Antigen-binding proteins against serum albumin and uses thereof

Abstract

The present application provides antigen-binding proteins (e.g., antibodies such as single- domain antibodies) that specifically bind to serum albumin. The application also provides fusion proteins and conjugates comprising the antigen-binding proteins, polynucleotides and recombinant vectors encoding the antigen-binding proteins, as well as host cells and methods for preparing the antigen-binding proteins. The application further provides pharmaceutical compositions comprising the antigen-binding proteins and uses thereof.

Description

Attorney Docket No: 260525.000077 ANTIGEN-BINDING PROTEINS AGAINST SERUM ALBUMIN AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 657,191, filed June 7, 2024, the disclosure of which is herein incorporated by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 03, 2025, is named 260525_000077_SL.xml and is 2,311,747 bytes in size. FIELD OF THE INVENTION

[0003] The present application relates to antigen-binding proteins (e.g., antibodies such as single-domain antibodies) that specifically bind to serum albumin, methods for their preparation, and uses thereof. BACKGROUND

[0004] Numerous chemical compounds and protein therapeutics, such as hormones, cytokines, coagulation factors, growth factors, enzymes, antibody, antibody-fragments have limited therapeutic efficacy due to their poor in vivo stabilities. Therapeutic and diagnostic efficacies of such protein molecules and chemical compounds are hampered by the short half-lives. Protein therapeutics generally benefit from long in vivo serum half-lives to maximize their efficacy and reduce dosing requirements. Smaller proteins are rapidly cleared from the circulation in mammals via renal filtration. Various methods have been developed to increase pharmacokinetic availability of small biologics with short half-lives, including fusion to an antibody fragment crystallizable (Fc) region, PEGylation, glycosylation, fusion to serum albumin or albumin-binding proteins and peptides, and fusion to other small polypeptides to increase size and hydrodynamic radius. Small albumin-binding molecules offer advantage in terms of functionality that allow for fusion to many biologics without compensating target binding affinities. It would be advantageous to find serum albumin binding agents or polypeptides with sufficiently high affinity to human albumin to extend the half-life of a therapeutic protein, with the highest affinity or longest half-life in frequently used 1 315736224v1Attorney Docket No: 260525.000077 testing animals, such as rodents, to increase the success rate and translatability of pre-clinical results. SUMMARY OF THE INVENTION

[0005] As mentioned in the background section above, there is a need in the art to develop molecules that stabilize therapeutic molecules in vivo with their optimal efficacy. This application provides compositions and methods to address this and other related needs.

[0006] In one aspect, provided herein is an antigen-binding protein that specifically binds serum albumin, comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from (a) (Q / L)AYRQ(K / S)GS(N / K / Q / A)(A / S)PLI(V / I) (SEQ ID NO: 41), (b) KAWYLTTTY (SEQ ID NO: 2), (c) AAAYGAGRYRMVKQYDY (SEQ ID NO: 3), (d) AADLWAVCPTGASGRGSDYDW (SEQ ID NO: 4), (e) NAF(T / V)SWPLEDY(D / N)Y (SEQ ID NO: 45), (f) NAGNS(G / W)G(L / W)G(P / T)FDY (SEQ ID NO: 46), (g) APTSSWYL (SEQ ID NO: 7), and (h) HAYR(A / Q)KTSSGKL(H / I)I (SEQ ID NO: 48).

[0007] In some embodiments, the antigen-binding comprises a CDR3 comprising an amino acid sequence selected from LAYRQKGSNSPLIV (SEQ ID NO: 1), LAYRQKGSKSPLIV (SEQ ID NO: 209), LAYRQSGSNSPLIV (SEQ ID NO: 120), LAYRQKGSNSPLII (SEQ ID NO: 91), LAYRQKGSNAPLII (SEQ ID NO: 2378), LAYRQKGSQSPLII (SEQ ID NO: 2379), LAYRQKGSASPLII (SEQ ID NO: 2380), KAWYLTTTY (SEQ ID NO: 2), AAAYGAGRYRMVKQYDY (SEQ ID NO: 3), AADLWAVCPTGASGRGSDYDW (SEQ ID NO: 4), NAFVSWPLEDYNY (SEQ ID NO: 5), NAGNSWGLGTFDY (SEQ ID NO: 6), APTSSWYL (SEQ ID NO: 7), and HAYRQKTSSGKLHI (SEQ ID NO: 8).

[0008] In some embodiments, the antigen-binding protein further comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from (a) GFTF(S / T)(D / F)Y(A / S) (SEQ ID NO: 49), (b) GFTFRNYV (SEQ ID NO: 10), (c) G(G / R)(S / T)F(N / S)(I / T)Y(T / V), (d) G(F / L)TLANYS (SEQ ID NO: 52), (e) G(F / L)T(F / L)(I / S)SY(A / D) (SEQ ID NO: 53), (f) GFTFS(V / Y)Y(A / R) (SEQ ID NO: 54), (g) GFTFSSY(A / P) (SEQ ID NO: 55), and (h) (E / G)FTFS(G / R / S)Y(A / S) (SEQ ID NO: 56).

[0009] In some embodiments, the antigen-binding protein comprises a CDR1 comprising an amino acid sequence selected from GFTFTDYS (SEQ ID NO: 9), GFTFSFYS (SEQ ID NO: 488), GFTFRNYV (SEQ ID NO: 10), GGSFNIYT (SEQ ID NO: 11), GFTLANYS (SEQ ID NO: 12), 2 315736224v1Attorney Docket No: 260525.000077 GFTFISYD (SEQ ID NO: 13), GFTFSVYA (SEQ ID NO: 14), GFTFSSYA (SEQ ID NO: 15), and EFTFSSYA (SEQ ID NO: 16).

[0010] In some embodiments, the antigen-binding protein further comprises a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from (a) TRTTGG(N / S)I (SEQ ID NO: 57), (b) (I / V)TSV(D / G)DST (SEQ ID NO: 58), (c) (I / V)(S / T) (R / W)SG(N / R / S)(G / R / S)(L / T), (d) ISRSGGST (SEQ ID NO: 20), (e) I(S / T) S(T / A) GR(N / T)T (SEQ ID NO: 61), (f) ITST(G / S)(G / S)(R / S)(L / T) (SEQ ID NO: 62), (g) ITSGG(E / G)ST (SEQ ID NO: 63), and (h) ITTT(D / G)(G / N / S)(S / T)(T / -) (SEQ ID NO: 64).

[0011] In some embodiments, the antigen-binding protein comprises a CDR2 comprising an amino acid sequence selected from TRTTGGSI (SEQ ID NO: 17), TRTTGGNI (SEQ ID NO: 856), ITSVDDST (SEQ ID NO: 18), ITWSGSRL (SEQ ID NO: 19), ISRSGGST (SEQ ID NO: 20), ITSTGRNT (SEQ ID NO: 21), ITSTSSRL (SEQ ID NO: 22), ITSGGGST (SEQ ID NO: 23), and ITTTDNT (SEQ ID NO: 24).

[0012] In some embodiments, an antigen-binding protein of the present disclosure comprises: i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR2 comprising an amino acid sequence of SEQ ID NO: 57, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 41; ii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence of SEQ ID NO: 58, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2; iii) a CDR1 comprising the amino acid sequence of G(G / R)(S / T)F(N / S)(I / T)Y(T / V), a CDR2 comprising an amino acid sequence of (R / W)SG(N / R / S)(G / R / S)(L / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3; iv) a CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR2 comprising an amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4; v) a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR2 comprising an amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 45; vi) a CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a CDR2 comprising an amino acid sequence of SEQ ID NO: 62, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 46; vii) a CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR2 comprising an amino acid sequence of SEQ ID NO: 63, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7; and / or viii) a CDR1 comprising the amino acid sequence 3 315736224v1Attorney Docket No: 260525.000077 of SEQ ID NO: 56, a CDR2 comprising an amino acid sequence of SEQ ID NO: 64, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 48.

[0013] In one aspect, provided herein is an antigen-binding protein that specifically binds serum albumin, comprising a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 9- 16 and 433-800, a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 17-24 and 801-1168, and / or a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 1- 8, 65-432, and 2378-2380.

[0014] In some embodiments, an antigen-binding protein of the present disclosure comprises (a) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNSPLIV (SEQ ID NO: 1); (b) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSKSPLIV (SEQ ID NO: 209); (c) a CDR1 comprising an amino acid sequence of GFTFSFYS (SEQ ID NO: 488), a CDR2 comprising an amino acid sequence of TRTTGGNI (SEQ ID NO: 856), and a CDR3 comprising an amino acid sequence of LAYRQSGSNSPLIV (SEQ ID NO: 120); (d) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNSPLII (SEQ ID NO: 91); (e) a CDR1 comprising an amino acid sequence of GFTFRNYV (SEQ ID NO: 10), a CDR2 comprising an amino acid sequence of ITSVDDST (SEQ ID NO: 18), and a CDR3 comprising an amino acid sequence of KAWYLTTTY (SEQ ID NO: 2); (f) a CDR1 comprising an amino acid sequence of GGSFNIYT (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ITWSGSRL (SEQ ID NO: 19), and a CDR3 comprising an amino acid sequence of AAAYGAGRYRMVKQYDY (SEQ ID NO: 3); (g) a CDR1 comprising an amino acid sequence of GFTLANYS (SEQ ID NO: 12), a CDR2 comprising an amino acid sequence of ISRSGGST (SEQ ID NO: 20), and a CDR3 comprising an amino acid sequence of AADLWAVCPTGASGRGSDYDW (SEQ ID NO: 4); (h) a CDR1 comprising an amino acid sequence of GFTFISYD (SEQ ID NO: 13), a CDR2 comprising an amino acid sequence of ITSTGRNT (SEQ ID NO: 21), and a CDR3 comprising an amino acid sequence of NAFVSWPLEDYNY (SEQ ID NO: 5); (i) a CDR1 comprising an amino acid sequence of GFTFSVYA (SEQ ID NO: 14), a CDR2 comprising an amino acid sequence of 4 315736224v1Attorney Docket No: 260525.000077 ITSTSSRL (SEQ ID NO: 22), and a CDR3 comprising an amino acid sequence of NAGNSWGLGTFDY (SEQ ID NO: 6); (j) a CDR1 comprising an amino acid sequence of GFTFSSYA (SEQ ID NO: 15), a CDR2 comprising an amino acid sequence of ITSGGGST (SEQ ID NO: 23), and a CDR3 comprising an amino acid sequence of APTSSWYL (SEQ ID NO: 7); (k) a CDR1 comprising an amino acid sequence of EFTFSSYA (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of ITTTDNT (SEQ ID NO: 24), and a CDR3 comprising an amino acid sequence of HAYRQKTSSGKLHI (SEQ ID NO: 8); (l) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNAPLII (SEQ ID NO: 2378); (m) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSQSPLII (SEQ ID NO: 2379); and / or (n) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSASPLII (SEQ ID NO: 2380).

[0015] In some embodiments, the antigen-binding protein comprises i) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNSPLII (SEQ ID NO: 91); ii) a CDR1 comprising an amino acid sequence of GFTFRNYV (SEQ ID NO: 10), a CDR2 comprising an amino acid sequence of ITSVDDST (SEQ ID NO: 18), and a CDR3 comprising an amino acid sequence of KAWYLTTTY (SEQ ID NO: 2); (iii) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNAPLII (SEQ ID NO: 2378); (iv) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSQSPLII (SEQ ID NO: 2379); and / or (v) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSASPLII (SEQ ID NO: 2380).

[0016] In some embodiments, the antigen-binding protein is a single-domain antibody. 5 315736224v1Attorney Docket No: 260525.000077

[0017] In some embodiments, the antigen-binding protein is a VHH, a VNAR, or a VH domain.

[0018] In some embodiments, the antigen-binding protein is a VHH.

[0019] In some embodiments, the VHH is a camelid VHH. In some embodiments, the VHH comprises an amino acid sequence selected from SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, and 1169-1536, or an amino acid sequence having at least 75% identity thereto. In some embodiments, the VHH comprises an amino acid sequence selected from SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 1195, 1224, 1313, and 1420, or an amino acid sequence having at least 75% identity thereto. In one embodiment, the VHH comprises an amino acid sequence of SEQ ID NO: 26 or 1195, or an amino acid sequence having at least 75% identity thereto.

[0020] In some embodiments, the VHH is a humanized VHH. In some embodiments, the humanized VHH comprises an amino acid sequence selected from SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 1537-1904, and 2381-2384, or an amino acid sequence having at least 75% identity thereto. In some embodiments, the humanized VHH comprises an amino acid sequence selected from SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 1563, 1592, 1681, 1788, 2381, 2382, 2383, and 2384, or an amino acid sequence having at least 75% identity thereto. In some embodiments, the humanized VHH comprises an amino acid sequence of SEQ ID NO: 34, 1563, 2381, 2382, 2383 and / or 2384, or an amino acid sequence having at least 75% identity thereto.

[0021] In one aspect, provided herein is an antigen-binding protein that competes for binding to serum albumin with the antigen-binding protein described above.

[0022] In one aspect, provided herein is an antigen-binding protein that binds to the same epitope as the antigen-binding protein described above.

[0023] In some embodiments, the antigen-binding protein binds to domain I and / or domain II of human serum albumin. In some embodiments, the antigen-binding protein binds to domain II of human serum albumin. In some embodiments, the antigen-binding protein binds to domain III of human serum albumin.

[0024] In one aspect, provided herein is an antigen-binding protein that specifically binds human serum albumin (HSA), wherein the antigen-binding protein binds human serum albumin at an epitope comprising or consisting essentially of one or more amino acid residues selected from a) Glu227, Phe228, Ala229, Glu230, Lys233, Lys240, Asp308, Glu321, Val325, and / or Met329; and / or b) Glu542, Asp549, Asp550, Ala553, Phe554, Glu556, Lys557, Lys560, Glu570, Glu571, and / or Leu575. In some embodiments, the epitope further comprises one or more amino acid 6 315736224v1Attorney Docket No: 260525.000077 residues selected from: a) Ala226, Ser232, Thr236, Tyr263, Ser270, Asp301, Pro303, Phe309, Asn318, Ala322, Lys323, Asp324, Tyr332, Glu333, and / or Arg336; and / or b) Lys545, Ala546, Ala552, Ala561, Asp563, Thr566, Cys567, Lys574, and / or Ala578.

[0025] In one embodiment, the antigen-binding protein binds human serum albumin at an epitope comprising or consisting essentially of one or more amino acid residues selected from Glu227, Phe228, Ala229, Glu230, Lys233, Lys240, Asp308, Glu321, Val325, and / or Met329. In one embodiment, the epitope further comprises one or more amino acid residues selected from: Ala226, Ser232, Thr236, Tyr263, Ser270, Asp301, Pro303, Phe309, Asn318, Ala322, Lys323, Asp324, Tyr332, Glu333, and / or Arg336. In one embodiment, the antigen-binding protein binds human serum albumin at an epitope comprising or consisting essentially of one or more amino acid residues selected from Glu227, Phe228, Ala229, Glu230, Lys233, Lys240, Asp308, Glu321, Val325, Met329, Ala226, Ser232, Thr236, Tyr263, Ser270, Asp301, Pro303, Phe309, Asn318, Ala322, Lys323, Asp324, Tyr332, Glu333, and / or Arg336.

