, for example, Borrebaeck, CAK; Ohlin, M. (2002). “Antibody evolution beyond Nature”. Nature Biotechnology 20 (12): 1189-90). If affinity can be maintained after monomerization, the single-domain antibody can similarly be produced in Escherichia coli, Saccharomyces cerevisiae, or other suitable organisms.

[0183] A "humanized antibody" refers to a genetically modified chimeric antibody in which the amino acid sequence (typically CDR) derived from an antibody (donor antibody), such as a camelid antibody, is grafted onto a human antibody (acceptor antibody). Thus, a humanized antibody typically includes a CDR derived from the donor antibody and a variable region framework and (if present) a constant region derived from the human antibody. Therefore, a "humanized VHH" includes a CDR that is "humanized" to correspond to the CDR of a naturally occurring VHH domain (e.g., camelid VHH). Humanized VHH can be prepared by substituting one or more amino acid residues in the amino acid sequence of a naturally occurring VHH sequence (particularly in the framework sequence) with one or more amino acid residues that occur at the corresponding positions in a VH domain derived from a conventional four-chain human antibody. Such humanized VHH can be obtained by any suitable method known to those skilled in the art, and therefore not strictly limited to the methods described herein.

[0184] Humanization of VHH can be achieved using resurfacing or CDR grafting. Resurfacing strategies are described, for example, in Conrath et al., 2005 J Mol Biol; Kazemi-Lomedasht et al., 2018; and Vincke et al., 2009 J Biol Chem. CDR grafting strategies are described, for example, in 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 these references is incorporated in its entirety by reference).

[0185] To humanize camelid VHHs using a resurfacing approach, the human germline reference most similar to the camelid germline sequence of the selected VHH may be identified. Most isolated camelid VHHs in the literature belong to the camelid IGHV3 subfamily 2 (Nguyen et al., 2000, EMBO J), and DP-47 / VH3-23 of the IGHV3 family is commonly used as a human reference. Next, the framework of the camelid VHH can be compared to the human reference sequence. Surface-exposed residues are considered to contribute relatively little to protein stability and are therefore replaced with corresponding human residues. However, embedded residues are likely to contribute to the overall stability of the VHH and are therefore retained as camelid-derived residues. Humanization of framework regions 1, 3, and 4 usually does not affect the biochemical properties of the VHH, but overall humanization of framework 2 significantly increases local hydrophobicity. The residues H37, H44, H45, and H47 (Chothia numbering) within framework 2, i.e., so-called tetrade or hallmark residues, are partially embedded in human VH (VGLW) and are involved in VH / VL pairing, thus exhibiting some hydrophobicity. However, in camelid VHH, these residues are partially charged (FERG), significantly increasing the solubility of VHH and inhibiting pairing with camelid VL (Soler et al., 2021, Biomolecule; Conrath et al., 2005 J Mol Biol). Furthermore, residues H37 and H47 are known to contribute to antigen binding affinity by stabilizing the three-dimensional structure of many VHH by interacting with the CDR-H3 loop. Furthermore, a significant number of VHHs utilize the framework residues H44, H45, and H47 for antigen binding (Zavrtanik et al., 2018, J Mol Biol). Therefore, complete humanization of these residues often leads to decreased solubility or aggregation of VHHs, and decreased or complete loss of binding affinity to target antigens (van Faassen et al., 2020, Vincke et al., 2009).As a result, when humanizing VHH, all or at least some of these hallmark residues within framework 2 remain of camelid origin.

[0186] Another approach that can be applied to humanize VHH is CDR grafting. The CDR of a selected VHH can be transplanted into a partially or fully humanized universal VHH framework (Saerens et al., 2009 J Biol Chem, Soler et al., 2021, Vincke et al., 2009 J Biol Chem). While CDR grafting has worked well in some cases, it has failed in several others, with the VHH often losing its ability to bind to the desired antigen and / or becoming structurally unstable and more prone to aggregation (van Faassen et al., 2020, FASEB). This is mainly due to the interaction between CDR3 and specific residues in framework 2 that are important for the conformation of CDR3, the overall stability of the VHH, and the overall hydrophobicity, which are impaired by this approach. Inverse mutations of the camelid family can sometimes be introduced into the framework to compensate for these effects (van Faassen et al., 2020, FASEB).

[0187] An alternative strategy to mitigate the need to humanize selected VHH sequences is to use libraries of fully or partially humanized synthetic VHHs instead of camelid immunolibraries 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 derived from camelids for the reasons mentioned above.

[0188] Other suitable humanization substitutions are described in International Publication No. 09 / 138519 and International Publication No. 08 / 020079, and in Tables A-3 to A-8 of International Publication No. 08 / 020079 (list of possible humanization substitutions) (each document is incorporated herein by reference in its entirety). Non-limiting examples of such humanization substitutions include Q108L and A14P. Such humanization substitutions may be appropriately combined with one or more other mutations described herein (e.g., one or more mutations that reduce binding by existing antibodies).

[0189] In some embodiments, the humanized VHH sequence still retains residues associated with binding to protein A. In some embodiments, manipulation can be performed during humanization to confer protein A binding properties to VHH that did not interact with protein A (Graille et al., 2000, PNAS).

[0190] Similar to "humanized antibodies," a "camelized antibody" refers to an antibody that has an amino acid sequence (typically CDR) derived from a donor antibody, such as a human antibody, and a variable region framework and (if present) a constant region derived from a camelid antibody. Therefore, "camelized VH" includes an amino acid sequence that is "camelized" to the amino acid sequence of a naturally occurring VH domain. Camelized VH can be prepared by substituting one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain derived from a conventional four-chain antibody with one or more amino acid residues that occur at the corresponding positions in the VHH domain of a heavy-chain antibody. This can be carried out, for example, as described in International Publication No. 2008 / 020079. Such "camelizing" substitutions are typically inserted at amino acid positions that form and / or exist at the VH-VL interface, and / or at so-called camelid hallmark residues, e.g., F37, E44, R45, and F47 (see, for example, International Publication No. 94 / 04678, Davies and Riechmann (1994 and 1996)). In one embodiment, the VH sequence used as a starting material or starting point for the generation or design of camelized VH is a mammalian VH sequence or a human antibody VH sequence. However, such camelized VH can be obtained by any suitable method known to those skilled in the art, and is therefore not strictly limited to polypeptides obtained using naturally occurring VH domain-containing polypeptides as starting materials.

[0191] The amino acid residues of single-domain antibodies can be numbered according to the general numbering for VH domains provided by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91), similar to how it is applied to VHH domains derived from camelids, as described by Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see, for example, Figure 2 of this publication). The total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by Kabat's numbering. For example, one or more positions indicated by Kabat's numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat's numbering. As a result, Kabat's numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in the VH domain and VHH domain is typically 110–120, and often 112–115. However, shorter and longer sequences may be more suitable for the purposes described herein.

[0192] The determination of the CDR region of single-domain antibodies is based on the following studies: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme); 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 MP 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(1):55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun 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.This can be achieved using various methods, including those described in “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 ("AbM" numbering scheme) (each reference cited herein is incorporated herein by reference in its entirety).

[0193] The boundaries of a particular CDR or framework (FR) can vary depending on the scheme used. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the length of the most common antibody region sequence, with insertions represented by insertion letters such as "30a," and deletions found in some antibodies. These two schemes place specific insertions and deletions ("indels") in different locations, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions and is based on the definitions used by Oxford Molecular's AbM antibody modeling software.

[0194] In some embodiments, the single-domain antibodies described herein (e.g., VHH) are numbered according to Chothia's scheme (see Figure 2).

[0195] In some embodiments, a CDR can be defined according to one of the following: 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 includes three CDRs named CDR1, CDR2, and CDR3. Tables 1-3 below enumerate exemplary positional boundaries of CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. In CDR-H1, residue numbers are shown using both the Kabat and Chothia numbering schemes. FRs are located between CDRs; for example, FR-H1 is located before CDR-H1, FR-H2 is located between CDR-H1 and CDR-H2, FR-H3 is located between CDR-H2 and CDR-H3, and FR-H4 is located after CDR-H3. Furthermore, in the Kabat numbering scheme shown, insertions are located at H35A and H35B. Therefore, the end of a Chothia CDR-H1 loop numbered using the Kabat numbering rules shown will vary between H32 and H34 depending on the length of the loop.

[0196] [Table 1-3]

[0197] Therefore, unless otherwise specified, a “CDR” or “complementarity-determining region” of an antibody or its variable region, or any specified individual CDR (e.g., CDR-H1, CDR-H2, CDR-H3), is understood to encompass a certain (or specific) CDR as defined by any of the schemes described above. For example, if it is stated that a particular CDR (e.g., CDR-H3) contains the amino acid sequence of the corresponding CDR in a certain VHH amino acid sequence, such a CDR is understood to have the sequence of the corresponding CDR (e.g., CDR-H3) in the VHH as defined by any of the schemes described above. In some embodiments, a specific CDR sequence is specified. CDR sequences can be described using various numbering schemes (see, for example, Table 1-3), but it is understood that an antibody may contain a CDR described according to any other numbering scheme described above, or any other numbering scheme known to those skilled in the art.

[0198] In the single-domain antibody sequences of this disclosure, the framework sequence may be any suitable framework sequence. For example, the framework sequence may be a framework sequence derived from a heavy chain variable domain (e.g., a VH sequence or a VHH sequence). In some embodiments, the framework sequence is a framework sequence derived from a VHH sequence (in which case the framework sequence may optionally be partially or completely humanized) or a conventional VH sequence (in which case the framework sequence may optionally be partially or completely camelized).

[0199] Antigen-binding fragments (or combinations of fragments) of any of the single-domain antibodies described herein, such as fragments comprising one or more CDR sequences and appropriately sandwiched and / or appropriately linked via one or more framework sequences, are also included within the scope of this disclosure.

[0200] However, it should be noted that this disclosure is not limited to the origin of single-domain antibodies (or the nucleotide sequences used to express them) or to methods for producing or obtaining such single-domain antibodies or nucleotide sequences. Therefore, the single-domain antibodies of this disclosure may include naturally occurring sequences, recombinant sequences, or synthetic or semi-synthetic sequences (derived from a suitable species). Similarly, the nucleotide sequences encoding the single-domain antibodies of this disclosure may include naturally occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (e.g., sequences prepared by PCR or sequences isolated from a library).