[0026] In one embodiment, the antigen-binding protein binds human serum albumin at an epitope comprising or consisting essentially of one or more amino acid residues selected from Glu542, Asp549, Asp550, Ala553, Phe554, Glu556, Lys557, Lys560, Glu570, Glu571, and / or Leu575. In one embodiment, the epitope further comprises one or more amino acid residues selected from: Lys545, Ala546, Ala552, Ala561, Asp563, Thr566, Cys567, Lys574, and / or Ala578. In one embodiment, the antigen-binding protein binds human serum albumin at an epitope comprising or consisting essentially of one or more amino acid residues selected from Glu542, Asp549, Asp550, Ala553, Phe554, Glu556, Lys557, Lys560, Glu570, Glu571, Leu575, Lys545, Ala546, Ala552, Ala561, Asp563, Thr566, Cys567, Lys574, and / or Ala578.

[0027] In some embodiments, the epitope numbering described above is with respect to amino acids 25-609 of SEQ ID NO: 42.

[0028] In one aspect, provided herein is an antigen-binding protein that comprises a paratope that binds human serum albumin (HSA), wherein the paratope comprises or consists essentially of one or more amino acid residues selected from: a) TyrH37, ArgH45, ArgH52, ThrH52A, TyrH59, AspH61, TyrH95, ArgH96, GlnH97, LeuH100D, and / or TrpH103; and / or b) ValH2, GlyH26, PheH27, ThrH28, ValH31, TyrH32, SerH52A, SerH55, AsnH96, SerH97, TrpH98, GlyH100A, ThrH100B, AspH100D, and / or TyrH102, according to Chothia numbering. In some embodiments, the paratope further comprises one or more amino acid residues selected from: a) SerH33, GluH44, 7 315736224v1Attorney Docket No: 260525.000077 LeuH47, AlaH50, ThrH53, IleH57, ValH58, LysH64, LeuH93, GlyH99, SerH100, and / or IleH101; and / or b) SerH30, ThrH53, LeuH100, and / or PheH100C, according to Chothia numbering.

[0029] In one embodiment, the antigen-binding protein comprises a paratope that binds human serum albumin (HSA), wherein the paratope comprises or consists essentially of one or more amino acid residues selected from: TyrH37, ArgH45, ArgH52, ThrH52A, TyrH59, AspH61, TyrH95, ArgH96, GlnH97, LeuH100D, and / or TrpH103, according to Chothia numbering. In some embodiments, the paratope further comprises one or more amino acid residues selected from: SerH33, GluH44, LeuH47, AlaH50, ThrH53, IleH57, ValH58, LysH64, LeuH93, GlyH99, SerH100, and / or IleH101, according to Chothia numbering. In one embodiment, the paratope comprises or consists essentially of one or more amino acid residues selected from: TyrH37, ArgH45, ArgH52, ThrH52A, TyrH59, AspH61, TyrH95, ArgH96, GlnH97, LeuH100D, TrpH103, SerH33, GluH44, LeuH47, AlaH50, ThrH53, IleH57, ValH58, LysH64, LeuH93, GlyH99, SerH100, and / or IleH101, according to Chothia numbering.

[0030] In one embodiment, the antigen-binding protein comprises a paratope that binds human serum albumin (HSA), wherein the paratope comprises or consists essentially of one or more amino acid residues selected from: ValH2, GlyH26, PheH27, ThrH28, ValH31, TyrH32, SerH52A, SerH55, AsnH96, SerH97, TrpH98, GlyH100A, ThrH100B, AspH100D, and / or TyrH102, according to Chothia numbering. In some embodiments, the paratope further comprises one or more amino acid residues selected from: SerH30, ThrH53, LeuH100, and / or PheH100C, according to Chothia numbering. In one embodiment, the paratope comprises or consists essentially of one or more amino acid residues selected from: ValH2, GlyH26, PheH27, ThrH28, ValH31, TyrH32, SerH52A, SerH55, AsnH96, SerH97, TrpH98, GlyH100A, ThrH100B, AspH100D, SerH30, ThrH53, LeuH100, and / or PheH100C, according to Chothia numbering.

[0031] In one aspect, provided herein is an antigen-binding protein that comprises a paratope that binds human serum albumin (HSA), wherein the paratope comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence a). (Q / L)AYRQ(K / S)GS(N / K / Q / A)(A / S)PLI(V / I) (SEQ ID NO: 41), or b). NAGNS(G / W)G(L / W) G(P / T)FD Y (SEQ ID NO: 46).

[0032] In some embodiments, the CDR3 comprises an amino acid sequence selected from a) LAYRQKGSNSPLIV (SEQ ID NO: 1), LAYRQKGSKSPLIV (SEQ ID NO: 209), 8 315736224v1Attorney Docket No: 260525.000077 LAYRQSGSNSPLIV (SEQ ID NO: 120), LAYRQKGSNSPLII (SEQ ID NO: 91), LAYRQKGSNAPLII (SEQ ID NO: 2378), LAYRQKGSQSPLII (SEQ ID NO: 2379), or LAYRQKGSASPLII (SEQ ID NO: 2380), or b) NAGNSWGLGTFDY(SEQ ID NO: 6).

[0033] In some embodiments, the paratope further comprises one or more amino acid residues selected from: a). TyrH37, ArgH45, ArgH52, ThrH52A, TyrH59, AspH61, and / or TrpH103; or b). ValH2, GlyH26, PheH27, ThrH28, ValH31, TyrH32, SerH52A, and / or SerH55, according to Chothia numbering. In some embodiments, the paratope further comprises one or more amino acid residues selected from: a). SerH33, GluH44, LeuH47, AlaH50, ThrH53, IleH57, ValH58, and / or LysH64; or b). SerH30 and / or ThrH53, according to Chothia numbering.

[0034] In some embodiments, the antigen-binding protein comprises a paratope that binds human serum albumin (HSA), wherein the paratope comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence (Q / L)AYRQ(K / S)GS(N / K / Q / A)(A / S)PLI(V / I) (SEQ ID NO: 41). In some embodiments, the antigen-binding protein comprises a paratope that binds human serum albumin (HSA), wherein the paratope comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence (Q / L)AYRQ(K / S)GS(N / K)SPLI(V / I) (SEQ ID NO: 2385). In some embodiments, the CDR3 comprises an amino acid sequence selected from a) LAYRQKGSNSPLIV (SEQ ID NO: 1), LAYRQKGSKSPLIV (SEQ ID NO: 209), LAYRQSGSNSPLIV (SEQ ID NO: 120), LAYRQKGSNSPLII (SEQ ID NO: 91), LAYRQKGSNAPLII (SEQ ID NO: 2378), LAYRQKGSQSPLII (SEQ ID NO: 2379), or LAYRQKGSASPLII (SEQ ID NO: 2380). In some embodiments, the paratope further comprises one or more amino acid residues selected from: TyrH37, ArgH45, ArgH52, ThrH52A, TyrH59, AspH61, and / or TrpH103. In some embodiments, the paratope further comprises one or more amino acid residues selected from: SerH33, GluH44, LeuH47, AlaH50, ThrH53, IleH57, ValH58, and / or LysH64. In some embodiments, the paratope further comprises one or more amino acid residues selected from: TyrH37, ArgH45, ArgH52, ThrH52A, TyrH59, AspH61, TrpH103, SerH33, GluH44, LeuH47, AlaH50, ThrH53, IleH57, ValH58, and / or LysH64.

[0035] In one aspect, provided herein is an antigen-binding protein that comprises a paratope that binds human serum albumin (HSA), wherein the paratope comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence NAGNS(G / W)G(L / W)G(P / T)FDY (SEQ ID NO: 46). In some embodiments, the CDR3 comprises 9 315736224v1Attorney Docket No: 260525.000077 an amino acid sequence NAGNSWGLGTFDY (SEQ ID NO: 6). In some embodiments, the paratope further comprises one or more amino acid residues selected from: ValH2, GlyH26, PheH27, ThrH28, ValH31, TyrH32, SerH52A, and / or SerH55. In some embodiments, the paratope further comprises one or more amino acid residues selected from: SerH30 and / or ThrH53. In some embodiments, the paratope further comprises one or more amino acid residues selected from: ValH2, GlyH26, PheH27, ThrH28, ValH31, TyrH32, SerH52A, SerH55, SerH30, and / or ThrH53.

[0036] In some embodiments, the antigen-binding protein is a single-domain antibody.

[0037] In some embodiments, the single-domain antibody is a VHH, a VNAR, or a VH domain.

[0038] In some embodiments, the single-domain antibody is a VHH.

[0039] In some embodiments, the VHH is a camelid VHH.

[0040] In some embodiments, the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 25, 1313, 1224, 1195, and 30, or an amino acid sequence having at least 75% identity thereto.

[0041] In some embodiments, the VHH is a humanized VHH.

[0042] In some embodiments, the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 33, 1563, 1592, 168138, 2381, 2382, 2383, and 2384, or an amino acid sequence having at least 75% identity thereto.

[0043] In some embodiments, the antigen-binding protein binds to HSA at neutral pH.

[0044] In some embodiments, the antigen-binding protein binds to HSA at endosomal pH.

[0045] In some embodiments, the antigen-binding protein binds to HSA at both neutral and endosomal pH.

[0046] In some embodiments, the antigen-binding protein binds to HSA with a KDof less than about 3×10−7M at neutral pH.

[0047] In some embodiments, the antigen-binding protein binds to HSA with a KD of about 1×10−9to 5×10−8M at neutral pH.

[0048] In some embodiments, the antigen-binding protein binds to HSA with a KD of about 5.9×10−9M at neutral pH.

[0049] In some embodiments, the antigen-binding protein binds to HSA with a KDof about 3×10−7M at endosomal pH. In some embodiments, the antigen-binding protein binds to HSA with a KD of about 1×10−9x 7×10−8M at endosomal pH. 10 315736224v1Attorney Docket No: 260525.000077

[0050] In some embodiments, the antigen-binding protein binds to cyno serum albumin at neutral pH and / or endosomal pH.

[0051] In some embodiments, the antigen-binding protein binds to mouse serum albumin at neutral pH and / or endosomal pH.

[0052] In some embodiments, the antigen-binding protein binds to rat serum albumin at neutral pH and / or endosomal pH.

[0053] In some embodiments, the endosomal pH is in the range from about pH 5.3 to about pH 6.3. In some embodiments, the antigen-binding protein binds to HSA at pH 5.8.

[0054] In some embodiments, the neutral pH is in the range from about 7.2 to about 7.8. In some embodiments, the antigen-binding protein binds to HSA at pH 7.4.

[0055] In some embodiments, the antigen-binding protein binds to serum albumin without disrupting the interaction of serum albumin with neonatal Fc receptor / beta-2 microglobulin (FcRn / β2M).

[0056] In some embodiments, the antigen-binding protein binds to serum albumin at the FcRn / β2M binding site on serum albumin.

[0057] In some embodiments, the antigen-binding protein comprises one or more modifications that reduce binding of the antigen-binding protein by pre-existing antibodies found in human blood or serum.

[0058] In some embodiments, the single-domain antibody comprises one or more modifications at the amino-terminus and / or the carboxy-terminus. In some embodiments, the single-domain antibody comprises the amino acid sequence VPAG (SEQ ID NO: 4698) or VAGG (SEQ ID NO: 4697) at the carboxy-terminus starting from position 111 according to Chothia. In some embodiments, the single-domain antibody comprises a substitution of amino acid residue Glu with Asp (E1D) at the first position of the amino-terminus.

[0059] In some embodiments, the single-domain antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2381, 2382, 2383, and 2384, or an amino acid sequence having at least 75% identity thereto.

[0060] In another aspect, provided herein is an antigen-binding protein that specifically binds serum albumin, comprising a means for binding an epitope within human serum albumin bound by an antibody selected from H-001, H-002, H-002Hu1.DVQ.VPAG, H- 11 315736224v1Attorney Docket No: 260525.000077 002Hu1.DVQ.VPAG.NA, H-002Hu1.DVQ.VPAG.QS, H-002Hu1.DVQ.VPAG.AS, H-003, H- 004, H-005, H-006, H-007, H-008, H-009, H-010, H-011, and H-012.

[0061] In some embodiments, provided herein is a fusion protein comprising one or more of the antigen-binding proteins described above.

[0062] In some embodiments, the fusion protein comprises one or more of the described antigen- binding proteins operably linked to a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, or combinations thereof.

[0063] In some embodiments, the one or more antigen-binding proteins in the fusion protein bind to the same epitope on serum albumin.

[0064] In some embodiments, the one or more antigen-binding proteins in the fusion protein bind to different epitopes on serum albumin.

[0065] In some embodiments, the one or more antigen-binding proteins in the fusion protein are one or more single-domain antibodies.

[0066] In some embodiments, the one or more antigen-binding proteins in the fusion protein are one or more VHHs.

[0067] In some embodiments, the fusion protein further comprises an immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is the Fc region of a human immunoglobulin. In some embodiments, the immunoglobulin Fc region is a Fc variant that has reduced or abolished binding to FcRn. In some embodiments, the immunoglobulin Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof. In some embodiments, the immunoglobulin Fc region is the Fc region of human IgG1, or a variant thereof. In some embodiments, the Fc region of human IgG1 comprises one or more mutations selected from Ile253Ala (I253A), His310Ala (H310A), His435Ala (H435A), and / or His435Gln (H435Q), according to EU numbering.

[0068] In some embodiments, the Fc region of human IgG1 comprises one or more mutations selected from 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) 12 315736224v1Attorney Docket No: 260525.000077 according to EU numbering. In some embodiments, the Fc region of human IgG1 comprises a set of mutations selected from 1). L234A and L235A; 2). L234A, L235A, and P329A; 3). D265A, N297A and P329A; 4). L234A, L235A, and G237A; 5). L234G, L235S, and G236R; 6). L234S, L235T, and G236R; 7). L234S, L235V, and G236R; 8). L234T, L235Q, and G236R; 9). L234T, L235T, and G236R; 10). L234A, L235A, and P329G; and 11). M252Y, S254T, and T256E. In some embodiments, the immunoglobulin Fc region is an Fc region of human IgG4, or a variant thereof. In some embodiments, the Fc region of human IgG4 comprises one or more mutations selected from Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and / or Pro329Gly (P329G) according to EU numbering. In some embodiments, the Fc region of human IgG4 comprises a set of mutations selected from 1). S228P and L235E; 2). S228P and L235A; 3). S228P, F234A, and L235E; 4). S228P, F234A, and L235A; and 5). P329G, S228P, and L235E.

[0069] In one aspect, provided herein is a conjugate comprising the antigen-binding protein described above or the fusion protein described above, wherein the antigen-binding protein or the fusion protein is conjugated to a second moiety. In some embodiments, the second moiety is selected from a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a detectable label, a drug, a toxin, a radionuclide, an immunomodulatory agent, a cytokine, a cytotoxic agent, a chemotherapeutic agent, a diagnostic agent, or a combination thereof.

[0070] In one aspect, provided herein is a polynucleotide molecule encoding the antigen-binding protein described above or the fusion protein described above. In some embodiments, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 1905- 2272, or a nucleotide sequence having at least 70% identity thereto. In some embodiments, the polynucleotide molecule comprises the nucleotide sequence of any one of SEQ ID NOs: 1905, 1931, 1960, 2049, 2110, 2152, 2156, 2157, 2185, 2221, 2229, 2230, or a nucleotide sequence having at least 70% identity thereto.

[0071] In one aspect, provided herein is a recombinant vector comprising the polynucleotide molecule described above.

[0072] In one aspect, provided herein is a host cell comprising the polynucleotide molecule described above, or the recombinant vector described above.

[0073] In one aspect, provided herein is a kit comprising the antigen-binding protein described above, the fusion protein described above, the conjugate described above, the polynucleotide 13 315736224v1Attorney Docket No: 260525.000077 molecule described above, or the recombinant vector described above, and optionally, instructions and / or packaging for the same.

[0074] In one aspect, provided herein is a pharmaceutical composition comprising the antigen- binding protein described above, the fusion protein described above, the conjugate described above, the polynucleotide molecule described above, or the recombinant vector described above and a pharmaceutically acceptable carrier and / or excipient.

[0075] In one aspect, provided herein is a method for preparing an antigen-binding protein or a fusion protein that specifically binds to serum albumin, comprising the steps of: (a) culturing the host cell in a culture medium under conditions suitable for the expression of the antigen-binding protein or the fusion protein, and (b) isolating the antigen-binding protein or the fusion protein from the host cell and / or culture medium.

[0076] In one aspect, provided herein is a method for increasing the serum half-life of a biological molecule comprising attaching to the biological molecule an antigen-binding protein, the fusion protein, or the conjugate described above.

[0077] In one aspect, provided herein is a method of removing a target molecule from serum of a subject, comprising administering the fusion protein or the conjugate to the subject, wherein the antigen-binding protein is fused or conjugated to a biological molecule that binds to the target molecule.

[0078] In some embodiments, the biological molecule is a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a small molecule, or combination thereof. In some embodiments, the biological molecule binds to a target molecule in a subject.

[0079] In one aspect, provided herein is a method of delivering a therapeutic or imaging agent to a tumor in a subject in need thereof, comprising administering the antigen-binding protein or the fusion protein described above to the subject, wherein the antigen-binding protein or the fusion protein is fused or conjugated to the therapeutic or imaging agent.

[0080] In some embodiments, the therapeutic agent is a drug, a chemotherapeutic agent, a growth inhibitory agent, a toxin, or a radioactive isotope.

[0081] In one aspect, provided herein is a solid or semi-solid support for isolating albumin, a derivative, or a fragment thereof, comprising attached thereon the antigen-binding protein, the fusion protein, or the conjugate described above. 14 315736224v1Attorney Docket No: 260525.000077

[0082] In one aspect, provided herein is a method of isolating albumin, or a derivative or fragment thereof, comprising contacting a sample comprising albumin with the solid or semi-solid support.

[0083] In one aspect, provided herein is a method of isolating the antigen-binding protein, the fusion protein, or the conjugate described above from a sample comprising contacting the sample to a solid or semi-solid support, wherein the solid or semi-solid support comprises attached thereon albumin, or a derivative or fragment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 depicts immune library construction and panning strategy for discovery of serum albumin binders. Sample selection for next generation sequencing throughout the phage display process is shown. The libraries of the two alpacas, 12 human serum albumin (HSA) samples of the first panning round, and 40 HSA samples of the second panning round were sequenced with 20 million, 5 million, and 5 million reads, respectively. Comparison of V-body enrichment from the initial library to the first and second round of panning enabled the identification of potential V-body candidates.

[0085] Figure 2 depicts a schematic diagram of next generation sequencing (NGS) workflow. Following phage display, the VHH region of the phage elutions was PCR amplified, unique and sample-specific barcodes were fused, and NGS was performed with the Illumina NovaSeq platform from Genewiz. The raw data were demultiplexed, and then processed by the Pipebio NGS analysis pipeline. Forward and reverse sequence pairs were merged via overlapping regions and the VHH, including CDRs were annotated. Based on CDR3 identity, V-body sequences were clustered, allowing for a detailed analysis of V-body enrichment during phage display, sequence diversity, CDR3 length distribution and cluster abundance. Based on these analyses, up to ~500 candidates were selected for DNA synthesis by Twist and further characterization.

[0086] Figures 3A-3K depict high throughput kinetic analysis. Binding affinities of indicated V-bodies to human serum albumin were analyzed. Fitted binding curves and calculated dissociation constants (KD) are included. The top, middle and bottom panels show binding affinities of single V-bodies to human serum albumin, cyno serum albumin, and mouse serum albumin, respectively. Each tile represents the global analysis of serum albumin binding to a single V-body coupled to a discrete spot, with KD values reported. The grey shaded lines (representing 15 315736224v1Attorney Docket No: 260525.000077 ascending albumin concentration) are fitted globally (grey line) indicating that the binding kinetics are well-described by a simple Langmuir mode. Spots with non-ideal behaviors are displayed in grey (e.g., insufficient on-rate / off-rate information, inactive or barely binding). Binding affinities were measured under physiological (50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% (v / v) Tween20, 25°C) and slightly acidic conditions (50 mM MES pH 5.8, 150 mM NaCl, 0.05% (v / v) Tween20) using 8 different antigen concentrations ((Figures 3A-3J) 2-fold serial dilution, start at 200 nM; (Figure 3K) 3-fold serial dilution, start at 500 nM).

[0087] Figures 4A-4B depict high throughput kinetic analysis. Binding affinities of indicated V-bodies to human serum albumin were analyzed. Each tile represents the global analysis of serum albumin binding to a single V-body coupled to a discrete spot, with KDvalues reported. The grey shaded lines (representing ascending albumin concentration) are fitted globally (grey line) indicating that the binding kinetics are well-described by a simple Langmuir mode. Binding affinities were measured under physiological (50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% (v / v) Tween20, 25°C) (Figure 4A) and slightly acidic conditions (50 mM MES pH 5.8, 150 mM NaCl, 0.05% (v / v) Tween20) (Figure 4B) using 12 different antigen concentrations (2-fold serial dilution, start at 500 nM).

[0088] Figure 5 depicts results of ligand competition as determined by surface plasmon resonance (SPR). Each tile represents the sensorgram overlay plot for a single V-body captured onto a discrete spot. The sensorgrams display FcRn / β2M competition: association of human serum albumin to the V-body followed either by additional binding by FcRn / β2M (extracellular domain) indicating an unoccupied epitope (non-overlapping epitopes) or no FcRn / β2M binding indicating epitope blocking (overlapping epitopes) and a buffer control - association and dissociation of human serum albumin in the absence of FcRn / β2M. HSA was injected (500 nM) under acidic conditions (50 mM MES pH 5.8, 150 mM NaCl, 0.05% (v / v) Tween20, 25°C) followed by human FcRn / β2M (extracellular domain; 500 nM).

[0089] Figure 6 depicts a three-dimensional model of human serum albumin (HSA) protein. HSA domains (I / II, II, and III) and FcRn / β2M interaction site are indicated.

[0090] Figures 7A-7D depict domain mapping of anti-serum albumin VHHs. Binding of VHHs to distinct domains of human serum albumin was determined. Interactions were measured under physiological condition (50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% (v / v) Tween20, 25°C) 16 315736224v1Attorney Docket No: 260525.000077 using 8 different antigen concentrations ((Figures 7A-7B) 2-fold serial dilution, start at 1µM; (Figures 7C-7D) 3-fold serial dilution, start at 600nM).

[0091] Figure 8A-8E depict epitope mapping of anti-human serum albumin VHHs. Figure 8A depicts structural model of VHHs bound to HSA-FcRn complex. HSA, heavy chain of FcRn, light chain of FcRn, VHH1 (H-001) and VHH2 (H-010Hu1) with CDRs are indicated. Model was built based on density from a cryo-EM map. Figure 8B depicts paratope of VHH1. Residues within 3.5 Angstroms of HSA are labeled and their side chains are shown as sticks. VHH backbone is colored maroon and CDRs are highlighted in red, blue, and yellow. Figure 8C depicts epitope of VHH1. Residues within 3.5 Angstroms of VHH1 are labeled and their side chains are shown as sticks. Figure 8D depicts paratope of VHH2. Residues within 3.5 Angstroms of HSA are labeled and their side chains are shown as sticks. VHH backbone is colored pink and CDRs are highlighted in red, blue, and yellow. Figure 8E depicts epitope of VHH2. Residues within 3.5 Angstroms of VHH2 are labeled and their side chains are shown as sticks. DETAILED DESCRIPTION Definitions

[0092] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.

[0093] As used herein, the term “about” when used in reference to a particular recited numerical value, means that the value may vary from the recited value by no more than 5%. For example, as used herein, the expression “about 100” includes 95 and 105 and all values in between (e.g., 96, 97, 98, 99, etc.).

[0094] The term “antigen” encompasses any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleotide, portions thereof, or combinations thereof) that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody 17 315736224v1Attorney Docket No: 260525.000077 molecule or T-cell receptor. In various embodiments of the present disclosure, the antigen described herein is serum albumin, including human, cynomolgus, and / or mouse serum albumin.

[0095] The term “epitope” can refer to an antigenic determinant on the surface of an antigen to which an antibody molecule binds. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is formed by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is formed by adjacent amino acid residues in a polypeptide chain. In some cases, an epitope may include non-peptidic moieties on the antigen, such as saccharides, phosphoryl groups, or sulfonyl groups.

[0096] The term “antigen-binding protein” refers in its broadest sense to a protein that specifically binds an antigen (e.g., serum albumin). In certain embodiments, an antigen-binding protein is an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody, a camelid antibody, a chimeric antibody, a recombinant antibody, a heavy chain antibody, a single-domain antibody (e.g., VHH), a single chain antibody (e.g., single chain fragment variable (scFv)), a diabody, a triabody, a tetrabody, a Fab fragment, a F(ab’) 2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody, and fragments thereof. The term “antigen-binding protein” also encompasses, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen-binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. In addition, peptide antibody mimetics can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components (e.g., fibronectin type III domain (FN3)) as a scaffold.

[0097] The term “antibody” and “immunoglobulin” or “Ig” are used interchangeably herein, and is used in the broadest sense and encompasses, for example, individual monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length, or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies (e.g., VHH), single chain antibodies, intrabodies, 18 315736224v1Attorney Docket No: 260525.000077 anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of antibodies, as described below. An antibody can be human, humanized, camelized, recombinantly produced, chimeric, synthetic, affinity de-matured and / or affinity matured as well as an antibody from other species, for example mouse, camel, llama, rabbit, etc. In specific embodiments, the specific target antigen that can be bound by an antibody provided herein includes a serum albumin polypeptide, serum albumin fragment or serum albumin epitope. An “antigen-binding fragment” generally refers a portion of an antibody heavy and / or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of antigen- binding fragments include single-domain antibody (e.g., VHH), single-chain Fvs (scFv), Fab fragments, F(ab’) fragments, F(ab)2 fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody, or a chemically modified derivative thereof. In particular, antibodies provided herein include immunoglobulin molecules and molecules that contain immunologically active portion(s) of an immunoglobulin molecule, for example, one or more complementarity determining regions (CDRs) of an antibody that binds to serum albumin. Such antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E.D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.

[0098] The term “single-domain antibody” or “sdAb” as used herein, refers to an antibody or antibody fragment containing a single antibody variable domain that is able to bind to a specific antigen alone, without the requirement of another antibody variable domain. The complementarity determining regions (CDRs) of a single-domain antibody are part of a single antibody variable domain. Examples of single-domain antibodies include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional four-chain antibodies, engineered antibodies, variable domains derived from the aforementioned antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, 19 315736224v1Attorney Docket No: 260525.000077 human, camel, llama, shark, goat, rabbit, and / or bovine. In some embodiments, a single domain antibody as used herein is a naturally occurring single domain antibody known as heavy chain antibody devoid of light chains. For clarity reasons, the variable domain derived from a heavy chain antibody naturally devoid of light chain is known herein as a VHH to distinguish it from the conventional VH of four-chain immunoglobulins. Such a VHH molecule can be derived from antibodies raised in Camelidae species, e.g., camel, llama, dromedary, alpaca, and guanaco. Other species besides Camelidae may produce heavy chain antibodies naturally devoid of light chain, which are also within the scope of the invention. For example, cartilaginous fishes such as sharks can produce immunoglobulin-like structures known as VNAR. In some embodiments, a single- domain antibody may be obtained from a Camelidae VH domain. In some embodiments, a single- domain antibody may be obtained from human VH by camelization. See Saerens et al., Current Opinion in Pharmacology, 2008, 8:600-608, the disclosure of which being incorporated by reference, for review of single-domain antibodies.

[0099] The term “specifically binds” as used herein means that an antigen-binding protein forms a complex with a target antigen that is relatively stable under physiological conditions. Specific binding can be characterized by a dissociation constant (KD) of about 1x10-6M or less (e.g., less than 10-6M, less than 5x10-7M, less than 10-7M, less than 5x10-8M, less than 10-8M, less than 5x10-9M, less than 10-9 M, or less than 10-10M). Methods for determining the binding affinity of an antigen-binding protein, e.g., an antibody or an antibody fragment, to a target antigen are well known in the art and include, e.g., surface plasmon resonance (e.g., BIACORE®assays), bio-layer interferometry, ligand binding assays (e.g., enzyme-linked immunosorbent assay (ELISA)), equilibrium dialysis, fluorescent-activated cell sorting (FACS), or flow cytometry-based binding assays and the like. Specific binding to a particular target antigen from a certain species does not exclude that the antigen-binding protein can also specifically bind to the analogous target from a different species. For example, specific binding to human serum albumin (HSA) does not exclude that the antigen-binding protein can also specifically bind to serum albumin from cynomolgus monkeys (“cyno”), mouse, or rat.

[0100] The term “isolated” when used in the context of antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides, and vectors, means the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies), polypeptides, polynucleotides and vectors are at least partially free of other biological molecules from the cells 20 315736224v1Attorney Docket No: 260525.000077 or cell culture from which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antigen-binding protein may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules (e.g., minor, or insignificant amounts of impurity may remain) or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antigen-binding proteins (e.g., antibodies, such as single-domain antibodies).