[0201] In some embodiments, the single-domain antibodies described herein bind to therapeutic targets, such as antigens. The binding affinity of molecular interactions between two molecules can be measured by various techniques, including surface plasmon resonance (SPR), bio-layer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescence-activated cell sorting (FACS), or flow cytometry-linked assays. Surface plasmon resonance is a biosensor technique that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, in which one molecule is immobilized on a biosensor chip, and another molecule passes over this immobilized molecule under flow conditions (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559). SPR can be performed, for example, using the BIACORE® system or the Carterra LSA system. Another biosensor technique that can be used to determine the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377: 209-217). Biolayer interferometry is a label-free optical technique that analyzes the interference patterns of light reflected from two surfaces, namely the internal reference layer and the layer of immobilized proteins at the biosensor tip (reference beam and signal beam, respectively). A change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern, which is reported as a wavelength shift (nm), and its magnitude is a direct measure of the number of molecules bound to the biosensor tip surface. Because interactions can be measured in real time, association and dissociation rates, as well as affinity, can be determined. BLI can be performed, for example, using Octet® Systems.Alternatively, affinity can be measured using the Kinetic Exclusion Assay (KinExA), a solution-based method for determining the true equilibrium binding affinity and kinetics of unmodified molecules (see, for example, Drake et al. 2004, Anal. Biochem., 328: 35-43). The equilibrium solution of the antibody / antigen complex is passed through a column containing beads pre-coated with the antigen (or antibody) to bind the free antibody (or antigen) to the coated molecule. Detection of the thus captured antibody (or antigen) is achieved by a fluorescently labeled protein that binds to the antibody (or antigen).

[0202] The single-domain antibodies of this disclosure may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR region of the heavy chain variable domain compared to the exemplary antibody sequences provided herein. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The antigen-binding molecules of this disclosure may contain an antigen-binding domain derived from any of the exemplary amino acid sequences disclosed herein, where one or more amino acids in the framework and / or CDR region are mutated to the corresponding one or more residues in another germline sequence, or to a conserved amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as “germline mutations”). Those skilled in the art can readily generate a number of antibodies and antigen-binding fragments containing one or more individual germline mutations or combinations thereof, starting from the heavy chain variable region sequences disclosed herein. In certain embodiments, all framework and / or CDR residues within the VHH domain are mutated back to residues found in the original germline sequence from which the antigen-binding domain originally derives. In other embodiments, only specific residues, for example, only mutated residues found in the first eight amino acids of FR1 or the last eight amino acids of FR4, or only mutated residues found in CDR1, CDR2, or CDR3 are mutated back to the original germline sequence. In other embodiments, one or more of the framework and / or CDR residues are mutated back to the corresponding one or more residues in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antigen-binding domain originally derives).

[0203] Furthermore, the antigen-binding domain may contain any combination of two or more germline mutations within the framework and / or CDR region, for example, individual specific residues being mutated to corresponding residues in a particular germline sequence, while other specific residues different from the original germline sequence are maintained or mutated to corresponding residues in a different germline sequence. Once an antigen-binding domain containing one or more germline mutations is obtained, it can be readily tested for one or more desired properties, such as improved binding specificity, enhanced binding affinity, improved or enhanced biological properties (e.g., agonist effect), or reduced immunogenicity. Single-domain antibodies containing one or more antigen-binding domains obtained by such a general method are included within the scope of this disclosure.

[0204] This specification considers single-domain antibodies comprising any variant of a VHH and / or CDR amino acid sequence having one or more amino acid substitutions. For example, this disclosure includes single-domain antibodies having a VHH and / or CDR amino acid sequence having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2, or 1 amino acid substitution to any VHH and / or CDR amino acid sequence of the original germline sequence. The amino acid substitutions may be introduced into a single-domain antibody of interest, and the resulting variant can be screened for desired activities, such as retained / improved antigen binding, reduced immunogenicity, or reduced ADCC or CDC.

[0205] Amino acids can be grouped according to their common side-chain properties as follows: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. In some embodiments, amino acid substitutions are conserved substitutions, meaning that one amino acid is replaced with another amino acid of the same class. In some embodiments, amino acid substitutions may also include non-conservative substitutions, meaning that one amino acid is replaced with an amino acid of a different class. Other exemplary amino acid substitutions are shown in Table 1-4.

[0206] [Table 1-4]

[0207] In some embodiments, the single-domain antibodies of this disclosure (e.g., VHH) are modified to enhance binding to Staphylococcus aureus protein A (SpA) or Streptococcus aureus protein G (SpG). Binding of SpA and SpG to an antibody or antibody fragment may be useful in the manufacturing process of the antibody or antibody fragment. High-affinity interactions between the IgG Fc region and SpA and SpG are widely used and have become the gold standard for monoclonal antibody purification (Bjoerck and Kronvall, 1984). Other non-Fc-containing antibody fragments, such as VHH and Fab, do not have the ability to bind to SpA or SpG via their Fc region. However, these non-Fc-containing antibody fragments have been demonstrated to interact with SpA in a sequence-dependent manner (Graille et al., 2000; Henry et al., 2016). This feature avoids the possibility of using affinity tags fused to drug candidates for affinity chromatography that have drawbacks considered unfavorable to the sequence. This is because it can affect the immunogenicity of the protein, as well as its structure and stability, potentially impairing its functionality. The interaction of single-domain antibodies (e.g., VHH) with SpA depends on a different binding mode, with affinities of 1–5 μM, comparable to the 0.2–3 μM measured for VH-SpA interactions (To et al., JBC, 2005; Henry et al., Plos One, 2016).

[0208] In some embodiments, the single-domain antibodies of this disclosure (e.g., VHH) have or are modified to have a SpA-binding motif. For example, the VHH-SpA interface is mapped to 13 residues clustered within a framework on the back side of the V-body, separate from the CDR (Graille et al., 2000, Henry et al., 2016). In the absence of the VHH-SpA costructure, the binding mode can be visualized by superimposing the SpA-Fab crystal structure with VHH. Based on structural and functional analysis, the 13 residues at the VHH-SpA interface have been characterized as being inresistant to substitutions (residues Gly15, Arg19, Tyr59, Gly65, and Arg66), resistant to specific substitutions (residues Thr / Lys / Arg57, Thr68, Gln81, Asn82a, and Ser82b), or resistant to various substitutions overall (residues Ser17, Lys64, and Ser70) (all residue positions refer to Kabat numbering) (Henry et al., Plos One, 2016). Therefore, the SpA-binding motif contained in the single-domain antibody (e.g., VHH) of this disclosure may contain one, more, or all of the above 13 residues.

[0209] Fusion proteins and conjugates In some embodiments, what is provided herein are fusion proteins and conjugates comprising one or more single-domain antibodies described herein. In some embodiments, at least one single-domain antibody is located at the carboxyl terminus of the fusion protein. In some embodiments, one or more single-domain antibodies may be directly or indirectly ligated to one or more additional domains or portions. In some embodiments, one or more single-domain antibodies may be conjugated to a second portion. In some embodiments, the fusion protein or conjugate of the Disclosure comprises a single polypeptide. In other embodiments, the fusion protein or conjugate of the Disclosure comprises two or more polypeptides. In some embodiments, the fusion protein or conjugate of the Disclosure comprises two polypeptides.

[0210] In some embodiments, the fusion protein or conjugate of the Disclosure comprises at least one single-domain antibody as described herein. In some embodiments, the fusion protein or conjugate is polyvalent. For example, the fusion protein or conjugate of the Disclosure may be at least bivalent, but may also be trivalent, tetravalent, pentavalent, hexavalent, etc. The terms “bivalent,” “trivalent,” “tetravalent,” “pentavalent,” or “hexavalent” are all included in the term “polyvalent,” indicating the presence of 2, 3, 4, 5, or 6 binding units (e.g., VHH), respectively.

[0211] In certain embodiments, the fusion protein or conjugate is multispecific. For example, one or more additional domains or portions may be one or more additional binding domains that bind to one or more further antigens or proteins. The fusion protein or conjugate of this disclosure may be, for example, bispecific, trispecific, tetraspecific, pentaspecific, etc. The terms "bispecific," "trispecific," "tetraspecific," and "pentaspecific" are all included in the term "multispecific," meaning that they bind to two, three, four, five, etc., different target molecules, respectively.

[0212] When two or more single-domain antibodies are included in a fusion protein or conjugate, these two or more single-domain antibodies may contain identical sequences or different sequences. In such embodiments, the two or more single-domain antibodies may bind to the same epitope on the target antigen or to multiple different epitopes on the target antigen. For example, the fusion protein or conjugate of this disclosure may be biparatopic if, for example, two VHHs bind to two different epitopes on a single target antigen.

[0213] Fusion or conjugation into the FC domain In some embodiments, the fusion protein or conjugate of the Disclosure comprises at least one single-domain antibody provided herein, operably linked to an immunoglobulin Fc region. The immunoglobulin Fc region may be indirectly or directly linked to at least one single-domain antibody. In some embodiments, the fusion protein or conjugate of the Disclosure comprises one, two, three, four, five, six, or more single-domain antibodies provided herein, operably linked to an Fc region.

[0214] As used herein, “Fc region” refers to a portion of the heavy chain constant region containing CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In various embodiments, if the Fc region includes a hinge, this hinge can mediate dimerization between two Fc-containing polypeptides. In various embodiments, the Fc region contained in the fusion protein or conjugate of this disclosure is either a human immunoglobulin Fc region or derived from a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is of the IgG, IgE, IgM, IgD, IgA, or IgY isotype. In some embodiments, the immunoglobulin Fc region is an IgG isotype, such as the IgG1, IgG2, IgG3, or IgG4 subclass. The immunoglobulin Fc region may include variants or fragments of a native IgG Fc region.

[0215] Native Fc regions typically possess effector functions, including but not limited to Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cellular cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Such effector functions generally require the combination of the Fc region with a binding domain (e.g., an antibody-variable domain) and can be evaluated using various assays.

[0216] In some embodiments, the fusion protein or conjugate of the present disclosure may contain dimers of the Fc region. In some embodiments, the Fc region mediates the dimerization of antigen-binding units under physiological conditions, such as during cell expression, thereby forming a dimer that doubles the number of antigen-binding units. For example, a fusion polypeptide containing one VHH domain that binds to both an antigen and an Fc region is monovalent as a monomer, but because the Fc region can mediate dimerization, the resulting fusion protein is bivalent (i.e., has two VHH domains per molecule). Similarly, in some embodiments, two VHH domains (2x) fuse to one IgG Fc region, and as a result of dimerization, the fusion protein becomes tetravalent (i.e., has four VHH domains per molecule). In some embodiments, three VHH domains (3×) fuse to one IgG Fc region, and as a result of dimerization, the fusion protein becomes pentavalent (i.e., has six VHH domains per molecule).

[0217] In some embodiments, the 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, where n and m can independently be any integer (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.

[0218] For example, the 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 following structure: (VHH)-linker-Fc-linker-(VHH). The multiple linkers used in the fusion protein are not necessarily identical.

[0219] As another example, the fusion protein or conjugate of the present 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 identical.

[0220] In some embodiments, the CH3 domain of the Fc region can be used as a homodimerization domain, and the resulting fusion protein can be formed from two identical polypeptides. In other cases, heterodimerization can be enabled by mutating the CH3 dimer interface region of the Fc region. For example, by incorporating a heterodimerization domain into the fusion protein, the construct can be made into a heterodimer fusion protein.