[0101] The term “operably linked” as used herein can refer to a functional relationship between two or more regions of a polypeptide chain in which the two or more regions are linked so as to produce a functional polypeptide.

[0102] As used herein, the term “variant”, “derivative”, or “derived from” in the context of proteins or polypeptides (e.g., antigen-binding proteins or domains thereof), refer to: (a) a polypeptide that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the polypeptide it is a variant or derivative of; (b) a polypeptide encoded by a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a nucleotide sequence encoding the polypeptide it is a variant or derivative of; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid mutations (i.e., additions, deletions and / or substitutions) relative to the polypeptide it is a variant or derivative of; (d) a polypeptide encoded by nucleic acids can hybridize under high, moderate or typical stringency hybridization conditions to nucleic acids encoding the polypeptide it is a variant or derivative of; (e) a polypeptide encoded by a nucleotide sequence that can hybridize under high, moderate or typical stringency hybridization conditions to a nucleotide sequence encoding a fragment of the polypeptide, it is a variant or derivative of, of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of the polypeptide it is a variant or derivative of. The terms also encompass a fusion protein or polypeptide comprising the polypeptide it is a variant or derivative of. 21 315736224v1Attorney Docket No: 260525.000077

[0103] The term “substantial identity” or “substantially identical” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 95%, and more preferably at least about 96%, 97%, 98%, or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0104] As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains: serine, and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et 22 315736224v1Attorney Docket No: 260525.000077 al. (1992) Science 256: 1443-1445, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0105] Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-402, each herein incorporated by reference.

[0106] The terms “enhance”, or “promote,” or “increase,” or “expand,” or “improve” refer generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response compared to the response caused by either vehicle or a control molecule / composition. A measurable physiological response may include an increase in serum half-life, among others apparent from the understanding in the art and the description herein. In certain embodiments, an “increased” or “enhanced” amount can be a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7., 1.8, etc.) the response produced by vehicle or a control composition.

[0107] The terms “decrease”, or “lower,” or “lessen,” or “reduce,” or “abate” refer generally to the ability of composition contemplated herein to produce, elicit, or cause a lesser physiological response compared to the response caused by either vehicle or a control molecule / composition. In certain embodiments, a “decrease” or “reduced” amount can be a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times 23 315736224v1Attorney Docket No: 260525.000077 (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc.) the response (reference response) produced by vehicle or a control composition.

[0108] The terms “treat” or “treatment” of a state, disease, disorder, or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disease, disorder, or condition developing in a subject that may be afflicted with or predisposed to the state, disease, disorder, or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disease, disorder, or condition; or (2) inhibiting the state, disease, disorder, or condition, e.g., arresting, reducing or delaying the development of the state, disease, disorder, or condition, or a relapse thereof or at least one clinical or sub-clinical symptom of the state, disease, disorder, or condition; or (3) relieving the state, disease, disorder, or condition, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0109] The terms “effective amount” or “therapeutically effective amount” refer to a quantity and / or concentration of a composition containing an active ingredient (e.g., anti-serum albumin antigen-binding protein) that when administered into a patient either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a significant decrease in disease, state, or disorder progression as, for example, by ameliorating or eliminating symptoms and / or the cause of the disease, state, or disorder. An effective amount may be an amount that relieves, lessens, or alleviates at least one symptom or biological response or effect associated with a disease, state, or disorder, prevents progression of the disease, state, or disorder, or improves physical functioning of the patient. A therapeutically effective amount of a composition containing an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the active agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount may be delivered in one or more administrations. A therapeutically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic and / or prophylactic result. 24 315736224v1Attorney Docket No: 260525.000077

[0110] The terms “individual”, “subject”, and “patient” are used interchangeably herein to refer to an animal, for example a mammal. The terms include human and veterinary subjects. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, a subject can be a subject in need of treatment for a disease or disorder. In particular embodiments, the subject is a human. Antigen-binding Proteins Against Serum Albumin

[0111] The present disclosure provides antigen-binding proteins (e.g., antibodies, such as single-domain antibodies) that bind to serum albumin.

[0112] Serum albumin is synthesized in the liver and its high concentration in plasma is the result of a cellular recycling pathway in which albumin is bound by the neonatal Fc receptor (FcRn) at endosomal pH and released at neutral pH at the cell surface.

[0113] The serum albumin referred to herein can be, but are not limited to, human serum albumin, cyno serum albumin, mouse serum albumin, or rat serum albumin.

[0114] Human serum albumin (HSA) is an abundant serum protein with an exceptionally long half-life of approximately three weeks (Larsen et al., Albumin-based drug delivery: harnessing nature to cure disease. Mol Cell Ther 4, 3, (2016)). Unlike small biomolecules (< 50 kDa) rapidly eliminated (e.g., from mins to a few hours) via glomerular filtration, HSA can be retained due to the relatively large size (66.5 kDa). Importantly, HSA can bind with the neonatal Fc receptor (FcRn) at acidic pH and is continuously recycled via the FcRn-mediated transcytosis (Sand et al., Frontiers in Immunology 5, 1-21, (2015); Merlot et al., Frontiers in Physiology 5, (2014)). Because of its stable interaction with the FcRn, it can escape the endo-lysosomal degradation pathway and obtain outstanding serum stability.

[0115] In some embodiments, a human serum albumin can be encoded by human albumin (ALB) gene (NCBI Gene ID 213) and has the amino acid sequence MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQC PFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCA KQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYA 25 315736224v1Attorney Docket No: 260525.000077 PELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGER AFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEP QNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEA KRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEF NAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGKKLVAASQAALGL (UniProtKB Accession No. P02768) (SEQ ID NO: 42). (The part of the sequence indicated in bold is a signal peptide.)

[0116] In some embodiments, a cyno serum albumin can be encoded by a cyno albumin gene (NCBI Gene ID: 102130757) and has the amino acid sequence MKWVTFISLLFLFSSAYSRGVFRRDTHKSEVAHRFKDLGEEHFKGLVLVAFSQYLQQ CPFEEHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCC AKQEPERNECFLQHKDDNPNLPPLVRPEVDVMCTAFHDNEATFLKKYLYEVARRHPYF YAPELLFFAARYKAAFAECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFG DRAFKAWAVARLSQKFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYM CENQDSISSKLKECCDKPLLEKSHCLAEVENDEMPADLPSLAADYVESKDVCKNYAEA KDVFLGMFLYEYARRHPDYSVMLLLRLAKAYEATLEKCCAAADPHECYAKVFDEFQP LVEEPQNLVKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGAKC CKLPEAKRMPCAEDYLSVVLNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALELDEA YVPKAFNAETFTFHADMCTLSEKEKQVKKQTALVELVKHKPKATKEQLKGVMDNFAA FVEKCCKADDKEACFAEEGPKFVAASQAALA (UniProtKB Accession No. A2V9Z4) (SEQ ID NO: 43). (The part of the sequence indicated in bold is a signal peptide.)

[0117] In some embodiments, a mouse serum albumin can be encoded by mouse albumin (Alb) gene (NCBI Gene ID: 11657) and has the amino acid sequence MKWVTFLLLLFVSGSAFSRGVFRREAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQK CSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCC TKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFY APELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGE RAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCE NQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKD 26 315736224v1Attorney Docket No: 260525.000077 VFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEE PKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPE DQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEF KAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKA ADKDTCFSTEGPNLVTRCKDALA (UniProtKB Accession No. P07724) (SEQ ID NO: 44) (The part of the sequence indicated in bold is a signal peptide.).

[0118] In some embodiments, a rat serum albumin can be encoded by rat albumin (Alb) gene (NCBI Gene ID: 24186) and has the amino acid sequence MKWVTFLLLLFISGSAFSRGVFRREAHKSEIAHRFKDLGEQHFKGLVLIAFSQYLQKC PYEEHIKLVQEVTDFAKTCVADENAENCDKSIHTLFGDKLCAIPKLRDNYGELADCCAK QEPERNECFLQHKDDNPNLPPFQRPEAEAMCTSFQENPTSFLGHYLHEVARRHPYFYAP ELLYYAEKYNEVLTQCCTESDKAACLTPKLDAVKEKALVAAVRQRMKCSSMQRFGER AFKAWAVARMSQRFPNAEFAEITKLATDVTKINKECCHGDLLECADDRAELAKYMCEN QATISSKLQACCDKPVLQKSQCLAEIEHDNIPADLPSIAADFVEDKEVCKNYAEAKDVFL GTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEGDPPACYGTVLAEFQPLVEEPKN LVKTNCELYEKLGEYGFQNAVLVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEAQ RLPCVEDYLSAILNRLCVLHEKTPVSEKVTKCCSGSLVERRPCFSALTVDETYVPKEFKA ETFTFHSDICTLPDKEKQIKKQTALAELVKHKPKATEDQLKTVMGDFAQFVDKCCKAA DKDNCFATEGPNLVARSKEALA (UniProtKB Accession No. P02770) (SEQ ID NO: 2377) (The part of the sequence indicated in bold is a signal peptide.).

[0119] In some embodiments, a serum albumin-binding protein of the present disclosure binds to serum albumin at neutral pH and / or endosomal pH.

[0120] In some embodiments, a serum albumin-binding protein of the present disclosure binds to serum albumin at neutral pH. In some embodiments, a serum albumin-binding protein of the present disclosure binds to serum albumin at endosomal pH. In some embodiments, a serum albumin-binding protein of the present disclosure binds to serum albumin at both neutral pH and endosomal pH. In some embodiments, a serum albumin-binding protein of the present disclosure binds to serum albumin at neutral pH but not endosomal pH. In some embodiments, a serum 27 315736224v1Attorney Docket No: 260525.000077 albumin-binding protein of the present disclosure binds to serum albumin at endosomal pH but not neutral pH.

[0121] In some embodiments, the neutral pH is in the rage of about pH 7.2 to about pH 7.8. In some embodiments, a serum albumin-binding protein of the present disclosure binds to serum albumin at pH 7.2, at pH 7.2, at pH 7.3, at pH 7.4, at pH 7.5, at pH 7.6, at pH 7.7, or at pH 7.8. In specific embodiments, a serum albumin-binding protein of the present disclosure binds to serum albumin at pH 7.4.

[0122] In some embodiments, the endosomal pH is in the range of pH about 5.3 to about pH 6.3. In some embodiments, a serum albumin-binding protein of the present disclosure binds to serum albumin at pH 5.3, at pH 5.4, at pH 5.5, at pH 5.6, at pH 5.7, at pH 5.8, at pH 5.9, at pH 6.0, at pH 6.1, at pH 6.2, or at pH 6.3. In specific embodiments, the serum albumin-binding proteins of the present disclosure binds to serum albumin at pH 5.8.

[0123] In some embodiments, the antigen-binding proteins of the present disclosure bind to human serum albumin. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human serum albumin with a KD of less than about 1×10−6M, for example, less than about 5×10−7M, less than about 3×10−7M, less than about 1×10−7M, less than about 8×10−8M, less than about 5×10−8M, less than about 3×10−8M, less than about 1×10−8M, less than about 8×10−9M, less than about 5×10−9M, less than about 3×10−9M, or less than about 1×10−9M, or about 1×10−10to 1×10−9M, about 1×10−10to 5×10−9M, about 1×10−10to 1×10−8M, about 1×10−10to 5×10−8M, about 1×10−9to 1×10−8M, about 1×10−9to 5×10−8M, about 1×10−9to 1×10−7M, or about 1×10−8to 1×10−7M.

[0124] In some embodiments, the antigen-binding proteins (e.g., antibodies such as single- domain antibodies) of the present disclosure may bind to human serum albumin with a KDof less than about 3×10−7M at neutral pH. In certain embodiments, the antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human serum albumin with a KDof less than about 5×10−8M at neutral pH. In some embodiments, antigen- binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human serum albumin with a KD of about 1×10−9to 5×10−8M at neutral pH. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human serum albumin with a KDof about 1×10−9, about 2×10−9, 28 315736224v1Attorney Docket No: 260525.000077 about 3×10−9, about 4×10−9, about 5×10−9, about 6×10−9, about 7×10−9, about 8×10−9, about 9×10−9, about 1×10−8, about 2×10−8, about 3×10−8, about 4×10−8, or about 5×10−8M at neutral pH.

[0125] In some embodiments, the antigen-binding proteins (e.g., antibodies such as single- domain antibodies) of the present disclosure may bind to human serum albumin with a KD of less than about 3×10−7M at endosomal pH. In certain embodiments, the antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human serum albumin with a KDof less than about 1×10−7M at endosomal pH. In certain embodiments, the antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human serum albumin with a KDof less than about 7×10−8M at endosomal pH. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human serum albumin with a KD of about 1×10−9to 7×10−8M at endosomal pH. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to human serum albumin with a KD of about 1×10−9, about 2×10−9, about 3×10−9, about 4×10−9, about 5×10−9, about 6×10−9, about 7×10−9, about 8×10−9, about 9×10−9, about 1×10−8, about 2×10−8, about 3×10−8, about 4×10−8, about 5×10−8M, about 6×10−8, about 7×10−8M, about 8×10−8M, about 9×10−8M, or about 1×10−7M at endosomal pH.

[0126] In some embodiments, the antigen-binding proteins of the present disclosure bind to cynomolgus monkey (“cyno”) serum albumin. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno serum albumin with a KD of less than about 1×10−6M, for example, less than about 5×10−7M, less than about 3×10−7M, less than about 1×10−7M, less than about 8×10−8M, less than about 5×10−8M, less than about 3×10−8M, less than about 1×10−8M, less than about 8×10−9M, less than about 5×10−9M, less than about 3×10−9M, or less than about 1×10−9M, or about 1×10−10to 1×10−8M, about 1×10−10to 5×10−8M, about 1×10−9to 1×10−8M, about 1×10−9to 5×10−8M, about 1×10−9to 1×10−7M, about 1×10−9to 2×10−7M, about 1×10−9to 5×10−7M, about 1×10−8to 1×10−7M, about 1×10−8to 2×10−7M, about 1×10−8to 5×10−7M, or about 1×10−8to 1×10−6M.

[0127] In some embodiments, the antigen-binding proteins (e.g., antibodies such as single- domain antibodies) of the present disclosure may bind to cyno serum albumin with a KDof less than about 3×10−7M at neutral pH. In certain embodiments, the antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno serum 29 315736224v1Attorney Docket No: 260525.000077 albumin with a KD of less than about 8×10−8M at neutral pH. In some embodiments, antigen- binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno serum albumin with a KDof about 2×10−8to 8×10−8M at neutral pH. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno serum albumin with a KD of about 2×10−8, about 3×10−8, about 4×10−8, about 5×10−8, about 6×10−8, about 7×10−8, or about 8×10−8M at neutral pH.

[0128] In some embodiments, the antigen-binding proteins (e.g., antibodies such as single- domain antibodies) of the present disclosure may bind to cyno serum albumin with a KD of less than about 3×10−7M at endosomal pH. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno serum albumin with a KD of about 2×10−8to 1.7×10−7M at endosomal pH. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to cyno serum albumin with a KDof about 2×10−8, about 3×10−8, about 4×10−8, about 5×10−8, about 6×10−8, about 7×10−8, about 8×10−8M, about 1×10−7M, about 1.5×10−7M, or about 1.7×10−7M at endosomal pH.

[0129] In some embodiments, the antigen-binding proteins of the present disclosure bind to mouse serum albumin. In some embodiments, antigen-binding proteins of the present disclosure may bind to mouse serum albumin with a KD of less than about 1×10−6M, for example, less than about 5×10−7M, less than about 3×10−7M, less than about 1×10−7M, less than about 8×10−8M, less than about 5×10−8M, less than about 3×10−8M, less than about 1×10−8M, less than about 8×10−9M, less than about 5×10−9M, less than about 3×10−9M, or less than about 1×10−9M, or about 1×10−10to 1×10−9M, about 1×10−10to 5×10−9M, about 1×10−10to 1×10−8M, about 1×10−10to 5×10−8M, about 1×10−9to 1×10−8M, about 1×10−9to 5×10−8M, about 1×10−9to 1×10−7M, about 1×10−9to 2×10−7M, about 1×10−9to 5×10−7M, about 1×10−8to 1×10−7M, about 1×10−8to 2×10−7M, about 1×10−8to 5×10−7M, or about 1×10−8to 1×10−6M. In some embodiments, antigen- binding proteins of the present disclosure do not specifically bind to mouse serum albumin.

[0130] In some embodiments, the antigen-binding proteins (e.g., antibodies such as single- domain antibodies) of the present disclosure may bind to mouse serum albumin with a KD of less than about 3×10−7M at neutral pH. In certain embodiments, the antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to mouse serum albumin with a KD of less than about 1×10−7M at neutral pH. In certain embodiments, the antigen- 30 315736224v1Attorney Docket No: 260525.000077 binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to mouse serum albumin with a KD of less than about 2×10−8M at neutral pH. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to mouse serum albumin with a KD of about 7×10−9to 2×10−8M at neutral pH. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to mouse serum albumin with a KDof about 7×10−9, about 8×10−9, about 9×10−9, about 1×10−8, about 1.2×10−8, about 2×10−8, about 3×10−8, about 4×10−8, about 5×10−8M, about 6×10−8, about 7×10−8M, about 8×10−8M, about 9×10−8M, or about 1×10−7M at neutral pH.

[0131] In some embodiments, the antigen-binding proteins (e.g., antibodies such as single- domain antibodies) of the present disclosure may bind to mouse serum albumin with a KD of less than about 3×10−7M at endosomal pH. In certain embodiments, the antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to mouse serum albumin with a KD of less than about 2×10−7M at endosomal pH. In some embodiments, antigen- binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to mouse serum albumin with a KDof about 1×10−9to 2×10−7M at endosomal pH. In some embodiments, antigen-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may bind to mouse serum albumin with a KD of about 1×10−9, about 2×10−9, about 3×10−9, about 4×10−9, about 5×10−9, about 6×10−9, about 7×10−9, about 8×10−9, about 9×10−9, about 2.5×10−8, about 5×10−8, about 6×10−8, about 7×10−8, about 8×10−8, about 9×10−8, about 1×10−9, or about 1.8×10−7M at endosomal pH.

[0132] In some embodiments, the antigen-binding proteins of the present disclosure bind to rat serum albumin. In some embodiments, antigen-binding proteins of the present disclosure may bind to rat serum albumin with a KD of less than about 1×10−6M, for example, less than about 5×10−7M, less than about 3×10−7M, less than about 1×10−7M, less than about 8×10−8M, less than about 5×10−8M, less than about 3×10−8M, less than about 1×10−8M, less than about 8×10−9M, less than about 5×10−9M, less than about 3×10−9M, or less than about 1×10−9M, or about 1×10−10to 1×10−9M, about 1×10−10to 5×10−9M, about 1×10−10to 1×10−8M, about 1×10−10to 5×10−8M, about 1×10−9to 1×10−8M, about 1×10−9to 5×10−8M, about 1×10−9to 1×10−7M, about 1×10−9to 2×10−7M, about 1×10−9to 5×10−7M, about 1×10−8to 1×10−7M, about 1×10−8to 2×10−731 315736224v1Attorney Docket No: 260525.000077 M, about 1×10−8to 5×10−7M, or about 1×10−8to 1×10−6M. In some embodiments, antigen- binding proteins of the present disclosure do not specifically bind to rat serum albumin.

[0133] In some embodiments, a serum albumin-binding proteins of the present disclosure binds to domain I, domain II, and / or domain III of human serum albumin. In some embodiments, a serum albumin-binding protein of the present disclosure binds to domain I and / or domain II of human serum albumin. In some embodiments, a serum albumin-binding protein of the present disclosure binds to domain I of human serum albumin. In some embodiments, a serum albumin- binding protein of the present disclosure binds to domain II of human serum albumin. In some embodiments, a serum albumin-binding protein of the present disclosure binds to domain I and domain II of human serum albumin. In some embodiments, a serum albumin-binding protein of the present disclosure binds to domain III of human serum albumin.

[0134] In some embodiments, serum albumin-binding proteins of the present disclosure may be capable of binding to serum albumin without disrupting the interaction of serum albumin with neonatal Fc receptor / beta-2 microglobulin (FcRn / β2M).

[0135] In some embodiments, the serum albumin-binding proteins of the present disclosure may be capable of binding to serum albumin at FcRn / β2M binding site on serum albumin.

[0136] The present disclosure includes the antigen-binding proteins that bind serum albumin, e.g., antibodies such as single-domain antibodies, or antigen-binding fragments, that bind to the same epitope as serum albumin-binding proteins described herein (e.g., H-001, H-002, H-003, H- 004, H-005, H-006, H-007, H-008, H-009, H-010, H-011, and H-012, and / or humanized variants thereof such as H-002Hu1.DVQ.VPAG, H-002Hu1.DVQ.VPAG.NA, H- 002Hu1.DVQ.VPAG.QS, H-002Hu1.DVQ.VPAG.AS).

[0137] Methods for determining the epitope of an antigen-binding protein, e.g., antibody such as single-domain antibody or fragment or polypeptide, include alanine scanning mutational analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol.248: 443-63), peptide cleavage analysis, crystallographic studies, cryo-electronic microscopy, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9: 487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding protein (e.g., antibody such as single-domain antibody or fragment or polypeptide) interacts is hydrogen / deuterium exchange 32 315736224v1Attorney Docket No: 260525.000077 detected by mass spectrometry. See, e.g., Ehring (1999) Analytical Biochemistry 267: 252-259; Engen and Smith (2001) Anal. Chem.73: 256A-265A.

[0138] The present disclosure includes the antigen-binding protein that compete for binding to serum albumin, e.g., human serum albumin or fragment thereof as discussed herein, with an antigen-binding protein described herein, e.g., H-001, H-002, H-003, H-004, H-005, H-006, H- 007, H-008, H-009, H-010, H-011, and H-012, and / or humanized variants thereof such as H- 002Hu1.DVQ.VPAG, H-002Hu1.DVQ.VPAG.NA, H-002Hu1.DVQ.VPAG.QS, H- 002Hu1.DVQ.VPAG.AS. In some embodiments, an antigen-binding protein (e.g., antibody such as single-domain antibody or antigen-binding fragment thereof) that binds to an antigen (e.g., serum albumin) inhibits or blocks the binding of another antigen-binding protein (e.g., antibody such as single-domain antibody or antigen-binding fragment thereof) to the antigen. In some embodiments, the present disclosure includes competition between two antigen-binding proteins e.g., antibodies, in both orientations, i.e., a first antibody that binds antigen and blocks binding by a second antibody and vice versa. Thus, in an embodiment, competition occurs in one such orientation. In certain embodiments, the first antigen-binding protein (e.g., antibody such as single- domain antibody) and second antigen-binding protein (e.g., antibody such as single-domain antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies such as single-domain antibodies) may bind to different, but, for example, overlapping or non-overlapping epitopes, wherein binding of one inhibits or blocks the binding of the second antibody, e.g., via steric hindrance. Competition between antigen-binding proteins (e.g., antibodies such as single-domain antibodies) may be measured by methods known in the art, for example, by a real-time, label-free bio-layer interferometry assay. Also, binding competition between anti-serum albumin antigen-binding proteins (e.g., antibodies such as single-domain antibodies) can be determined using a real time, label-free bio-layer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp.).

[0139] In some embodiments, the serum albumin-binding proteins of the present disclosure bind to one or more residues of serum albumin while having minimal or no impact on the binding of FcRn to serum albumin. In some embodiments, the serum albumin-binding proteins of the present disclosure bind to one or more residues of serum albumin selected from: Glu227, Phe228, Ala229, Glu230, Lys233, Lys240, Asp308, Glu321, Val325, and / or Met329. In some embodiments, the serum albumin-binding proteins of the present disclosure bind to one or more 33 315736224v1Attorney Docket No: 260525.000077 residues of serum albumin selected from: Glu542, Asp549, Asp550, Ala553, Phe554, Glu556, Lys557, Lys560, Glu570, Glu571, and / or Leu575.

[0140] In some embodiments, the serum albumin-binding proteins of the present disclosure bind to one or more residues of serum albumin selected from: Ala226, Glu227, Phe228, Ala229, Glu230, Ser232, Lys233, Thr236, Lys240, Tyr263, Ser270, Asp301, Pro303, Asp308, Phe309, Asn318, Glu321, Ala322, Lys323, Asp324, Val325, Met329, Tyr332, Glu333, and / or Arg336. In some embodiments, the serum albumin-binding proteins of the present disclosure bind to one or more residues of serum albumin selected from: Glu542, Lys545, Ala546, Asp549, Asp550, Ala552, Ala553, Phe554, Glu556, Lys557, Lys560, Ala561, Asp563, Thr566, Cys567, Glu570, Glu571, Lys574, Leu575, and / or Ala578.

[0141] In some embodiments, the serum albumin-binding proteins of the present disclosure bind to one or more residues of domain II of human serum albumin selected from: Glu227, Phe228, Ala229, Glu230, Lys233, Lys240, Asp308, Glu321, Val325, and / or Met329. In such embodiments, the serum albumin-binding proteins of the present disclosure bind to one or more residues of domain II of human serum albumin selected from: Ala226, Glu227, Phe228, Ala229, Glu230, Ser232, Lys233, Thr236, Lys240, Tyr263, Ser270, Asp301, Pro303, Asp308, Phe309, Asn318, Glu321, Ala322, Lys323, Asp324, Val325, Met329, Tyr332, Glu333, and / or Arg336.

[0142] In some embodiments, the serum albumin-binding proteins of the present disclosure bind to one or more residues of domain III of human serum albumin selected from: Glu542, Asp549, Asp550, Ala553, Phe554, Glu556, Lys557, Lys560, Glu570, Glu571, and / or Leu575. In such embodiments, the serum albumin-binding proteins of the present disclosure bind to one or more residues of domain III of human serum albumin selected from: Glu542, Lys545, Ala546, Asp549, Asp550, Ala552, Ala553, Phe554, Glu556, Lys557, Lys560, Ala561, Asp563, Thr566, Cys567, Glu570, Glu571, Lys574, Leu575, and / or Ala578.

[0143] In some embodiments, the antigen-binding proteins that bind to serum albumin comprise a paratope comprising one or more residues selected from: TyrH37, ArgH45, ArgH52, ThrH52A, TyrH59, AspH61, TyrH95, ArgH96, GlnH97, LeuH100D, and / or TrpH103. In some embodiments, the antigen- binding proteins that bind to serum albumin comprise a paratope comprising one or more residues selected from: ValH2, GlyH26, PheH27, ThrH28, ValH31, TyrH32, SerH52A, SerH55, AsnH96, SerH97, TrpH98, GlyH100A, ThrH100B, AspH100D, and / or TyrH102. 34 315736224v1Attorney Docket No: 260525.000077

[0144] In some embodiments, the antigen-binding proteins that bind to serum albumin comprise a paratope comprising one or more residues selected from: SerH33, TyrH37, GluH44, ArgH45, LeuH47, AlaH50, ArgH52, Thr52A, ThrH53, IleH57, ValH58, TyrH59, AspH61, LysH64, LeuH93, TyrH95, ArgH96, GlnH97, GlyH99, SerH100, LeuH100D, IleH101, and / or TrpH103. In some embodiments, the antigen-binding proteins that bind to serum albumin comprise a paratope comprising one or more residues selected from: ValH2, GlyH26, PheH27, ThrH28, SerH30, ValH31, TyrH32, SerH52A, ThrH53, SerH55, AsnH96, SerH97, TrpH98, LeuH100, GlyH100A, ThrH100B, PheH100C, AspH100D, and / or TyrH102.

[0145] In some embodiments, the one or more residues of serum albumin-binding proteins of the present disclosure bind to serum albumin while having minimal or no impact on the binding of FcRn to serum albumin. In some embodiments, a serum albumin-binding protein of the present disclosure binds to domain II of human serum albumin, wherein the serum albumin-binding protein comprises a paratope comprising one or more residues selected from TyrH37, ArgH45, ArgH52, ThrH52A, TyrH59, AspH61, TyrH95, ArgH96, GlnH97, LeuH100D, and / or TrpH103. In such embodiments, a serum albumin-binding protein of the present disclosure binds to domain II of human serum albumin, wherein the serum albumin-binding protein comprises a paratope comprising one or more residues selected from SerH33, TyrH37, GluH44, ArgH45, LeuH47, AlaH50, ArgH52, Thr52A, ThrH53, IleH57, ValH58, TyrH59, AspH61, LysH64, LeuH93, TyrH95, ArgH96, GlnH97, GlyH99, SerH100, LeuH100D, IleH101, and / or TrpH103.

[0146] In some embodiments, a serum albumin-binding protein of the present disclosure binds to domain III of human serum albumin, wherein the serum albumin-binding protein comprises comprising one or more residues selected from ValH2, GlyH26, PheH27, ThrH28, ValH31, TyrH32, SerH52A, SerH55, AsnH96, SerH97, TrpH98, GlyH100A, ThrH100B, AspH100D, and / or TyrH102. In such embodiments, the serum albumin-binding proteins of the present disclosure bind to domain III of human serum albumin, wherein the serum albumin-binding protein comprises a paratope comprising one or more residues selected from ValH2, GlyH26, PheH27, ThrH28, SerH30, ValH31, TyrH32, SerH52A, ThrH53, SerH55, AsnH96, SerH97, TrpH98, LeuH100, GlyH100A, ThrH100B, PheH100C, AspH100D, and / or TyrH102.