[0221] When a dimer of an Fc region 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, for example, IgG1 / IgG1, IgG2 / IgG2, or IgG4 / IgG4. Alternatively, the first and second Fc regions may be of different IgG isotypes, for example, IgG1 / IgG2, IgG1 / IgG4, or IgG2 / IgG4.

[0222] In some embodiments, the Fc region contained in the 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 the effector function of the Fc region. Various examples of mutations to the Fc region that alter, for example, reduce, the effector function are known and include any of the following. Generally, the numbering of residues in immunoglobulin heavy chains or parts thereof such as the Fc region is by EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0223] In some embodiments, the human IgG Fc region is modified to alter antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cell-mediated cytotoxicity (CDC). Non-limiting examples of amino acid modifications that can alter ADCC and / or CDC include: 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 these references is incorporated herein by reference in its entirety).

[0224] In some embodiments, the Fc region contained in the fusion protein or conjugate of this disclosure exhibits reduced effector function (such as CDC and ADCC). Various in vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the fusion protein construct and / or its cleaved components lack binding to FcγR (and are therefore likely to lack ADCC activity), but retain FcRn binding ability. The main cells mediating ADCC are NK cells that express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Non-limiting examples of in vitro assays for evaluating the ADCC activity of molecules of interest are described, for example, in U.S. Patent No. 5,500,362; U.S. Patent No. 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, non-radioactive assay methods may be employed, such as the ACTI® non-radioactive cytotoxicity assay for flow cytometry or the CytoTox96® non-radioactive cytotoxicity assay. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).A C1q binding assay may be performed to confirm that the fusion protein construct or its cleavage components cannot bind to C1q and therefore lack CDC activity (see, for example, the C1q and C3c binding ELISAs in International Publication No. 2006 / 029879 and International Publication No. 2005 / 100402). A CDC assay may also be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Binding to FcRn and determination of in vivo clearance / half-life can also be carried out using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0225] Examples of mutations that enhance ADCC include mutations at Ser239 and Ile332, e.g., Ser239Asp and Ile332Glu (S239D, 1332E). Examples of mutations that enhance CDC include mutations at Lys326 and Glu333. In some embodiments, the Fc region is mutated at one or both of these locations, e.g., Lys326Ala and / or Glu333Ala (K326A and E333A) using the Kabat numbering system.

[0226] In some embodiments, Fc receptor binding is reduced by altering the Fc region of the fusion protein at one or more of the following positions: Leu234 (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 the Fc receptor γ receptor (FcγR) while minimizing the impact on binding to the neonatal Fc receptor (FcRn).

[0227] In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 to prevent glycosylation of the fusion protein. For example, 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 the interaction with the Fc receptor. For example, 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 the interaction with the Fc receptor. For example, Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat numbering) to alter the interaction with the Fc receptor. For example, Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235. For example, 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 234, 235, and 297. For example, Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329. For example, 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 the interaction with the Fc receptor. For example, Asp265Ala (D265A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat numbering) to alter the interaction with the Fc receptor. For example, Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fc region of the fusion protein is modified at both amino acids 265 and 329.For example, 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 234, 235, and 265. For example, Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329. For example, Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, 265, and 329. For example, 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, Gly235 is deleted from the fusion protein. In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 to enhance interaction with CD32A. For example, Gly236Ala (G236A). In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index from Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0228] In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236. For example, Leu234Gly, Leu235Ser, Gly236Arg (L234G / L235S / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236. For example, Leu234Ser, Leu235Thr, Gly236Arg (L234S / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236. For example, Leu234Ser, Leu235Val, Gly236Arg (L234S / L235V / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236. For example, Leu234Thr, Leu235Gln, Gly236Arg (L234T / L235Q / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 236. For example, Leu234Thr, Leu235Thr, Gly236Arg (L234T / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids 234, 235, and 329. For example, Leu234Thr, Leu235Thr, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids 252, 254, and 256. For example, Met252Tyr, Ser254Thr, Thr256Glu (M252Y / S254T / T256E).

[0229] In some embodiments, the Fc region of the fusion protein has reduced binding to the Fc receptor due to the absence of amino acids at one or more of the following positions: Glu233(E233), Leu234(L234), or Leu235(L235). In some embodiments, the Fc region of the fusion protein has reduced binding to the Fc receptor due to the absence of amino acids at one or more of the following positions: Glu233(E233), Leu234(L234), or Leu235(L235), and modification at one or more of Asp265(D265), Asn297(N297), or Pro329(P329). For example, the Fc region in the antigen-binding polypeptide is derived from a human Fc domain and contains deletions of three amino acids corresponding to IgG1 E233, L234, and L235 in the lower hinge. In some embodiments, such Fc polypeptides do not bind to FcγR and are therefore called "effector silent" or "effector null." For example, the deletion of these three amino acids in the Fc region reduces binding to the complement protein C1q. In some embodiments, polypeptides having an Fc region with these three amino acid Fc deletions maintain binding to FcRn, resulting in an extended half-life and transcytosis associated with FcRn-mediated recycling.

[0230] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000013.tif39169

[0231] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000014.tif40170

[0232] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000015.tif39170

[0233] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000016.tif40170

[0234] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000017.tif44170

[0235] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000018.tif42170

[0236] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000019.tif44170

[0237] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000020.tif45170

[0238] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000021.tif44170

[0239] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000022.tif46170

[0240] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG1 Fc region having the following amino acid sequence: TIFF2026521677000023.tif45170

[0241] In some embodiments, the human IgG Fc region is modified to enhance binding to FcRn. Examples of Fc mutations that enhance binding to FcRn include 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 Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L, respectively) (EU index in Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0242] In some embodiments, the Fc region lacks or has reduced fucose linked to the N297 N-linked glycan chain. Numerous methods exist to prevent fucosylation, including, but are not limited to, production in FUT8-deficient cell lines; addition of inhibitors such as castanospermine to mammalian cell culture media; and metabolic manipulation of producing cell lines.

[0243] In some embodiments, the Fc domains contained in the fusion proteins or conjugates of the Disclosure are derived from human Fc domains and include mutations M252Y and M428V. In some embodiments, the mutated or modified Fc polypeptides include 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 endosomes (around 6.5) but lose detectable binding at the neutral pH (around 7.2), enabling enhanced recycling via FcRn and extension of half-life.

[0244] In some embodiments, the Fc domains contained in the fusion protein or conjugate are derived from human Fc domains and include mutations to induce heterodimerization. In some embodiments, such mutations include those called "knob" and "hole" mutations. For example, if there is an amino acid modification at Thr366 in the CH3 domain, this CH3 domain can preferentially pair with a second CH3 domain having amino acid modifications at the positions of Thr366, Leu368, and Tyr407, such as Ser, Ala, and Val (T366S / L368A / Y407V), if a substitution with a bulkier amino acid, such as Try (T366W), is made. In some embodiments, the "knob" Fc domain includes the mutation T366W. In some embodiments, the "hole" Fc domain includes the mutations T366S, L368A, and Y407V. Heterodimerization by modification of CH3 can be further stabilized by introducing disulfide bonds, for example, by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) in the opposing CH3 domain (as described in Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, the Fc domain used for heterodimerization includes further mutations, such as the mutation S354C on the first member of the heterodimer Fc pair, which forms an asymmetric disulfide with the corresponding mutation Y349C on the second member of the heterodimer Fc pair. In some embodiments, one member of the heterodimer Fc pair includes a modified H435R or H435K to prevent binding to protein A while maintaining binding to FcRn. In some embodiments, one member of the heterodimer Fc pair includes modified H435R or H435K, while the second member of the heterodimer Fc pair is not modified in H435. In various embodiments, the whole Fc domain includes modified H435R or H435K (referred to as "whole R" in some examples where the modification is H435R), while the knob Fc domain does not include the modification.In some cases, the hole R mutation improves heterodimer purification by overriding any potentially present homodimeric hole Fc domain.

[0245] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the fusion protein of this disclosure is monomeric. For example, modification of the charged residue at residue Thr366, such as Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0246] In some embodiments, the immunoglobulin Fc region of the fusion protein is a human IgG3 isotype or a variant thereof. In one embodiment, the IgG3 Fc region is modified at amino acid Asn297 (Kabat numbering) to prevent antibody glycosylation, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG3 Fc region is modified at amino acid 435 to extend the half-life, e.g., Arg435His (R435H). In some embodiments, the human IgG3 Fc region lacks Lys447 (EU index in Kabat et al 1991).

[0247] In some embodiments, the immunoglobulin Fc region of the fusion protein is a human IgG4 isotype or a variant thereof. In one embodiment, the human IgG4 Fc region is modified at amino acid 235 to alter the interaction with the Fc receptor, e.g., Leu235Glu (L235E). In some embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (Kabat numbering) to prevent antibody glycosylation, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG4 Fc region lacks Lys447 (EU index in Kabat et al 1991).

[0248] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region having the following amino acid sequence: TIFF2026521677000024.tif35170

[0249] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region having the following amino acid sequence: TIFF2026521677000025.tif37170

[0250] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region having the following amino acid sequence: TIFF2026521677000026.tif36170

[0251] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region having the following amino acid sequence: TIFF2026521677000027.tif34170

[0252] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of the human IgG4 Fc region having the following amino acid sequence: TIFF2026521677000028.tif36170

[0253] Further modifications of the IgG4 heavy chain suitable for use in the fusion protein or conjugate 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).

[0254] In some embodiments, the fusion protein or conjugate includes an immunoglobulin hinge region. In some embodiments, the hinge region functions as a linker for connecting one or more antigen-binding units (e.g., VHH) to the Fc region. In other embodiments, the fusion protein may include a linker in addition to the hinge region for connecting one or more antigen-binding units (e.g., VHH) to the Fc region. The hinge region may also be selected from any of the human IgG subclasses. For example, the fusion protein may include a modified IgG1 hinge having the sequence EPKSSDKTHTCPPC (SEQ ID NO: 278), where Cys220, which 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 includes a truncated hinge having the sequence DKTHTCPPC (SEQ ID NO: 279).

[0255] In some embodiments, the fusion protein or conjugate has a modified hinge derived from IgG4 that has been modified (e.g., Ser228Pro(S228P)) to prevent or reduce chain exchange, and which has the sequence ESKYGPPCPPC (SEQ ID NO: 280).

[0256] In alternative embodiments, the fusion protein or conjugate of the present disclosure may include sequences other than the Fc region to achieve multimerization (e.g., dimerization). For example, including an amino acid sequence containing at least one cysteine ​​residue can facilitate dimerization of two polypeptides by forming a disulfide bond between them. In some embodiments, such a multimerizing domain may include one or more cysteine ​​residues or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides containing or comprising a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0257] Suitable Fc mutations for use in the fusion proteins or conjugates disclosed herein are discussed, for example, in Wilkinson et al., Fc-engineered antibodies with immune effector functions completely abolished. PLoS One. 2021; International Publication No. 2021234402; U.S. Patent No. 8,969,526; European Patent No. 3692065; and U.S. Patent No. 7,083,784 (each of which is incorporated herein by reference).

[0258] Fusion or conjugation of the half-life extension portion In some embodiments, the fusion protein or conjugate of the present disclosure may include one or more other parts that provide the fusion protein or conjugate with an increased (in vivo) half-life. An extended in vivo half-life means that the fusion protein or conjugate has an increased half-life in the body of a mammal, such as a human subject, after administration.

[0259] Non-limiting examples of half-life extension portions suitable for use in this disclosure include polyethylene glycol (PEG) molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind to serum proteins.

[0260] In some embodiments, the fusion protein or conjugate of the Disclosure may include a binding site that can bind to serum albumin, such as human serum albumin, or to serum immunoglobulin, such as IgG. In one embodiment, the fusion protein or conjugate of the Disclosure may include a binding site that can bind to human serum albumin. In one embodiment, the binding site is a single-domain antibody (e.g., VHH).

[0261] For example, but not limited to, albumin conjugates described in International Publication No. 04 / 041865, International Publication No. 06 / 122787, International Publication No. 2012 / 175400, International Publication No. 2012 / 175741, International Publication No. 2015 / 173325, International Publication No. 2017 / 080850, International Publication No. 2017 / 085172, International Publication No. 2018 / 104444, International Publication No. 2018 / 134235, and International Publication No. 2018 / 134234 (each of which is incorporated into this application in its entirety by reference) can be used in the fusion proteins or conjugates of this disclosure.

[0262] Fusion or conjugation with other parts The single-domain antibodies provided herein may be operably linked, directly or indirectly, to a second portion such as a detectable label, drug, toxin, radionuclide, enzyme, immunomodulator, cytokine, cytotoxic agent, small molecule drug, chemotherapeutic agent, therapeutic agent, diagnostic agent, or a combination thereof.

[0263] In some embodiments, the conjugates of the present disclosure include labels that can generate a detectable signal. Such conjugates can be used for research or diagnostic purposes, such as in vivo detection of cancer. Preferably, the labels can generate a detectable signal directly or indirectly. For example, the labels may be radiopaque or radioisotopes (e.g., 3H, 14C, 32P, 35S, 123I, 125I, 131I); fluorescent (fluorophore) or chemiluminescent (chromophore) compounds (e.g., fluorescein isothiocyanate, rhodamine, or luciferin); enzymes (e.g., β-galactosidase, alkaline phosphatase, or horseradish peroxidase); contrast agents; or metal ions. In some embodiments, the label is a radioactive atom for scintigraphy studies, such as 99Tc or 123I, 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. For example, for PET imaging, zirconium-89 can be complexed with various metal chelating agents and conjugated to an antibody (International Publication No. 2011 / 056983).

[0264] The single-domain antibodies of this disclosure may be conjugated to other parts, such as epitope tags, for purposes of purification or detection. Examples of such molecules useful for protein purification include molecules that present a structural epitope that can be 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 that can bind to the immobilized compound. Non-limiting examples of epitope tag molecules that can be conjugated to the single-domain antibodies of this disclosure for the purpose of molecular recognition include polyhistidine tags (His tags), myc tags, human influenza hemagglutinin (HA) tags, FLAG tags, maltose-binding proteins, glutathione-S-transferase, biotin, and streptavidin. The conjugates containing the epitopes presented by these molecules can be recognized by complementary molecules such as maltose, glutathione, nickel-containing complexes, anti-FLAG antibodies, anti-myc antibodies, anti-HA antibodies, streptavidin, or biotin. For example, the single-domain antibodies of this disclosure conjugated to an epitope tag can be purified 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 complementary molecules capable of selectively recognizing and binding to the epitope tag of the antigen antibody or its fragment. An example of a solid-phase resin is agarose beads suitable for purification in aqueous solution.

[0265] In some embodiments, the conjugates of the Disclosure may comprise one or more VHH domains described herein conjugated to a therapeutic agent, which may be cytotoxic, or inhibit cell proliferation, or provide some therapeutic benefit. In some embodiments, the cytotoxic agent may be a drug, a chemotherapeutic agent, a growth inhibitor, a toxin (e.g., an enzyme-active toxin or fragment thereof derived from bacteria, fungi, plants, or animals), or a radioisotope (e.g., a radioconjugate). Such conjugates may be applicable, for example, to the treatment or prevention of diseases associated with autoreactive cytotoxic T cell activity. In some embodiments, the antibody-drug conjugates described herein may enable targeted delivery of the drug portion to a target tissue (e.g., a tumor).

[0266] In some embodiments, the conjugates of this disclosure include toxins. In some embodiments, the toxins include, 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)), mytansinoids (European Patent No. 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calichemycin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res. Examples include 53:3336-3342 (1993). Toxins can exert their cytotoxic and cell proliferation inhibitory effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Examples of other therapeutic agents that can be conjugated to the single-domain antibodies of this disclosure are described herein.

[0267] In some embodiments, the single-domain antibodies of this disclosure may be fused or conjugated to one or more moieties that facilitate delivery to the central nervous system (CNS) / brain. The moieties that can facilitate delivery of the single-domain antibody to the CNS / brain may be, for example, peptides, polypeptides, small molecules, lipids, or synthetic polymers. Various approaches for delivering single-domain antibodies to the brain are described in Pothin et al., Pharmaceutics 2020, 12(10), 937 (which is incorporated herein by reference in its entirety).

[0268] As a non-limiting example, the single-domain antibodies of this disclosure may be fused to or conjugated to a transferrin receptor (TfR) or insulin receptor-binding moiety (e.g., an antibody). Transferrin receptors (TfRs) are highly expressed by brain capillary endothelial cells (BCECs) that form the blood-brain barrier (BBB) ​​and have been used as targets for drug delivery to the brain. Monoclonal antibodies that bind to TfRs, such as clone Ri7, have been shown to be internalized in BCECs in vivo. As another example, the single-domain antibodies of this disclosure may be conjugated to a hydrophobic fatty acid moiety such as a C18 fatty acid (stearic acid), a C16 fatty acid (palmitic acid), or a C8 fatty acid (octanoic acid); or to an amphiphilic block copolymer moiety such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (Pluronic® or Poloxamer) or poly(2-oxazoline). Various fatty acid moieties and block copolymer moieties that can be used for protein delivery to the brain are described, for example, in Yi and Kabanov, J Drug Target. 2013; 21(10): 940-955 (the entire work is incorporated herein by reference).

[0269] Examples of methods for attaching the label or other portion to the 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, NY (1983); and Colcher et al., Meth. Enzymol., 121:802-16 (1986). Further preferred methods for preparing the conjugates of this disclosure are described, for example, in International Publication No. 2009 / 067800, International Publication No. 2011 / 133886, and U.S. Patent Application Publication No. 2014322129 (which is incorporated herein by reference in its entirety).

[0270] In some embodiments, the binding between the single-domain antibody and the second portion may be covalent or non-covalent, for example, due to a non-covalent interaction between biotin and streptavidin. In some embodiments, the second portion can be conjugated to the single-domain antibody using any of the various molecular biological or chemical conjugation and linking methods known in the art and described below. In some embodiments, the second portion can be linked or conjugated to the single-domain antibody described herein using a linker such as a peptide linker, a cleavable linker, a non-cleavable linker, or a linker that assists the conjugation reaction.

[0271] In some embodiments, a single-domain antibody is optionally conjugated via a linker to, for example, about 1 to about 20 parts per molecule. In some embodiments, one or more second parts may be identical or different. The linker may consist of one or more linker components. For covalent bonding between the antibody and the second part, the linker typically has two reactive functional groups, i.e., is divalent in terms of reaction. Divalent linker reagents useful for conjugating two or more functional or bioactive moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups, are described, for example, in Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, pp. 234-242.

[0272] In some embodiments, linkers used in the conjugates of the present disclosure include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthiopentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-I carboxylate ("SMCC"), or N-succinimidyl (4-iodoacetyl)aminobenzoate ("STAB"), or combinations thereof.

[0273] In some embodiments, the linker used in the conjugate of this disclosure may include amino acid residues. Examples of amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Examples of dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Examples of tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues used in the amino acid linker components may include naturally occurring amino acids, trace amino acids, and non-naturally occurring amino acid analogs such as citrulline. The amino acid linker components can be designed and optimized with consideration for selectivity to enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0274] Conjugates of single-domain antibodies with a second portion (e.g., a cytotoxic agent) can be prepared using a variety of bifunctional protein conjugates, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl substrates), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bisactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0275] The conjugates of this disclosure can be prepared by a variety of methods. For example, conjugation methods include: (1) reacting the nucleophile of the VHH domain with a divalent linker reagent to form a VHH-linker by covalent bonding, and then reacting it with the drug moiety; or (2) reacting the nucleophile of the drug moiety with a divalent linker reagent to form a drug-linker by covalent bonding, and then reacting it with the nucleophile of the VHH domain.

[0276] Nucleophiles on proteins containing 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) glycosylated sugar hydroxyl or amino groups of the antibody. Amine, thiol, and hydroxyl groups are nucleophilic and can form covalent bonds with electrophiles 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; and (iii) aldehydes, ketones, carboxyls, and maleimide groups. Further nucleophiles can be introduced into proteins (e.g., antibodies such as VHH domains) by the reaction of lysine, which converts amines to thiols, with 2-iminothiolane (Trout's reagent). By introducing one, two, three, four, or even more cysteine ​​residues, reactive thiol groups can be introduced into proteins (e.g., antibodies such as VHH domains).

[0277] Conjugates, such as antibody-drug conjugates, can also be produced by modifying antibodies, such as VHH domains, to introduce electrophilic moieties that can react with nucleophilic substituents on linker reagents or drugs. By oxidizing the sugar of a glycosylated antibody, for example, using a periodic acid oxidation reagent, an aldehyde or ketone group can be formed, which can react with the amine group of the linker reagent or drug moiety. The resulting iminschiff base group can form a stable bond, or can form a stable amine bond by reduction, for example, with a boron hydride reagent. In one embodiment, a carbonyl (aldehyde and ketone) group that can react with an appropriate group on a drug can be generated in the protein by reacting the carbohydrate moiety of a glycosylated antibody with galactose oxidase or sodium metaperiodate (Hermanson, Bioconjugate Techniques). In another embodiment, a protein containing an N-terminal serine or threonine residue can react with sodium metaperiodate to generate an aldehyde in place of the first amino acid. This aldehyde can be reacted with the drug portion or the nucleophile of the linker.

[0278] Similarly, nucleophiles on the drug moiety include, but are not limited to, amine groups, thiol groups, hydroxyl groups, hydrazide groups, oxime groups, hydrazine groups, thiosemicarbazone groups, hydrazine carboxylate groups, and arylhydrazide groups, which can form covalent bonds with electrophiles on the linker moiety and linker reagent, including: (i) active esters such as NHS esters, HOBt esters, haloform esters, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; and (iii) aldehydes, ketones, carboxyl groups, and maleimide groups.