[0147] Binding affinity of a molecular interaction between two molecules can be measured via various techniques, such as surface plasmon resonance (SPR), bio-layer interferometry (BLI), enzyme-linked immunosorbent assay (ELISA), equilibrium dialysis, fluorescent-activated cell 35 315736224v1Attorney Docket No: 260525.000077 sorting (FACS), flow cytometry binding assays, or isothermal titration calorimetry (ITC), and the like. Surface plasmon resonance is a biosensor technique that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions (see e.g., Ober et al. 2001, Intern. Immunology 13: 1551-1559). SPR can for example be performed using the BIACORE®system or Carterra LSA system. Another biosensor technique that can be used to determine affinities of biomolecular interactions is bio-layer interferometry (BLI) (see e.g., Abdiche et al. 2008, Anal. Biochem. 377: 209-217). Bio-layer Interferometry is a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam). A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since the interactions can be measured in real-time, association and dissociation rates and affinities can be determined. BLI can for example be performed using the Octet®Systems. Alternatively, affinities can be measured in Kinetic Exclusion Assay (KinExA) (see e.g., Drake et al.2004, Anal. Biochem., 328: 35-43), which is a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules. Equilibrated solutions of an antibody / antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).

[0148] Antigen-binding proteins of the present disclosure can include an antibody or an antigen-binding fragment of an antibody, such as a human antibody, a humanized antibody, a camelid antibody, a chimeric antibody, a recombinant antibody, a heavy chain antibody, a single- domain antibody (e.g., VHH), a single chain antibody (e.g., single chain fragment variable (scFv)), a diabody, a triabody, a tetrabody, a Fab fragment, a F(ab’) 2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody, and fragments thereof.

[0149] In some embodiments, an antigen-binding protein that binds to serum albumin is a single-domain antibody (also termed as “sdAb”). The single-domain antibodies of the present 36 315736224v1Attorney Docket No: 260525.000077 disclosure can be derived from numerous sources, including but not limited to VHHs, VNARs, or VH domains (naturally occurring or engineered VH domains). VHHs can be generated from camelid heavy chain only antibodies and libraries (e.g., synthetic libraries) thereof. VNARs can be generated from cartilaginous fish heavy chain only antibodies and libraries (e.g., synthetic libraries) thereof. Various methods have been implemented to generate monomeric sdAbs from conventionally heterodimeric VH and VL domains, including interface engineering and selection of specific germline families. In some embodiments, the sdAb of the present invention are human or humanized.

[0150] In some embodiments, a single-domain antibody described herein is a VHH fragment (also known as a nanobody). VHH fragments are also referred to as “V-bodies” in the present disclosure. In some embodiments, the VHH is a camelid VHH, a humanized VHH ,or a camelized VH. In some embodiments, a single-domain antibody described herein is a VH domain. In some embodiments, a single-domain antibody described herein is a naturally occurring VH domain or engineered VH domain.

[0151] The variable domain of an antigen-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises at least three complementarity determining regions (CDRs) which determine its binding specificity. Preferably, in a variable domain, the CDRs are distributed between framework regions (FRs). The variable domain typically contains 4 framework regions interspaced by 3 CDR regions, resulting in the following typical antibody variable domain structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. CDRs and / or FRs of the single domain antibody of the invention may be fragments or derivatives from a naturally occurring antibody variable domain or may be synthetic.

[0152] Sequence identifiers corresponding to exemplary anti-serum albumin VHH antibodies provided herein are listed in Table 1-1. Table 1-1 sets forth the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DNA sequences of the full-length camelid VHH antibodies, as well as amino acid sequences of corresponding humanized VHH antibodies. Amino acid sequences of additional exemplary anti- serum albumin VHH antibodies and corresponding humanized VHH antibodies are provided in Table 1-2. In the “Antibody ID”, “DVQ” indicates that the N-terminal residue “E” in the full- length VHH antibody is replaced with “D”. “VPAG” indicates that the C-terminal residues “VSS” 37 315736224v1Attorney Docket No: 260525.000077 in the full-length VHH antibody is replaced with “VPAG”. “Hu1” indicates a humanized variant of the parent VHH antibody. Table 1-1. Sequence identifiers for exemplary anti-serum albumin VHH antibodies CDR1 CDR2 CDR3 Non-humanized VHHHumanizedAntibody ID ClusterVHHDNAAmino acidTable 1-2. Sequence identifiers for additional exemplary anti-serum albumin VHH antibodies Non-humanized Humanized VHH Cluster VHH Amino Acid Amino Acid315736224v1Attorney Docket No: 260525.000077 Non-humanized Humanized VHH Cluster VHH Amino Acid Amino Acid Sequence Sequence315736224v1Attorney Docket No: 260525.000077 Non-humanized Humanized VHH Cluster VHH Amino Acid Amino Acid Sequence Sequence315736224v1Attorney Docket No: 260525.000077 Non-humanized Humanized VHH Cluster VHH Amino Acid Amino Acid Sequence Sequence315736224v1Attorney Docket No: 260525.000077 Non-humanized Humanized VHH Cluster VHH Amino Acid Amino Acid Sequence Sequence315736224v1Attorney Docket No: 260525.000077 Non-humanized Humanized VHH Cluster VHH Amino Acid Amino Acid Sequence Sequence315736224v1Attorney Docket No: 260525.000077 Non-humanized Humanized VHH Cluster VHH Amino Acid Amino Acid Sequence Sequence315736224v1Attorney Docket No: 260525.000077 Non-humanized Humanized VHH Cluster VHH Amino Acid Amino Acid Sequence Sequence315736224v1Attorney Docket No: 260525.000077 Non-humanized Humanized VHH Cluster VHH Amino Acid Amino Acid Sequence Sequence315736224v1Attorney Docket No: 260525.000077 Non-humanized Humanized VHH Cluster VHH Amino Acid Amino Acid Sequence Sequence

[0153] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single- domain antibody) described herein may comprise a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a) GFTF(S / T)(D / F)Y(A / S) (SEQ ID NO: 49); b) GFTFRNYV (SEQ ID NO: 10); 47 315736224v1Attorney Docket No: 260525.000077 c) G(G / R)(S / T)F(N / S)(I / T)Y(T / V); d) G(F / L)TLANYS (SEQ ID NO: 52); e) G(F / L)T(F / L)(I / S)SY(A / D) (SEQ ID NO: 53); f) GFTFS(V / Y)Y(A / R) (SEQ ID NO: 54); g) GFTFSSY(A / P) (SEQ ID NO: 55); and h) (E / G)FTFS(G / R / S)Y(A / S) (SEQ ID NO: 56).

[0154] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a complementarity determining region 1 (CDR1) comprising an amino acid sequence selected from GFTFTDYS (SEQ ID NO: 9), GFTFSFYS (SEQ ID NO: 488), GFTFRNYV (SEQ ID NO: 10), GGSFNIYT (SEQ ID NO: 11), GFTLANYS (SEQ ID NO: 12), GFTFISYD (SEQ ID NO: 13), GFTFSVYA (SEQ ID NO: 14), GFTFSSYA (SEQ ID NO: 15), and EFTFSSYA (SEQ ID NO: 16).

[0155] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single- domain antibody) described herein may further comprise a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position; “-” represents an absence of amino acid at the particular position.) a) TRTTGG(N / S)I (SEQ ID NO: 57); b) (I / V)TSV(D / G)DST (SEQ ID NO: 58); c) (I / V)(S / T)(R / W)SG(N / R / S)(G / R / S)(L / T); d) ISRSGGST (SEQ ID NO: 20); e) I(S / T)S(T / A)GR(N / T)T (SEQ ID NO: 61); f) ITST(G / S)(G / S)(R / S)(L / T) (SEQ ID NO: 62); g) ITSGG(E / G)ST (SEQ ID NO: 63); and h) ITTT(D / G)(G / N / S)(S / T)(T / -) (SEQ ID NO: 64).

[0156] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a complementarity determining region 2 (CDR2) comprising an amino acid sequence selected from TRTTGGSI (SEQ ID NO: 17), TRTTGGNI (SEQ ID NO: 856), ITSVDDST (SEQ ID NO: 18), ITWSGSRL (SEQ ID NO: 19), ISRSGGST (SEQ ID NO: 20), ITSTGRNT (SEQ ID NO: 21), ITSTSSRL (SEQ ID NO: 22), ITSGGGST (SEQ ID NO: 23), ITTTDNT. (SEQ ID NO: 24), and ITTTDNTT. (SEQ ID NO: 60). 48 315736224v1Attorney Docket No: 260525.000077

[0157] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single- domain antibody) described herein may further comprise a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from (amino acids listed in a pair of brackets represent the possible amino acids at the particular position) a) (Q / L)AYRQ(K / S)GS(N / K / Q / A)(A / S)PLI(V / I) (SEQ ID NO: 41); b) (Q / L)AYRQ(K / S)GS(N / K)SPLI(V / I) (SEQ ID NO: 2385); c) KAWYLTTTY (SEQ ID NO: 2); d) AAAYGAGRYRMVKQYDY (SEQ ID NO: 3); e) AADLWAVCPTGASGRGSDYDW (SEQ ID NO: 4); f) NAF(T / V)SWPLEDY(D / N)Y (SEQ ID NO: 45); g) NAGNS(G / W)G(L / W)G(P / T)FDY (SEQ ID NO: 46); h) APTSSWYL (SEQ ID NO: 7); and i) HAYR(A / Q)KTSSGKL(H / I)I (SEQ ID NO: 48).

[0158] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from LAYRQKGSNSPLIV (SEQ ID NO: 1), LAYRQKGSKSPLIV (SEQ ID NO: 209), LAYRQSGSNSPLIV (SEQ ID NO: 120), LAYRQKGSNSPLII (SEQ ID NO: 91), LAYRQKGSNAPLII (SEQ ID NO: 2378), LAYRQKGSQSPLII (SEQ ID NO: 2379), LAYRQKGSASPLII (SEQ ID NO: 2380), KAWYLTTTY (SEQ ID NO: 2), AAAYGAGRYRMVKQYDY (SEQ ID NO: 3), AADLWAVCPTGASGRGSDYDW (SEQ ID NO: 4), NAFVSWPLEDYNY (SEQ ID NO: 5), NAGNSWGLGTFDY (SEQ ID NO: 6), APTSSWYL (SEQ ID NO: 7), and HAYRQKTSSGKLHI (SEQ ID NO: 8).

[0159] In some embodiments, a serum albumin-binding protein of the present disclosure comprises i. a CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR2 comprising an amino acid sequence of SEQ ID NO: 57, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 41; ii. a CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR2 comprising an amino acid sequence of SEQ ID NO: 57, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2385; 49 315736224v1Attorney Docket No: 260525.000077 iii. a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence of SEQ ID NO: 58, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2; iv. a CDR1 comprising the amino acid sequence of G(G / R)(S / T)F(N / S)(I / T)Y(T / V), a CDR2 comprising an amino acid sequence of (R / W)SG(N / R / S)(G / R / S)(L / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3; v. a CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR2 comprising an amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4; vi. a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR2 comprising an amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 45; vii. a CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a CDR2 comprising an amino acid sequence of SEQ ID NO: 62, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 46; viii. a CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR2 comprising an amino acid sequence of SEQ ID NO: 63, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7; and / or ix. a CDR1 comprising the amino acid sequence of SEQ ID NO: 56, a CDR2 comprising an amino acid sequence of SEQ ID NO: 64, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 48.

[0160] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of (Q / L)AYRQ(K / S)GS(N / K / Q / A)(A / S)PLI(V / I) (SEQ ID NO: 41); b) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of (Q / L)AYRQ(K / S)GS(N / K)SPLI(V / I) (SEQ ID NO: 2385); 50 315736224v1Attorney Docket No: 260525.000077 c) a CDR1 comprising an amino acid sequence of GFTFRNYV (SEQ ID NO: 10), a CDR2 comprising an amino acid sequence of ITSVDDST (SEQ ID NO: 18), and a CDR3 comprising an amino acid sequence of KAWYLTTTY (SEQ ID NO: 2); d) a CDR1 comprising an amino acid sequence of GGSFNIYT (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ITWSGSRL (SEQ ID NO: 19), and a CDR3 comprising an amino acid sequence of AAAYGAGRYRMVKQYDY (SEQ ID NO: 3); e) a CDR1 comprising an amino acid sequence of GFTLANYS (SEQ ID NO: 12), a CDR2 comprising an amino acid sequence of ISRSGGST (SEQ ID NO: 20), and a CDR3 comprising an amino acid sequence of AADLWAVCPTGASGRGSDYDW (SEQ ID NO: 4); f) a CDR1 comprising an amino acid sequence of GFTFISYD (SEQ ID NO: 13), a CDR2 comprising an amino acid sequence of ITSTGRNT (SEQ ID NO: 21), and a CDR3 comprising an amino acid sequence of NAF(T / V)SWPLEDY(D / N)Y (SEQ ID NO: 45); g) a CDR1 comprising an amino acid sequence of GFTFSVYA (SEQ ID NO: 14), a CDR2 comprising an amino acid sequence of ITSTSSRL (SEQ ID NO: 22), and a CDR3 comprising an amino acid sequence of NAGNS(G / W)G(L / W)G(P / T)FDY (SEQ ID NO: 46); h) a CDR1 comprising an amino acid sequence of GFTFSSYA (SEQ ID NO: 15), a CDR2 comprising an amino acid sequence of ITSGGGST (SEQ ID NO: 23), and a CDR3 comprising an amino acid sequence of APTSSWYL (SEQ ID NO: 7); i) a CDR1 comprising an amino acid sequence of EFTFSSYA (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of ITTTDNT (SEQ ID NO: 24), and a CDR3 comprising an amino acid sequence of HAYR(A / Q)KTSSGKL(H / I)I (SEQ ID NO: 48); j) a CDR1 comprising an amino acid sequence of GFTFSFYS (SEQ ID NO: 488), a CDR2 comprising an amino acid sequence of TRTTGGNI (SEQ ID NO: 856), and a CDR3 comprising an amino acid sequence of (Q / L)AYRQ(K / S)GS(N / K)SPLI(V / I) (SEQ ID NO: 2385); and / or 51 315736224v1Attorney Docket No: 260525.000077 k) a CDR1 comprising an amino acid sequence of GFTFSFYS (SEQ ID NO: 488), a CDR2 comprising an amino acid sequence of TRTTGGNI (SEQ ID NO: 856), and a CDR3 comprising an amino acid sequence of (Q / L)AYRQ(K / S)GS(N / K / Q / A)(A / S)PLI(V / I) (SEQ ID NO: 41).

[0161] In some embodiments, a serum albumin-binding protein of the present disclosure comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR2 comprising an amino acid sequence of SEQ ID NO: 57, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 41.

[0162] In some embodiments, a serum albumin-binding protein of the present disclosure comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 comprising an amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 41.

[0163] In some embodiments, a serum albumin-binding protein of the present disclosure comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a CDR2 comprising an amino acid sequence of SEQ ID NO: 856, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 41.

[0164] In some embodiments, a serum albumin-binding protein of the present disclosure comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence of SEQ ID NO: 58, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2.