[0279] Alternatively, a fusion protein containing a VHH domain and a cytotoxic agent may be prepared, for example, by recombinant DNA techniques or peptide synthesis. The DNA sequence may be recombined so that the regions encoding the two parts of the fusion protein are adjacent to each other, or separated by a region encoding a linker peptide that does not impair the desired properties of the fusion protein. This DNA sequence can then be transfected into host cells expressing the fusion protein. The fusion protein can be recovered from the cell culture and purified using techniques known in the art.

[0280] Linker In some embodiments, one or more polypeptides of the fusion protein of the present disclosure are operably linked via a peptide linker. The peptide linker can be 2 to 60 amino acids or longer, and in certain embodiments, the peptide linker may be 3 to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids in length.

[0281] In some embodiments, the peptide linker, which is, for example, a peptide linker separating two VHH domains or separating a VHH domain from the heavy chain constant region, is at least 5 amino acids long, at least 6 amino acids long, or at least 7 amino acids long, and optionally up to 30 amino acids long, up to 40 amino acids long, up to 50 amino acids long, or up to 60 amino acids long.

[0282] In some embodiments, the linker is from 5 to 50 amino acids in length, such as 5 - 50, 5 - 45, 5 - 40, 5 - 35, 5 - 30, 5 - 25, or 5 - 20 amino acids in length. In other embodiments, the linker is from 6 to 50 amino acids in length, such as 6 - 50, 6 - 45, 6 - 40, 6 - 35, 6 - 30, 6 - 25, or 6 - 20 amino acids in length. In still other embodiments, the linker is from 7 to 50 amino acids in length, such as 7 - 50, 7 - 45, 7 - 40, 7 - 35, 7 - 30, 7 - 25, or 7 - 20 amino acids in length.

[0283] In some embodiments, a charged linker (such as a charged hydrophilic linker) and / or a flexible linker is used. Examples of flexible linkers that can be used in the fusion proteins of the present disclosure include those described in Chen et al., 2013, Adv Drug Deliv Rev. 65(10): 1357 - 1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10): 325 - 330. Particularly useful flexible linkers are repeats of glycine and serine, such as G n S (SEQ ID NO: 254) or SG n (SEQ ID NO: 255) monomers or multimers, or include them (referred to herein as "GS linkers"), where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is a repeat of G4S (SEQ ID NO: 210), such as (GGGGS) n (SEQ ID NO: 256) monomers or multimers, or includes it.

[0284] Polyglycine linkers can be appropriately used in the fusion proteins of this disclosure. In some embodiments, the peptide linkers used herein include two consecutive glycine molecules (2Gly), three consecutive glycine molecules (3Gly), four consecutive glycine molecules (4Gly) (SEQ ID NO: 257), five consecutive glycine molecules (5Gly) (SEQ ID NO: 258), six consecutive glycine molecules (6Gly) (SEQ ID NO: 259), seven consecutive glycine molecules (7Gly) (SEQ ID NO: 260), eight consecutive glycine molecules (8Gly) (SEQ ID NO: 261), or nine consecutive glycine molecules (9Gly) (SEQ ID NO: 262).

[0285] In some embodiments, the 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 protein may include a combination of a GS linker and a glycine linker.

[0286] In one embodiment, two or more VHHs are linked via the GGGGSGGGGSGGGGS (Sequence ID 211) linker. In one embodiment, two or more VHHs are linked via the GGGGSGGGGS (Sequence ID 267) linker. In one embodiment, a VHH and an Fc region are linked via the GGGGSESKYGPPCPSCP (Sequence ID 249) linker. In one embodiment, a VHH and an Fc region are linked via the GGGGS (Sequence ID 210) linker.

[0287] In some embodiments, one or more polypeptides of the fusion protein of the present disclosure are operably linked via a “rigid” peptide linker. Such peptide linkers may contain proline-rich peptides. In one embodiment, the rigid peptide linker comprises PAPAPAPAPAPAPAPAP (SEQ ID NO: 250). In one embodiment, the rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 253). In one embodiment, the rigid peptide linker comprises A(EAAAK)nA (SEQ ID NO: 268), where n is any integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0288] Table 2 shows other exemplary peptide linkers that can be used with the fusion proteins described herein.

[0289] [Table 2]

[0290] Polynucleotide molecules In another embodiment, what is provided herein is a polynucleotide molecule encoding a single-domain antibody or fusion protein as described herein. A polynucleotide molecule encoding one or more polynucleotide portions of the conjugate of the disclosure is also within the scope of the disclosure.

[0291] Polynucleotide molecules can be used to transform / transfect host cells or host organisms for polypeptide expression and / or production. Suitable hosts or host cells for polypeptide production as described herein include any suitable fungal cells or cell lines, prokaryotic cells or cell lines, or eukaryotic cells or cell lines, or any suitable fungal organisms, prokaryotes, or eukaryotes. Hosts or host cells containing polynucleotide molecules encoding single-domain antibodies or fusion proteins as described herein are also included in this disclosure.

[0292] Polynucleotide molecules may be, for example, DNA, RNA, or hybrids thereof, and may also include (e.g., chemically) modified nucleotides, such as locked nucleic acid (LNA) or peptide nucleic acid (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 double-stranded DNA (e.g., plasmid). In some embodiments, the polynucleotide is in the form of single-stranded RNA (e.g., mRNA).

[0293] Techniques for generating polynucleotides include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combination of two or more naturally occurring sequences and / or synthetic sequences (or two or more parts thereof); introduction of mutations leading to the expression of a shortened expression product; introduction of one or more restriction sites (for example, to create cassettes and / or regions that can be readily digested and / or ligated using a suitable restriction enzyme); and / or introduction of mutations by PCR reaction using one or more "mismatch" primers. Alternatively, the polynucleotides of this disclosure may be isolated from a suitable natural source. By subjecting a polynucleotide sequence encoding a naturally occurring (poly)peptide to, for example, site-directed mutagenesis, polynucleotide molecules encoding polypeptides with sequence diversity can be generated.

[0294] vector Furthermore, provided herein are vectors comprising polynucleotide molecules encoding the single-domain antibodies, fusion proteins, or other related polypeptides of this disclosure. As used herein, “vector” is a vehicle suitable for delivering genetic material to a host cell. Examples of vectors include nucleic acid vectors such as plasmids or mRNA, or nucleic acids embedded in larger structures such as liposomes or viral vectors.

[0295] A vector may include one or more of the following elements: a starting point for replication, one or more regulatory sequences that regulate the expression of the polypeptide of interest (e.g., promoter, enhancer, terminator), and / or one or more select marker genes (e.g., drug resistance genes, and genes that can be used for colorimetric assays, e.g., β-galactosidase). In the case of DNA-based vectors, this typically includes the presence of elements for transcription (e.g., promoter and polyA signaling) and elements for translation (e.g., Kozak sequences). In some embodiments, the vector is an expression vector, i.e., a vector suitable for expressing an encoded polypeptide or construct under favorable conditions within a host cell.

[0296] To express the single-domain antibody or fusion protein (or fragment thereof) of this disclosure, a polynucleotide encoding a partial-length or full-length polypeptide chain (e.g., VHH, VHH-Fc-VHH) obtained, for example, as described above, can be inserted into an expression vector so that the gene can be functionally linked to one or more transcriptional and translational regulatory sequences. The expression vector and expression regulatory sequences are selected to be compatible with the expression host cell in which they are used. Polynucleotides encoding two or more (if any, distinct) polypeptide chains of the single-domain antibody or fusion protein of this disclosure can be inserted into separate vectors, or optionally incorporated into the same expression vector.

[0297] In addition to polynucleotides encoding one or more polypeptide chains of a single-domain antibody or fusion protein, the recombinant expression vector of the present invention may include regulatory sequences that control the expression of the genes encoding the one or more polypeptide chains in a host cell. The design of the expression vector, including the selection of regulatory sequences, may depend on the selection 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., adenovirus major late promoter (AdMLP)), and polyoma. Further examples of viral regulatory elements and their sequences are described, for example, in U.S. Patent No. 5,168,062; U.S. Patent No. 4,510,245; and U.S. Patent No. 4,968,615 (disclosures of each document are incorporated herein by reference).

[0298] The recombinant expression vectors of this disclosure may have additional sequences, such as sequences that regulate vector replication in host cells (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells into which the vector is introduced (see, for example, U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017 (the disclosures of each document are incorporated herein by reference in their entirety)). Typically, for example, selection marker genes confer resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or noseoslysine, or cytotoxic agents such as G418, puromycin, blastosidine, hygromycin, or methotrexate to host cells into which the vector is introduced. Suitable selection marker genes include dihydrofolate reductase (DHFR) genes (for use in DHFR-deficient host cells with methotrexate selection / amplification) and neo genes (for G418 selection).

[0299] The vectors of this disclosure may further include sequence elements that enhance the translation rate of these genes or improve the stability or nuclear transport of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosomal entry sites (IRESs), and polyadenylation signaling sites, for inducing efficient transcription of genes supported on the expression vector.

[0300] Viral vectors can be used to efficiently deliver foreign genes into the genome of cells (e.g., eukaryotic or prokaryotic cells). Viral vectors are particularly useful for gene delivery because the polynucleotides contained in such genomes are typically incorporated into the target cell by universal or specific transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of suitable viral vectors include: retroviruses; adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48); parvoviruses (e.g., adeno-associated viruses (AAVs) such as AAV2, AAV8, AAV9); negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), and paramyxoviruses (e.g., measles and Sendai virus); positive-strand RNA viruses such as picornaviruses and alphaviruses; and adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus). Examples of double-stranded DNA viruses include baculoviruses, coronaviruses, and poxviruses (e.g., vaccinia, modified vaccinia ankara (MVA), fowlpox, canarypox). Other viruses that can be used to deliver polynucleotides encoding the polypeptides of this disclosure include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis viruses. Examples of retroviruses include, but are not limited to, avian leukemia / sarcoma, mammalian type C, B, and D viruses, HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, JM1996. Fundamental Virology, DMKDN Fields, PM Howley, ed. (Philadelphia, Lippincott-Raven Publishers): 763-843 (disclosures in this document are incorporated herein by reference)).Other examples of viral genomes that can be used in the compositions and methods of this disclosure include mouse leukemia virus, mouse sarcoma virus, 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 leukemia virus, Mason-Pfizer monkey virus, monkey immunodeficiency virus, monkey sarcoma virus, Rous sarcoma virus, and lentiviruses.

[0301] host cell In some embodiments, the Disclosure also provides host cells or host organisms comprising polynucleotides or vectors encoding single-domain antibodies, fusion proteins, or other related polypeptides described herein. Suitable host cells or host organisms may be any suitable fungal cells or cell lines, prokaryotic cells or cell lines, or eukaryotic cells or cell lines, or any suitable fungal organisms, prokaryotes, or eukaryotes. Host cells may include offspring of a single host cell, which may not necessarily be completely identical (in terms of morphology or genomic DNA complementarity) to the original parent cell due to spontaneous, accidental, or intentional mutations. Host cells may also include cells transfected in vivo with one or more polynucleotides or vectors provided herein.

[0302] Examples of 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. Examples of non-exclusive and representative mammalian cells include, but are not limited to, NSO cells, PER.C6® cells (Crucell), COS cells, SP2 / 0 cells, and 293 and CHO cells, as well as their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Examples of prokaryotic cells include bacterial cells such as Escherichia coli.

[0303] Preparation method This disclosure also provides methods for producing single-domain antibodies, fusion proteins, or conjugates as described herein.

[0304] In some embodiments, the Disclosure provides a method for modifying a single-domain antibody (e.g., VHH) described herein to reduce the binding of an anti-drug antibody to its C-terminus, the method comprising modification of the amino acid sequence at the carboxyl terminus of the single-domain antibody (e.g., VHH). In some embodiments, the method may include mutations in the single-domain antibody in FR1, FR2, FR3, or FR4, or combinations thereof. One or more modifications of the amino acid sequence at the carboxyl terminus of the single-domain antibody (e.g., VHH) and one or more mutations in the amino acid sequence of the single-domain antibody in FR1, FR2, FR3, or FR4, or combinations thereof, can be combined (in any combination) in the practice of the method described herein. In some embodiments, the method for modifying a single-domain antibody (e.g., VHH) described herein may include, for example, any of the modifications and / or mutations described in Tables 1-1, 1-2, or 1-4, or combinations thereof.

[0305] In some embodiments, a method for generating a single-domain antibody, fusion protein, or conjugate as described herein may include the steps of: transforming / transfecting a host cell or host organism with a polypeptide encoding the single-domain antibody, fusion protein, or one or more related polypeptides as described herein; expressing the single-domain antibody, fusion protein, or one or more related polypeptides in the host; and any one or more subsequent isolation and / or purification steps.

[0306] When a recombinant expression vector encoding one or more polypeptides of the single-domain antibody, fusion protein, or conjugate of the present disclosure is introduced into mammalian host cells, the host cells are cultured for a period sufficient to express one or more proteins or one or more polypeptides in the host cells, or for a period sufficient to secrete one or more proteins or polypeptides into the culture medium in which the host cells are growing. One or more proteins or one or more polypeptides can be recovered from the culture medium using standard protein purification methods. The host cells can also be used to generate a portion of a complete antibody, such as a VHH domain.

[0307] After generating the proteins or polypeptides of this disclosure by recombinant expression, they can be purified by any method known in the art for the purification of proteins or polypeptides, such as chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly by affinity for selected antigens after protein A or protein G), and sizing column chromatography), centrifugation, solubility difference chromatography, or any other standard technique for protein purification. Furthermore, the proteins or polypeptides of this disclosure can be fused with heterologous polypeptide sequences described herein (e.g., His tags) or heterologous polypeptides known in the art to facilitate purification or to generate therapeutic conjugates as described below. After isolation, the proteins or polypeptides of this disclosure can be further purified as needed, for example by high-performance liquid chromatography or gel filtration chromatography on a Superdex® column.

[0308] Compositions and Formulations This disclosure also provides compositions comprising a single-domain antibody, fusion protein, or conjugate of the technology, at least one polynucleotide molecule encoding them, at least one vector containing the polynucleotide molecule, or at least one host cell containing 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 polypeptides and / or compounds having pharmaceutically active properties.

[0309] As used herein, the term “pharmaceutically acceptable carrier” is intended to include any solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent, and absorption retarder suitable for pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences (incorporated herein by reference). Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and non-volatile oils may also be used. Auxiliary active compounds may also be incorporated into the composition.

[0310] Suitable formulations include, but are not limited to, solutions, suspensions, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic)-containing vesicles (e.g., LIPOFECTIN®, Life Technologies (Carlsbad, California)), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al. "Compendium of excipients for parenteral formulations" PDA (1998) J Phdomain Sci Technol 52:238-311.

[0311] The pharmaceutical compositions of this disclosure may be formulated according to the intended route of administration. Examples of preferred routes of administration include, for example, intravenous, subcutaneous, intratumoral, oral (e.g., intrabuccal, sublingual), intranasal, inhalation, intraocular, intramuscular, intradermal, transdermal (i.e., topical), intraperitoneal, transmucosal, vaginal, and rectal administration, or injection into the CNS / brain (e.g., intraspinal, intracerebral, or intrathecal administration). Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may contain the following components: sterile diluents such as water for injection, physiological saline, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; non-volatile oils; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffering agents such as phosphates, acetates, or citrates; and tonic modifiers such as sodium chloride or glucose. The pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in plastic or glass ampoules, disposable syringes, or multi-dose vials.

[0312] Suitable pharmaceutical compositions for use by injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include, for example, physiological saline, sterilized water, Cremophor EL®, or phosphate-buffered saline (PBS). The compositions are preferably sterile and have appropriate fluidity. In most embodiments, the compositions are stable under manufacturing and storage conditions and can be protected from contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained by coating, for example, lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial contamination can be achieved by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents such as sugars, polyhydric alcohols such as mannitol and sorbitol, and sodium chloride in the composition. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents in the composition, such as aluminum monostearate and gelatin.

[0313] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent containing, as needed, one or a combination of the above-mentioned components, and then sterilizing by filtration. Generally, they are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method includes vacuum drying and / or freeze-drying, thereby obtaining a powder with the active ingredient and any desired additional components from the pre-sterilized filtered solution.

[0314] Oral compositions may contain inert diluents or food carriers. These may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be used in combination with excipients in the form of tablets, lozenges, capsules, or liquids. Tablet and liquid formulations can be used for protease-insensitive VHH. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is applied to the mouth, rinsed, and then spat out or swallowed. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds having similar properties: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; flow promoters such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0315] When administered by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as carbon dioxide or other gas, or from a nebulizer.

[0316] Systemic administration may also be carried out by mucosal or percutaneous means. For mucosal or percutaneous administration, an appropriate penetrating agent for the barrier to be penetrated is used in the formulation. Such penetrating agents are generally known in the art, and for mucosal administration, for example, surfactants, bile salts, and fusidic acid derivatives are used. Mucosal administration can be achieved by using nasal sprays or suppositories. In the case of percutaneous administration, the active compound is formulated as an ointment, salve, gel, or cream, as is generally known in the art.

[0317] The compound can also be prepared in the form of a suppository (for example, using conventional suppository bases such as cocoa butter and other glycerides) or as a retained enema for rectal delivery.

[0318] For brain delivery, the compounds of this disclosure may be formulated to facilitate crossing of the blood-brain barrier. For example, the single-domain antibodies, fusion proteins, or conjugates of this disclosure may be encapsulated in brain-targeted liposomes, lipid nanoparticles, lipid microparticles, or lipid macrocapsules for delivery to the brain. An exemplary liposome delivery system is described in Pothin et al., Pharmaceutics 2020, 12(10), 937 (the entire text of which is incorporated herein by reference).

[0319] In some embodiments, the active compound is prepared using a carrier that can protect the compound from rapid elimination from the body, such as a controlled-release formulation including implants and microencapsulation delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Liposome suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as those described in U.S. Patent No. 4,522,811 (which is incorporated herein by reference in its entirety).

[0320] To facilitate administration and ensure uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions into unit dosage forms. As used herein, a unit dosage form refers to a physically independent unit adapted as a unit dose for a target subject to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect, along with a desired pharmaceutical carrier. The specifications of the unit dosage forms in this disclosure depend on the unique properties of the active compound, the specific therapeutic effect to be achieved, and the inherent limitations of the technique for formulating such active compounds for the treatment of an individual.

[0321] Pharmaceutical compositions (or their components) may be included in kits, containers, packs, or dispensers, along with instructions for administration. These pharmaceutical compositions may also be included in diagnostic kits, along with instructions for use.

[0322] The pharmaceutical composition is administered in an amount effective for the treatment or prevention of a specific indication. The therapeutically effective dose typically depends on the weight of the person being treated, the physical or health condition of the person being treated, the severity of the condition being treated, or the age of the person being treated. In some embodiments, the pharmaceutical composition may be administered in amounts of about 50 μg / kg body weight to about 50 mg / kg body weight per single dose. In some embodiments, the pharmaceutical composition may be administered in amounts of about 100 μg / kg body weight to about 50 mg / kg body weight per single dose. In some embodiments, the pharmaceutical composition may be administered in amounts of about 100 μg / kg body weight to about 20 mg / kg body weight per single dose. In some embodiments, the pharmaceutical composition may be administered in amounts of about 0.5 mg / kg body weight to about 20 mg / kg body weight per single dose. The frequency and duration of treatment can be adjusted according to the severity of the condition. The effective dosage and schedule for administering the pharmaceutical composition of this disclosure can be determined empirically, for example, by monitoring the patient's progress through periodic evaluations and adjusting the dose accordingly. Furthermore, scaling of drug dosages between species can be performed using methods known in the art (e.g., Mordenti et al., 1991, Phdomainaceut. Res. 8:1351).

[0323] In some embodiments, the pharmaceutical composition may be administered in amounts of about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition may be administered in amounts of about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition may be administered in amounts of about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition may be administered in amounts of about 20 mg to about 200 mg per dose.

[0324] In some embodiments in which the single-domain antibody of this disclosure is administered as a viral vector (e.g., AAV), the dose range and frequency of administration of the viral vector described herein may vary depending on the properties of the viral vector and the medical condition, as well as the parameters of the specific patient and the route of administration used. In some embodiments, the viral vector composition may vary depending on the form of administration, route of administration, nature of the disease, and the condition of the subject, to about 1 × 10⁻⁶ 5 Plaque forming unit (PFU) ~ approximately 1 × 10⁻⁶ 15 It can be administered to the target at doses within the range of pfu. In some cases, the viral vector composition is approximately 1 × 10⁶. 8 pfu~approx. 1×10 15 PFU, or approximately 1 x 10 10 pfu~approx. 1×10 15 PFU, or approximately 1 x 10 8 pfu~approx. 1×10 12 It can be administered in doses within the range of pfu. A more precise dose may also depend on the recipient. For example, a relatively low dose may be required for a young subject, while a relatively high dose may be required for an adult subject. In certain embodiments, a more precise dose may depend on the subject's body weight. In certain embodiments, for example, for a young human subject, approximately 1 × 10⁻⁶ pfu can be administered. 8 pfu~approx. 1×10 10 While pfu can be administered, in adult human subjects it is approximately 1 × 10⁶ 10 pfu~approx. 1×10 12 A dose of PFU can be administered.