[0165] In some embodiments, a serum albumin-binding protein of the present disclosure comprises a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence of SEQ ID NO: 18, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2.

[0166] Provided herein are serum albumin-binding proteins (e.g., antibodies such as single- domain antibodies) comprising a CDR1 (CDR1) comprising an amino acid sequence selected from any of the CDR1 amino acid sequences listed in Table 1-1 or Table 4, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0167] In some embodiments, a serum albumin-binding protein (e.g., antibody such as single- domain antibody) comprises a CDR1 comprising an amino acid sequence selected from SEQ ID 52 315736224v1Attorney Docket No: 260525.000077 Nos: 9, 10, 11, 12, 13, 14, 15, 16, 488, 433-487, and 489-800, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0168] Provided herein are serum albumin-binding proteins (e.g., antibodies such as single- domain antibodies) comprising a CDR2 (CDR2) comprising an amino acid sequence selected from any of the CDR2 amino acid sequences listed in Table 1-1 or Table 4, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0169] In some embodiments, a serum albumin-binding protein (e.g., antibody such as single- domain antibody) comprises a CDR2 comprising an amino acid sequence selected from SEQ ID Nos: 17, 18, 19, 20, 21, 22, 23, 24, 60, 856, 801-855, and 857-1168, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0170] Provided herein are serum albumin-binding proteins (e.g., antibodies such as single- domain antibodies) comprising a CDR3 (CDR3) comprising an amino acid sequence selected from any of the CDR3 amino acid sequences listed in Table 1-1 or Table 4, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0171] In some embodiments, a serum albumin-binding protein (e.g., antibody such as single- domain antibody) comprises a CDR3 comprising an amino acid sequence selected from SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 91, 120, 209, 65-90, 92-119, 121-208, 210-432, and 2378-2380, or a similar sequence thereof having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity.

[0172] Provided herein are serum albumin-binding proteins (e.g., antibodies such as single- domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2-CDR3) contained within any of the exemplary anti-serum albumin VHH antibodies listed in Tables 1-1, Table 1-2, or Table 4. In certain embodiments, a serum albumin-binding protein (e.g., antibody such as a single- domain antibody) of the present disclosure comprises a) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNSPLIV (SEQ ID NO: 1); b) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSKSPLIV (SEQ ID NO: 209); 53 315736224v1Attorney Docket No: 260525.000077 c) a CDR1 comprising an amino acid sequence of GFTFSFYS (SEQ ID NO: 488), a CDR2 comprising an amino acid sequence of TRTTGGNI (SEQ ID NO: 856), and a CDR3 comprising an amino acid sequence of LAYRQSGSNSPLIV (SEQ ID NO: 120); d) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNSPLII (SEQ ID NO: 91); e) a CDR1 comprising an amino acid sequence of GFTFRNYV (SEQ ID NO: 10), a CDR2 comprising an amino acid sequence of ITSVDDST (SEQ ID NO: 18), and a CDR3 comprising an amino acid sequence of KAWYLTTTY (SEQ ID NO: 2); f) a CDR1 comprising an amino acid sequence of GGSFNIYT (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ITWSGSRL (SEQ ID NO: 19), and a CDR3 comprising an amino acid sequence of AAAYGAGRYRMVKQYDY (SEQ ID NO: 3); g) a CDR1 comprising an amino acid sequence of GFTLANYS (SEQ ID NO: 12), a CDR2 comprising an amino acid sequence of ISRSGGST (SEQ ID NO: 20), and a CDR3 comprising an amino acid sequence of AADLWAVCPTGASGRGSDYDW (SEQ ID NO: 4); h) a CDR1 comprising an amino acid sequence of GFTFISYD (SEQ ID NO: 13), a CDR2 comprising an amino acid sequence of ITSTGRNT (SEQ ID NO: 21), and a CDR3 comprising an amino acid sequence of NAFVSWPLEDYNY (SEQ ID NO: 5); i) a CDR1 comprising an amino acid sequence of GFTFSVYA (SEQ ID NO: 14), a CDR2 comprising an amino acid sequence of ITSTSSRL (SEQ ID NO: 22), and a CDR3 comprising an amino acid sequence of NAGNSWGLGTFDY (SEQ ID NO: 6); j) a CDR1 comprising an amino acid sequence of GFTFSSYA (SEQ ID NO: 15), a CDR2 comprising an amino acid sequence of ITSGGGST (SEQ ID NO: 23), and a CDR3 comprising an amino acid sequence of APTSSWYL (SEQ ID NO: 7); k) a CDR1 comprising an amino acid sequence of EFTFSSYA (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of ITTTDNT (SEQ ID NO: 24), and a CDR3 comprising an amino acid sequence of HAYRQKTSSGKLHI (SEQ ID NO: 8); 54 315736224v1Attorney Docket No: 260525.000077 l) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNAPLII (SEQ ID NO: 2378); m) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSQSPLII (SEQ ID NO: 2379); and / or n) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and / or a CDR3 comprising an amino acid sequence of LAYRQKGSASPLII (SEQ ID NO: 2380).

[0173] In certain embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNSPLIV (SEQ ID NO: 1).

[0174] In certain embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSKSPLIV (SEQ ID NO: 209).

[0175] In certain embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of GFTFSFYS (SEQ ID NO: 488), a CDR2 comprising an amino acid sequence of TRTTGGNI (SEQ ID NO: 856), and a CDR3 comprising an amino acid sequence of LAYRQSGSNSPLIV (SEQ ID NO: 120).

[0176] In certain embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNSPLII (SEQ ID NO: 91). 55 315736224v1Attorney Docket No: 260525.000077

[0177] In certain embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNAPLII (SEQ ID NO: 2378).

[0178] In certain embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSQSPLII (SEQ ID NO: 2379).

[0179] In certain embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and / or a CDR3 comprising an amino acid sequence of LAYRQKGSASPLII (SEQ ID NO: 2380).

[0180] In certain embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a CDR1 comprising an amino acid sequence of GFTFRNYV (SEQ ID NO: 10), a CDR2 comprising an amino acid sequence of ITSVDDST (SEQ ID NO: 18), and a CDR3 comprising an amino acid sequence of KAWYLTTTY (SEQ ID NO: 2).

[0181] In a related embodiment, provided herein are serum albumin-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2- CDR3) contained within a VHH amino acid sequence as defined by any of the exemplary anti- serum albumin VHH antibodies listed in Table 1-1, Table 1-2, or Table 4. For example, provided herein are antibodies, or antigen-binding fragments thereof, comprising the set of CDR1-CDR2- CDR3 amino acid sequences contained within a VHH amino acid sequence selected from SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, and 1169-1536.

[0182] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 25; 56 315736224v1Attorney Docket No: 260525.000077 b) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 26; c) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 27; d) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 28; e) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 29; f) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 30; g) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 31; h) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 32; i) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1195; j) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1224; k) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1313; or l) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1420.

[0183] In an embodiment provided herein, a serum albumin antigen-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected from SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, and 1169-1536, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0184] In an embodiment provided herein, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a VHH amino acid sequence selected from SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 1195, 1224, 1313, and 1420, 57 315736224v1Attorney Docket No: 260525.000077 or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0185] In some embodiments of any of the above-described antigen-binding proteins, the VHH may be a humanized VHH.

[0186] In an embodiment provided herein, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 1537-1904, and 2381- 2384, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0187] In an embodiment provided herein, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a humanized VHH amino acid sequence selected from SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 1563, 1592, 1681, 1788, 2381, 2382, 2383, and 2384, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0188] In a related embodiment, provided herein are serum albumin-binding proteins (e.g., antibodies such as single-domain antibodies) comprising a set of three CDRs (i.e., CDR1-CDR2- CDR3) contained within a humanized VHH amino acid sequence as defined by any of the exemplary anti-serum albumin VHH antibodies listed in Table 1-1, Table 1-2, or Table 4. For example, provided herein are antibodies, or antigen-binding fragments thereof, comprising the set of CDR1-CDR2-CDR3 amino acid sequences contained within a VHH amino acid sequence selected from SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 1537-1904, and 2381-2384.

[0189] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure can include a) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 33; b) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 34; 58 315736224v1Attorney Docket No: 260525.000077 c) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 35; d) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 36; e) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 37; f) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 38; g) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 39; h) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 40; i) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 1563; j) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 1592; k) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 1681; l) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 1788; m) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 2381; n) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 2382; o) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 2383; or p) a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a humanized VHH comprising the amino acid sequence of SEQ ID NO: 2384.

[0190] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a variable domain that comprises a 59 315736224v1Attorney Docket No: 260525.000077 CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 25 or 33.

[0191] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1313 or 1681.

[0192] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1224 or 1592.

[0193] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 1195 or 1563.

[0194] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2381.

[0195] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2382.

[0196] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2383.

[0197] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 2384. 60 315736224v1Attorney Docket No: 260525.000077

[0198] In some embodiments, a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) of the present disclosure comprises a variable domain that comprises a CDR1, CDR2, and CDR3 contained within a VHH comprising the amino acid sequence of SEQ ID NO: 26 or 34.

[0199] In some embodiments, the present disclosure also provides a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) that competes for binding to serum albumin with any one of the exemplary anti-serum albumin VHH antibodies listed in Table 1-1, Table 1-2, or Table 4.

[0200] In some embodiments, the present disclosure also provides a serum albumin-binding protein (e.g., antibody such as a single-domain antibody) that binds to the same epitope on serum albumin as any one of the exemplary anti-serum albumin VHH antibodies listed in Table 1-1, Table 1-2, or Table 4. Single-domain antibodies

[0201] A single-domain antibody (e.g., VHH) can be obtained by immunization of dromedaries, camels, llamas, alpacas, or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens. By reverse transcription and polymerase chain reaction (PCR), a gene library of single-domain antibodies containing several million clones can be produced. Screening techniques such as phage display, yeast display, and ribosome display help to identify the clones binding the antigen. Methods generation of heavy- chain antibody fragments are described in e.g., WO 94 / 04678; Hamers-Casterman et al. 1993; Muyldermans et al.2001; and Arbabi Ghahroudi, M. et al. (1997). FEBS Letters 414 (3): 521-526, each of which is incorporated herein by reference in its entirety.

[0202] A different method may use gene libraries from animals that have not been previously immunized. Such naïve libraries usually contain only antibodies with low affinity to the desired antigen, making it necessary to apply affinity maturation by random mutagenesis as an additional step. See e.g., Saerens, D.; et al. (2008). “Single-domain antibodies as building blocks for novel therapeutics”. Current Opinion in Pharmacology 8 (5): 600-608. 61 315736224v1Attorney Docket No: 260525.000077

[0203] Affinity maturation strategies can be categorized as either targeted / rational approaches or untargeted / random approaches. For targeted approaches information about the VHH of interest is needed, such as hot spots for affinity maturation or structural information on the VHH: antigen complex, whereas for untargeted approaches no prior information is needed. Targeted approaches that may be applied for affinity maturation of VHHs include site-directed in-vitro mutagenesis and in-silico / computational approaches. Common untargeted approaches used for affinity maturation of VHHs include random in-vitro mutagenesis, CDR swapping and autonomous hypermutation yeast surface display, with the latter two being novel, emerging and very time efficient techniques. Most of these strategies have in common, that after applying a certain randomization strategy to generate a mutational library, the resulting library can be screened by employing standard display techniques such as yeast, phage or ribosome display to select for the best binders. The choice of the display system is often guided by the library size to be displayed, with yeast display being able to handle library sizes of ~107-109, phage display ~108-1010and ribosome display ~1012-1013(Chan and Groves, 2021). Notably, during affinity maturation the number of highly interactive residues such as aromatic amino acids usually increase in the CDR regions. The selected affinity matured clones may be further evaluated by a developability assessment to test for undesired properties, such as unspecific binding to off-targets or VHH instability.

[0204] For targeted in vitro mutagenesis, a set of selected residues within the CDRs of a VHH may be mutated (Tiller et al., 2017; Yau et al., 2005). Pre-selection of these residues can be either performed using alanine scanning to identify hot spot residues for mutation or by using structural data of the antigen: VHH complex to identify positions to be mutated. These sites can then be either submitted to saturating mutagenesis to substitute a specific site with all possible amino acids or specific amino acid substitutions yielding several smaller libraries. After mutagenesis binders can be displayed to select the best matured candidate. Usually, several rounds of targeted mutagenesis are performed with separate sub-libraries to obtain combinations of individual mutations that cooperatively result in increased binding affinity.

[0205] Computer-aided / in silico methods are often used to guide targeted in vitro mutagenesis. Using homology modeling of the target: VHH complex or docking, hotspots for mutations can be identified that are then submitted to in vitro mutagenesis (Bert Schepens et al., 2021; Cheng et al., 2019; Inoue et al., 2013; Mahajan et al., 2018). Further, in silico methods can search all designed variants in a virtual library (˜1040members) in a rather short amount of time to identify a feasible 62 315736224v1Attorney Docket No: 260525.000077 number of promising candidates to be tested experimentally. These techniques can be especially valuable if structural data on the drug-target interaction are available.

[0206] Untargeted / random affinity maturation strategies that can be applied to affinity mature VHHs include random in vitro mutagenesis, CDR shuffling / swapping and in vivo affinity maturation via yeast display. For random in vitro mutagenesis the sequence of either the entire VHH or only the CDRs are mutated randomly (Chen et al., 2021; Ye et al., 2021; Zupancic et al., 2021). The most commonly used technique is error prone PCR employing a DNA polymerase that lacks proof reading activity and PCR conditions that increase the polymerase error rate even further. This technique can be applied without further structural knowledge or information on the importance of residues that contribute to antigen: VHH interaction. The resulting mutational library can then be displayed to select the best matured candidate. This technique may also be combined with NGS sequencing of the display elutions to get an in-depth readout of all obtained candidates, enabling the identification of low abundant but still promising clones (Chen et al., 2021).

[0207] In some embodiments, CDR shuffling or swapping is applied for VHH affinity maturation, such as described in Zupancic et al., 2021. For CDR swapping, enriched libraries can be used as input material for a PCR reaction to individually amplify the CDR of the VHHs. The PCR products can then be mixed and reassembled using overlapping PCR to generate the entire plasmid for further rounds of display to select for the best matured binder. One limitation of this approach is that it can only be used for VHHs comprising the same framework as it is the case for synthetic libraries.