[0325] Various delivery systems are known and can be used for administering the pharmaceutical compositions of this disclosure. Examples include liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, intraspinal, intracerebral, intrathecal, and oral routes. The compositions can be administered by any convenient route, such as infusion or bolus injection, absorption through the epithelium or inner mucocutaneous tissue (e.g., oral mucosa, rectal mucosa, and intestinal mucosa), and may be administered together with other bioactive agents. Administration can be systemic or topical.

[0326] The pharmaceutical compositions of this disclosure can be delivered subcutaneously or intravenously using standard needles and syringes. Furthermore, with respect to subcutaneous delivery, pen-type delivery devices can be readily applied to the delivery of the pharmaceutical compositions of this disclosure. Such pen-type delivery devices may be reusable or disposable. Reusable pen-type delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. When all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there are no replaceable cartridges. Instead, disposable pen-type delivery devices are provided pre-filled with the pharmaceutical composition held in a reservoir within the device. When the pharmaceutical composition in the reservoir is empty, the entire device is discarded.

[0327] Under certain circumstances, pharmaceutical compositions can be delivered using controlled-release systems. In one embodiment, a pump may be used (see Langer, cited above; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymer materials may be used. See Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, the controlled-release system can be positioned near the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, 1984, in Medical Applications of Controlled Release, cited above, vol. 2, pp. 115-138). Other controlled-release systems are described in the review by Langer, 1990, Science 249:1527-1533.

[0328] The preparations for injection may include dosage forms for intravenous, subcutaneous, intradermal, intramuscular, intratumoral, intraperitoneal, intraspinal, intracerebral, and intrathecal injection, intradrip infusion, etc. In one embodiment, the preparations for injection can be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or salt thereof in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline and isotonic solutions containing glucose and other adjuvants, which can be used in combination with appropriate solubilizers such as alcohol (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene alkyl ether (50 mol) adduct of hydrogenated castor oil)). Examples of oily media include sesame oil and soybean oil, which can be used in combination with appropriate solubilizers such as benzyl benzoate and benzyl alcohol. The injections thus prepared are preferably filled into appropriate ampoules.

[0329] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into unit dose dosage forms adapted to the dose of the active ingredient. Examples of such unit dose dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of single-domain antibody described herein may be about 5 to about 500 mg per unit dose dosage form, and in particular, in the form of an injection, the single-domain antibody described herein may contain only about 5 to about 100 mg, and in other dosage forms, it may contain only about 10 to about 250 mg.

[0330] The pharmaceutical composition can be administered to the subject as needed. In some embodiments, an effective dose of the pharmaceutical composition is administered to the subject once 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, 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, for example, every two weeks, every week, twice a week, three times a week, daily, or multiple times a day. An effective amount of the pharmaceutical composition is administered to the subject at least once. In some embodiments, an effective amount of the pharmaceutical composition can be administered multiple times, including a period of at least one month, at least six months, or at least one year. In some embodiments, the pharmaceutical composition is administered to the subject as needed to alleviate one or more symptoms of a medical condition.

[0331] In some embodiments, the pharmaceutical composition of the present disclosure may be administered to a subject at a level lower than necessary to achieve the desired therapeutic effect, and the dosage may be gradually increased until the desired effect is achieved. Alternatively, the pharmaceutical composition of the present disclosure may be administered at a high dose, and then the dose may be gradually decreased until the therapeutic effect is achieved. Generally, a preferred daily dose of the single-domain antibody of the present invention is the amount of antibody that is the minimum effective dose to produce a therapeutic effect.

[0332] The pharmaceutical compositions of this disclosure may optionally contain two or more activators.

[0333] kit The Disclosure further includes a kit comprising any of the various compositions of the Disclosure, including a single-domain antibody of the Disclosure, a fusion protein or conjugate containing the single-domain antibody, a polynucleotide molecule, a vector, or a cell. In some embodiments, a kit comprising any of the compositions described herein may optionally include instructions and / or packaging for the kit.

[0334] In various embodiments, the kit of this disclosure may include lyophilized formulations of one or more compositions disclosed herein, and / or instructions for their reconstitution and / or use, in a suitable container. Examples of suitable containers include vials, syringes, bottles, flasks, and test tubes. Containers can be manufactured from a variety of materials, such as plastic or glass.

[0335] The kit and / or container may include instructions associated with or on the container, which may include, for example, instructions for reconstituting the lyophilized formulation and / or using the kit. In some embodiments, the label may indicate that the lyophilized formulation is reconstituteable to appropriate compositional concentrations. The label may indicate that the formulation can be used or is intended for any route of administration, e.g., parenteral routes (including subcutaneous, intramuscular, or intravenous), enteral routes (including oral or rectal), inhalation, or nasal routes.

[0336] In some embodiments, the kit may include a device (e.g., one or more needles, syringes, eye drop devices, pipettes, etc.) that can enable the administration of the activating agent of the present disclosure, which is a component of the kit.

[0337] The container holding the formulation may be a multi-use vial. In some embodiments, this multi-use vial can enable repeated administration (e.g., 2 to 20 doses) of the reconstituted formulation. The kit may further include a second container containing a suitable diluent (e.g., physiological saline, sterile water for injection, or a 5% dextrose aqueous solution).

[0338] By mixing the diluent with the lyophilized formulation, the final concentration of the composition in the reconstituted formulation can be achieved. The kit may further include additional articles that may be desirable from the user and / or from a commercial standpoint. Such additional articles may include a syringe, needle, diluent, buffer, filter, and / or accompanying documentation, which may include, for example, instructions for use.

[0339] The kits described herein may have a single container containing the formulation of the composition according to this disclosure together with or without other components (e.g., other compounds or pharmaceutical compositions of such compounds), or each component may have a separate container. In some embodiments, if two or more components are present, the kit may include a second vial or other container to enable individual dosing. The kit may also include another container for pharmaceutically acceptable liquids.

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

[0341] The components of the kit can be provided as one or more liquid solutions. The liquid solutions described herein may be aqueous solutions, which may be, for example, sterile aqueous solutions. The components of the kit may, in some cases, be provided as solids, which can be converted into liquids by adding a suitable solvent, which may be provided in a separate container. [Examples]

[0342] The following examples are provided to further illustrate some of the embodiments disclosed herein. These examples are intended to illustrate, rather than limit, the embodiments of the present disclosure.

[0343] Example 1. Expression and purification of VHH Constructs with manipulated C-terminuses (Table 1-1) and framework-modified constructs (Table 1-2) were designed. All designed bacterial expression plasmids encoding VHH were transformed into chemically competent E. coli T7 Shuffle subcolumns. The transformed bacteria were used for overnight protein expression in self-inducible Terrific Broth (TB) medium at 30°C in 96-well plate and / or 1-liter permeable flask formats. The following morning, E. coli cells were harvested by centrifugation and treated with a combination of high-sucrose and low-sucrose buffers to release VHH from the bacterial periplasm by osmotic shock. The periplasm extract was then clarified by centrifugation and purified by Ni-NTA affinity chromatography using standard protocols and buffers. After elution from Ni-NTA resin, VHH was rebuffered in 1x phosphate-buffered saline (PBS) and rapidly frozen in liquid nitrogen for further storage.

[0344] Example 2. Anti-drug antibody (ADA) assay To measure the binding of existing anti-drug antibodies (ADAs) to manipulated VHH, MaxiSorp plates were coated with affinity-purified VHH overnight at 4°C (10 μl VHH solution and 40 μl PBS were added per well). Subsequently, the samples were washed twice with 0.05% (v / v) PBS-Tween20 and blocked on a plate shaker with casein solution (Thermo Fisher, catalog number: 37528) for 1 hour at room temperature (RT). After washing twice with 0.05% (v / v) PBS-Tween20, the samples were incubated on a plate shaker with human intravenous IgG (IVIg) (Creative Biomart, catalog number: THP-0108) diluted 1:3,150 in 0.01% (v / v) casein-PBS for 1 hour at RT. Next, the samples were washed five times with 0.05% (v / v) PBS-Tween20 and incubated on a plate shaker with anti-human IgG (Fc-specific)-HRP (Sigma-Aldrich, catalog number: A0170) diluted 1:60,000 in 0.01% (v / v) casein-PBS for 1 hour under light protection. After washing five times with 0.05% (v / v) PBS-Tween20 and twice with PBS, 50 μL of TMB One Component HRP Microwell Substrate (Tebubio) was added per well. The plates were incubated in the dark for 10 minutes under light protection, and the reaction was stopped by adding H2SO4. Absorbance was measured at 450 nm and at 620 nm as a reference. Figure 3 shows a bar graph of the binding of existing anti-drug antibodies (pre-ADA) (human IgG) to the purified V bodies described herein, and Figure 4 shows a summary of the corresponding normalized data against the control VHH, ODY-349. To generate this data, a total of 452 manipulated V bodies were incubated with human intravenous IgG (IVIg), and the binding of human IgG was detected. The results are also summarized in Figures 5A-5D.

[0345] Example 3. Binding of existing ADA derived from human plasma to the modified V-body. The binding of existing anti-drug antibodies (ADAs) to C-terminally modified V bodies was evaluated in an ADA assay using individual serum samples from 50 healthy human donors. WT VHH (ODY-349) with the C-terminal amino acid sequence VSS was used as a control. To measure the binding of existing ADAs to the modified VHH, MaxiSorp plates were coated with affinity-purified VHH (1 μg of VHH per well in PBS, 0.1% BSA, and 0.05% Tween20). The samples were then washed and blocked on a plate shaker for 2 hours at room temperature (RT). Serum samples from healthy naive donors were added (diluted 1:20 in PBS, 0.1% BSA, and 0.05% Tween20) and incubated with shaking at room temperature for 2 hours. Next, the samples were washed and incubated at RT on a plate shaker with anti-human IgG (Fc-specific)-HRP (Sigma-Aldrich, catalog number: A0170) diluted 1:100,000 in PBS, 0.1% BSA, and 0.05% Tween 20. After washing, (3,3',5,5'-tetramethylbenzidine)(TMB) horseradish peroxidase (HRP) substrate was added, and the plate was incubated at RT in the dark. The reaction was stopped by adding H2SO4, and the absorbance was measured at 450 nm.

[0346] Figure 6 shows a heatmap of optical density (OD) values ​​for existing ADA binding analysis for 24 V-body variants screened in individual serum samples from 50 healthy human donors. The intensity of the color represents the binding of existing ADA to the purified V-bodies.

[0347] Example 4. C-terminal modification of a tetravalent V-body agonist targeting hepatocyte membrane proteins significantly reduced hepatotoxicity. One quadrivalent unmodified V-body agonist (wt-Vtetra) and two C-terminally modified versions of this molecule targeting hepatocyte membrane proteins (Vtetra3-VPAG and Vtetra3-VAGG) were incubated on primary hepatocytes at concentrations of 0.1–1000 pM in the presence and absence of an existing anti-drug antibody (ADA) containing human intravenous IgG (IVIg). A summary of the data generated in this example is shown in Figures 7A–7B. In Figure 7A, the relative viability of primary hepatocytes, measured by the CellTiter-Glo assay (CTG), is plotted on the y-axis against the indicated concentrations (x-axis) of the wild-type V-body agonist (wt-Vtetra3) and modified Vtetra3-VPAG in the presence and absence of human IVIg after 24 hours of incubation. In Figure 7B, the relative viability of primary hepatocytes, measured by the CellTiter-Glo assay (CTG), is plotted on the y-axis against the indicated concentrations (x-axis) of the wild-type V-body agonist (wt-Vtetra3) and modified Vtetra3-VAGG in the presence and absence of IVIg after 24-hour incubation. The horizontal dashed lines in Figures 7A-7B highlight the hepatocyte viability for the highest concentration agonist tested in the absence of IVIg for comparison. Structural models of the V-body agonist and the modification (C-terminal end) are shown at the bottom of the figure panel. The results showed that the unmodified tetravalent V-body agonist caused existing ADA-induced hepatotoxicity, while the C-terminally modified versions of this molecule (VPAG (SEQ ID NO: 94) and VAGG (SEQ ID NO: 5)) significantly reduced hepatotoxicity.

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

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

Claims

1. It is a single-domain antibody, and according to Chothia, the carboxyl terminus starts at position 111, (1) V, (2)VX １ 、 (3)VX １ X ２ 、 (4) VX １ X ２ G, or (5)VX １ X ２ P [X １ It is selected from the amino acids Ala (A), Asp (D), Glu (E), Gly (G), Ile (I), Lys (K), Leu (L), Asn (N), Pro (P), Arg (R), Ser (S), Thr (T), and Val (V), X ２ is selected from the amino acids Ala (A), Asp (D), Glu (E), Gly (G), Ile (I), Lys (K), Leu (L), Asn (N), Pro (P), Gln (Q), Arg (R), Ser (S), Thr (T), and Val (V)] A single-domain antibody modified to contain an amino acid sequence selected from the following.

2. X １ is selected from the amino acids Ala (A), Gly (G), Pro (P), Asp (D), and Leu (L); and / or X ２ The single-domain antibody according to claim 1, wherein is selected from the amino acids Ala(A), Gly(G), Pro(P), Asp(D), Gln(Q), and Leu(L).

3. X １ is selected from the amino acids Ala (A), Gly (G), and Pro (P); and / or X ２ The single-domain antibody according to claim 2, wherein is selected from the amino acids Ala(A), Gly(G), Gln(Q), and Pro(P).

4. The single-domain antibody according to any one of claims 1 to 3, wherein the amino acid sequence is not 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, VGPG (SEQ ID NO: 25), VSSP (SEQ ID NO: 295), and VSSG (SEQ ID NO: 286).

5. According to Chothia, the carboxyl terminus starting from position 111, (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, VVE, VVG, VVI, VVK, VVL, VVN, VVP, VVQ, VVR, VVS, VVT, or VVV; (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 (Sequence ID 39), VGAG (Sequence ID 41), VGDG (Sequence ID 43), VGEG (Sequence ID 45), VGGG (Sequence ID 47), VGIG (Sequence ID 49), VGKG (Sequence ID 51), VGLG (Sequence ID 53), VGNG (Sequence ID 55), VGPG (Sequence ID 57), VGQG (Sequence ID 59), VGRG (Sequence ID 61), VGSG (Sequence ID 63), VGTG (Sequence ID 65), VGVG (Sequence ID 67), VIGG (Sequence ID 69), VIPG (Sequence ID 71), VISG (Sequence ID 73), VITG (Sequence ID 75), VLGG (Sequence ID Number 77), VLPG (Sequence ID 79), VLSG (Sequence ID 283), VLTG (Sequence ID 82), VNAG (Sequence ID 84), VNGG (Sequence ID 86), VNPG (Sequence ID 88), VNSG (Sequence ID 90), VNTG (Sequence ID 92), VPAG (Sequence ID 94), VPDG (Sequence ID 96), VPEG (Sequence ID 98), VPGG (Sequence ID 100), VPIG (Sequence ID 102), VPKG (Sequence ID 104), VPLG (Sequence ID 106), VPNG (Sequence ID 108), VPPG (Sequence ID 110), VPQG (Sequence ID 112), VP RG (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), VVPG (SEQ ID NO: 186), VVSG (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), VET P (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), VL Column number 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), VP RP (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), VVPP (SEQ ID NO: 187), VVSP (SEQ ID NO: 189), or VVTP (SEQ ID NO: 191), The single-domain antibody according to claim 1, comprising an amino acid sequence selected from

6. A single-domain antibody according to claim 5, comprising 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 VGAP (SEQ ID NO: 22) at the carboxy terminus starting at position 111 according to Chothia.

7. A single-domain antibody according to claim 5 or 6, comprising 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), VGGG (SEQ ID NO: 23), or VGAP (SEQ ID NO: 22) at the carboxy terminus starting at position 111 according to Chothia.

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

9. A single-domain antibody according to any one of claims 1 to 8, further comprising one or more amino acid substitutions at positions 11, 13, 87, 88, 89, and / or 108.

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

11. A single-domain antibody according to claim 9, comprising at least one amino acid substitution at position 87.

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

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

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

15. A single-domain antibody according to claim 14, comprising at least one amino acid substitution at position 87.

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

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

18. A single-domain antibody according to any one of claims 1 to 17, wherein binding to its C-terminus by an anti-drug antibody (ADA) is reduced compared to an unmodified single-domain antibody.

19. The single-domain antibody according to claim 18, wherein the unmodified single-domain antibody contains a VSS at its C-terminus.

20. A single-domain antibody according to claim 18 or 19, wherein ADA binding to its C-terminus is reduced by at least about 80% compared to an unmodified single-domain antibody.

21. The single-domain antibody according to claim 20, wherein ADA binding to its C-terminus is reduced by at least about 85% compared to an unmodified single-domain antibody.

22. The single-domain antibody according to claim 21, wherein ADA binding to its C-terminus is reduced by at least about 90% compared to an unmodified single-domain antibody.

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

24. A single-domain antibody according to any one of claims 1 to 23, wherein the domain is VHH or VH domain.

25. A single-domain antibody according to claim 24, which is a VHH of a camelid animal.

26. A single-domain antibody according to claim 24, which is a humanized VHH.

27. The single-domain antibody according to claim 24, which is camelized VH.

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

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

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

31. A recombinant vector comprising the polynucleotide molecule described in claim 30.

32. A host cell comprising the polynucleotide molecule described in claim 30 or the recombinant vector described in claim 31.

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

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

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

36. A method for modifying a single-domain antibody, wherein the method modifies the amino acid sequence at the carboxyl terminus of a single-domain antibody starting at position 111 according to Chothia, (1) V, (2)VX １ 、 (3)VX １ X ２ 、 (4) VX １ X ２ G, or (5)VX １ X ２ P [X １ It is selected from the amino acids Ala (A), Asp (D), Glu (E), Gly (G), Ile (I), Lys (K), Leu (L), Asn (N), Pro (P), Arg (R), Ser (S), Thr (T), and Val (V), X ２ [The amino acids Ala (A), Asp (D), Glu (E), Gly (G), Ile (I), Lys (K), Leu (L), Asn (N), Pro (P), Gln (Q), Arg (R), Ser (S), Thr (T), and Val (V) are selected.] The method comprising mutating to an amino acid sequence selected from the following.

37. X １ is selected from the amino acids Ala (A), Gly (G), Pro (P), Asp (D), and Leu (L); and / or X ２ The method according to claim 36, wherein is selected from the amino acids Ala(A), Gly(G), Pro(P), Asp(D), Gln(Q), and Leu(L).

38. X １ is selected from the amino acids Ala (A), Gly (G), and Pro (P); and / or X ２ The method according to claim 37, wherein is selected from the amino acids Ala(A), Gly(G), Gln(Q), and Pro(P).

39. The method according to any one of claims 36 to 38, wherein the amino acid sequence is not 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), VDD, VDDPG (SEQ ID NO: 25), VSSP (SEQ ID NO: 295), and VSSG (SEQ ID NO: 286).

40. The aforementioned amino acid sequence is, (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, VVE, VVG, VVI, VVK, VVL, VVN, VVP, VVQ, VVR, VVS, VVT, or VVV; (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 (Sequence ID 39), VGAG (Sequence ID 41), VGDG (Sequence ID 43), VGEG (Sequence ID 45), VGGG (Sequence ID 47), VGIG (Sequence ID 49), VGKG (Sequence ID 51), VGLG (Sequence ID 53), VGNG (Sequence ID 55), VGPG (Sequence ID 57), VGQG (Sequence ID 59), VGRG (Sequence ID 61), VGSG (Sequence ID 63), VGTG (Sequence ID 65), VGVG (Sequence ID 67), VIGG (Sequence ID 69), VIPG (Sequence ID 71), VISG (Sequence ID 73), VITG (Sequence ID 75), VLGG (Sequence ID Number 77), VLPG (Sequence ID 79), VLSG (Sequence ID 283), VLTG (Sequence ID 82), VNAG (Sequence ID 84), VNGG (Sequence ID 86), VNPG (Sequence ID 88), VNSG (Sequence ID 90), VNTG (Sequence ID 92), VPAG (Sequence ID 94), VPDG (Sequence ID 96), VPEG (Sequence ID 98), VPGG (Sequence ID 100), VPIG (Sequence ID 102), VPKG (Sequence ID 104), VPLG (Sequence ID 106), VPNG (Sequence ID 108), VPPG (Sequence ID 110), VPQG (Sequence ID 112), VP RG (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), VVPG (SEQ ID NO: 186), VVSG (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), VET P (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), VL Column number 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), VP RP (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), VVPP (SEQ ID NO: 187), VVSP (SEQ ID NO: 189), or VVTP (SEQ ID NO: 191), The method according to claim 36, selected from

41. The method according to 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), 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 method according to claim 40 or 41, wherein the single-domain antibody comprises 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), VGGG (SEQ ID NO: 23), or VGAP (SEQ ID NO: 22) at the carboxy terminus starting at position 111 according to Chothia.

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

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

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

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

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

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

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

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

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

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

53. The method according to any one of claims 36 to 52, wherein the single-domain antibody exhibits reduced binding to its C-terminus by an anti-drug antibody (ADA) compared to an unmodified single-domain antibody.

54. The method according to claim 53, wherein the unmodified single-domain antibody contains VSS at the C-terminus.

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

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

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

58. The method according to any one of claims 55 to 57, wherein ADA binding is measured using enzyme immunoassay (ELISA).

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

60. The method according to claim 59, wherein the single-domain antibody is Camelidae VHH.

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

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

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

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