[0208] In some embodiments, in vivo affinity maturation via yeast display is applied for VHH affinity maturation, such as described in Wellner et al., 2021. The method is based on an autonomous hypermutation yeast surface display (AHEAD), which imitates somatic hypermutation during VHH selection using engineered yeast strains. The yeast’s error prone orthogonal DNA replication system can generate new variants during plasmid replication by randomly introducing mutations. The new variants can then be displayed and selected using yeast surface display to identify the best binders. This enables the production of high affinity clones in very little time (about 2 weeks), which is significantly faster than classical affinity maturation procedures. The method can be applied using synthetic or immune libraries using unenriched libraries enriched libraries or a subset of preselected clones. 63 315736224v1Attorney Docket No: 260525.000077

[0209] In case binders with medium affinity are required, as it is the case for the anti-serum albumin V-bodies and the affinity of the identified candidates need to be decreased, very similar techniques can be applied. For example, mutations that are aiming at lowering the affinity can be introduced using the same targeted or untargeted approaches as described for the affinity maturation. The selection afterwards can be adapted accordingly. If larger libraries are generated that need to be screened via a display technique, the selection strategy can be adapted to enrich medium affinity binders while excluding high affinity candidates. This could, for example be a pre-panning in phage display with low antigen concentration to remove all higher affinity candidates, followed by a selection with high antigen concentration to obtain medium affinity VHHs. For library sizes of up to 1000 candidates a kinetic off-rate characterization can be used to get immediate information about the kinetic behavior of the candidates.

[0210] When the most potent clones have been identified, their DNA sequence can be optimized, for example to improve their stability towards enzymes. Another goal is humanization to prevent immunological reactions of the human organism against the antibody. Humanization can be achieved based on the homology between camelid VHH and human VH fragments, which is described in further detail below. Finally, the optimized single-domain antibody can be translated and expressed in suitable organisms such as E. coli or Saccharomyces cerevisiae.

[0211] Single-domain antibodies can also be derived from conventional antibodies. In some embodiments, single-domain antibodies can be made from conventional murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naïve donors and display techniques for identification of the most specific antigens. However, the binding region of a conventional IgG consists of two domains (VH and VL), which tend to dimerize or aggregate because of their lipophilicity. Monomerization can be accomplished by replacing lipophilic by hydrophilic amino acids. (See e.g., Borrebaeck, C. A. K.; Ohlin, M. (2002). “Antibody evolution beyond Nature”. Nature Biotechnology 20 (12): 1189-90.) If affinity can be retained after monomerization, the single-domain antibodies can likewise be produced in E. coli, S. cerevisiae or other suitable organisms.

[0212] A “humanized antibody” refers to a chimeric, genetically engineered, antibody in which the amino acid sequences (typically CDRs) from an antibody (donor antibody), e.g., a camelid antibody, are grafted onto a human antibody (acceptor antibody). Thus, a humanized antibody typically comprises CDRs from a donor antibody and variable region framework and 64 315736224v1Attorney Docket No: 260525.000077 constant regions, when present, from a human antibody. Accordingly, a “humanized VHH” comprises CDRs that corresponds to the CDRs of a naturally occurring VHH domain (e.g., a camelid VHH), but that has been “humanized”. Humanized VHH may be prepared by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (particularly in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain human antibody. Such humanized VHHs can be obtained in any suitable manner known to a skilled person in the art and thus not strictly limited to methods described herein.

[0213] Humanization of VHHs can achieved using resurfacing or CDR grafting. Resurfacing strategies have been described in e.g., Conrath et al., 2005 J Mol Biol; Kazemi-Lomedasht et al., 2018; Vincke et al., 2009 J Biol Chem, and CDR grafting strategies have been described in e.g., ben Abderrazek et al., 2011; van Faassen et al., 2020 FASEB; Li et al., 2018; Vaneycken et al., 2010; Vincke et al., 2009 J Biol Chem; and Yu et al., 2017, each of which is incorporated herein by reference in its entirety.

[0214] To humanize a camelid VHH using a resurfacing approach, a human germline reference that is most similar to the camelid germline sequence of the selected VHH may be identified. Most of the isolated camelid VHHs in literature belong to the camelid IGHV3 subfamily 2 (Nguyen et al., 2000, EMBO J) with DP-47 / VH3-23 from the IGHV3 family commonly used as human reference. The framework of the camelid VHH can then be compared to the human reference sequence. Surface exposed residues are substituted to their human counterpart as it is assumed that their contribution to protein stability is rather low. Buried residues however remain of camelid origin, as they likely contribute to the overall VHH stability. Humanization of framework regions 1, 3 and 4 usually does not impact the physicochemical properties of the VHHs, whereas a general humanization of framework 2 would significantly increase local hydrophobicity. Residues H37, H44, H45, and H47 (Chothia numbering) in framework 2, the so called tetrade or hallmark residues, have a rather hydrophobic nature in human VHs (VGLW) as they are partially buried and involved in VH / VL paring, while in camelid VHHs these residues are partially charged (FERG), which significantly increases VHH solubility and inhibits paring of camelid VL (Soler et al., 2021, Biomolecules, Conrath et al., 2005 J Mol Biol). Further, residues H37 and H47 are known to interact with the CDR-H3 loop in many VHHs, stabilizing its conformation and thereby contributing to antigen binding affinity. In addition, a significant number of VHHs use framework 65 315736224v1Attorney Docket No: 260525.000077 2 residues H44, H45, and H47 for antigen binding (Zavrtanik et al., 2018, J Mol Biol). A full humanization of these residues hence frequently results in reduced solubility or aggregation of the VHHs and a reduced or complete loss of binding affinity for the target antigen (van Faassen et al., 2020, Vincke et al., 2009). In consequence, all or at least some of these hallmark residues in framework 2 remain of camelid origin when humanizing VHHs.

[0215] Another approach that may be applied to humanize VHHs is CDR grafting. CDRs of the selected VHHs can be transplanted onto a universal VHH framework that has been partially or fully humanized (Saerens et al., 2009 J Biol Chem, Soler et al., 2021, Vincke et al., 2009 J Biol Chem). CDR grafting has been successfully used in some cases but failed for several others, with VHHs frequently losing their potential to bind to the desired antigen and / or becoming structurally instable with a high tendency to aggregate (van Faassen et al., 2020, FASEB). This is mostly attributed to interactions of CDR3 with specific residues in framework 2 that are important for CDR3 conformation, general VHH stability and overall hydrophobicity, which are impaired by this approach. Sometimes camelid backmutations are introduced into the framework to compensate for these effects (van Faassen et al., 2020, FASEB).

[0216] An alternative strategy to mitigate the need of humanizing the selected VHH sequences is to use fully or partially humanized synthetic VHH libraries instead of camelid immune libraries for VHH discovery (Moutel et al.2016, eLife; McMahon, 2018, NSMB; Zimmermann et al., 2018, eLife). In many of these libraries the hallmark residues are still of camelid origin for reasons discussed above.

[0217] Other suitable humanizing substitutions are described in WO 09 / 138519 and WO 08 / 020079, as well as Tables A-3 to A-8 from WO 08 / 020079 (which are lists showing possible humanizing substitutions), each of which is incorporated herein by reference in its entirety. Non- limiting examples of such humanizing substitutions include Q108L and A14P. Such humanizing substitutions may also be suitably combined with one or more other mutations as described herein (such as with one or more mutations that reduce binding by pre-existing antibodies).

[0218] In some embodiments, humanized VHH sequences still retain the residues that are relevant for protein A binding. In some embodiments, the engineering activities during humanization may be applied to engineer protein A binding properties into a VHH that did previously not interact with protein A (Graille et al., 2000, PNAS). 66 315736224v1Attorney Docket No: 260525.000077

[0219] Like a “humanized antibody”, a “camelized antibody” refers to an antibody having amino acid sequences (typically CDRs) from a donor antibody, e.g., a human antibody, and variable region framework and constant regions, when present, from a camelid antibody. Accordingly, a “camelized VH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been “camelized”. Camelized VH may be prepared by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner, for example as described in WO 2008 / 020079. Such “camelizing” substitutions are usually inserted at amino acid positions that form and / or are present at the VH-VL interface, and / or at the so-called Camelidae hallmark residues, e.g., F37, E44, R45, and F47 (see for example WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). In one embodiment, the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, or the VH sequence of a human antibody. However, such camelized VH can be obtained in any suitable manner known to a skilled person in the art and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.

[0220] The amino acid residues of a single-domain antibody can be numbered according to the general numbering for VH domains given by Kabat et al. (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, Md., Publication No.91), as applied to VHH domains from Camelids described in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for example FIG.2 of this publication). The total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering. For example, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering. As a result, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain is usually in the range of from 110 to 120, often between 112 and 115. However, smaller, and longer sequences may also be suitable for the purposes described herein. 67 315736224v1Attorney Docket No: 260525.000077

[0221] Determination of CDR regions in a single-domain antibody may be accomplished using different methods, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” 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 M P et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 January; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun.8; 309(3):657-70, (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268- 9272, (“AbM” numbering scheme), each reference cited herein is incorporated by reference in its entirety.

[0222] The boundaries of a given CDR or framework (FR) may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular’s AbM antibody modeling software.

[0223] In some embodiments, CDRs can be defined in accordance with any of the Kabat numbering scheme, the Chothia numbering scheme, a combination of Kabat and Chothia, the AbM numbering scheme, and / or the Contact numbering scheme. A VHH typically comprises three CDRs, designated CDR1, CDR2, and CDR3. Table 1-3, below, lists exemplary position boundaries of CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-H1 located before 68 315736224v1Attorney Docket No: 260525.000077 CDR-H1, FR-H2 located between CDR-H1 and CDR-H2, FR-H3 located between CDR-H2 and CDR-H3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop. An example of alignment according to Kabat, Chothia and IMGT numbering schemes is shown in Figure 4 of Dondelinger M, et al., (2018) Front. Immunol.9:2278, which is incorporated herein by reference in its entirety. Table 1-3. CDRs definitions according to various numbering schemes. CDR Kabat Chothia AbM Contact CDR-H1 H31 - H35B H26 - H32…34 H26 - H35B H30 - H35B

[0000] us, un ess ot erw se spec ed, a C or comp ementarty determ n ng reg on,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) CDR as defined by any of the above-mentioned schemes. For example, where it is stated that a particular CDR (e.g., CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VHH amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the VHH, as defined by any of the above-mentioned schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes (see e.g., Table 1-3), although it is understood that a provided antibody can include CDRs as described according to any of the other above-mentioned numbering schemes or other numbering schemes known to a person of ordinary skill in the art.1Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD2Al-Lazikani et al., (1997) JMB 273, 927-948 69 315736224v1Attorney Docket No: 260525.000077

[0225] In a single-domain antibody sequence of the present disclosure, the framework sequences may be any suitable framework sequences. For example, the framework sequences may be framework sequences derived from a heavy chain variable domain (e.g., a VH sequence or VHH sequence). In some embodiments, the framework sequences are either framework sequences that have been derived from a VHH sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences (in which said framework sequences may optionally have been partially or fully camelized).

[0226] Antigen-binding fragments (or combinations of fragments) of any of single-domain antibodies described herein, such as fragments that contain one or more CDR sequences, suitably flanked by and / or linked via one or more framework sequences, are also encompassed within the present disclosure.

[0227] It should be noted, however, that the present disclosure is not limited to the origin of the single-domain antibody (or of the nucleotide sequence used to express it), nor to the way that the single-domain antibody or nucleotide sequence is generated or obtained. Thus, an antigen- binding protein of the present disclosure may comprise naturally occurring sequences (from a suitable species), recombinant sequences, or synthetic or semi-synthetic sequences. Similarly, nucleotide sequences encoding antigen-binding proteins of the present disclosure may comprise naturally occurring nucleotide sequences, recombinant sequences, or synthetic or semi-synthetic sequences (for example, sequences that are prepared by PCR or isolated from a library).

[0228] Serum albumin-binding proteins (e.g., antibodies such single-domain antibodies) of the present disclosure may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy chain variable domains as compared to the exemplary antibody sequences provided herein. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available from, for example, public antibody sequence databases. The antigen-binding molecules of the present disclosure may comprise antigen-binding domains which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A 70 315736224v1Attorney Docket No: 260525.000077 person of ordinary skill in the art, starting with the heavy chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VHH domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germline sequence from which the antigen-binding domain was originally derived).

[0229] Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germline sequence while certain other residues that differ from the original germline sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen- binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved, or enhanced biological properties (e.g., agonistic effect), reduced immunogenicity, etc. Antigen- binding proteins comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.

[0230] Provided herein are serum albumin-binding proteins comprising variants of any of the VHH and / or CDR amino acid sequences disclosed herein having one or more amino acid substitutions. For example, the present disclosure includes serum albumin-binding proteins having VHH and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2, or 1 amino acid substitutions relative to any of the VHH and / or CDR amino acid sequences set forth in Tables 1-1, 1-2, or 4 herein. Amino acid substitutions may be introduced into an antigen-binding protein of interest and the resultant variants can screened for a desired activity, for example, retained / improved antigen binding, decreased immunogenicity, or reduced ADCC or CDC. 71 315736224v1Attorney Docket No: 260525.000077

[0231] Amino acids may be grouped according to common side-chain properties: (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 influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. In some embodiments, an amino acid substitution is a conservative substitution, meaning exchanging an amino acid with another amino acid of the same class. In some embodiments, amino acid substitutions may also include a non-conservative substitution, meaning exchanging an amino acid with an amino acid of a different class. Other exemplary amino acid substitutions are shown in Table 1-4. Table 1-4. Exemplary amino acid substitutions Original Residue Exemplary Substitutions Ala (A) Val; Leu; Ile

[0232] In some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations to reduce oxidation levels of oxidation-labile residues such as Met (M). In certain embodiments, it may be desirable to address Met (M) oxidation liability by mutation of a Met (M) residue. In some embodiments, the single-domain antibodies (e.g., VHH) of the present disclosure may comprise one or more mutations (e.g., substitution mutations) of a Met residue to reduce oxidation. As a non-limiting example, a Met residue may be substituted in 72 315736224v1Attorney Docket No: 260525.000077 any of the single-domain antibodies described herein with e.g., Ile (I), Ala (A), or Leu (L), to reduce oxidation.

[0233] In some embodiments, the serum albumin-binding proteins described herein may comprise one or more modifications that reduce binding of the serum albumin-binding protein by pre-existing antibodies found in human blood or serum. In some embodiments, the serum albumin- binding proteins described herein may comprise one or more modifications that reduce binding of the serum albumin-binding protein by pre-existing antibodies from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 10% to about 50%, from about 10% to about 60%, from about 10% to about 70%, from about 10% to about 80%, from about 10% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The serum albumin-binding proteins described herein may comprise one or more modifications that reduce binding of the serum albumin-binding protein by pre-existing antibodies by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100%.

[0234] In some embodiments, single-domain antibodies (e.g., VHHs) of the present disclosure are modified by mutation of amino acid position 11, for example Leu11Glu (L11E), Leu11Lys (L11K), or Leu11Val (L11V). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure may comprise a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering). As another example, a single-domain antibody (e.g., VHH) of the present disclosure may comprise an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the single-domain antibody (e.g., VHH). The C-terminus of a VHH is normally VTVSS (SEQ ID NO: 2273). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering). In another embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering). Accordingly, the C-terminus of a single- 73 315736224v1Attorney Docket No: 260525.000077 domain antibody (e.g., VHH) can be any one of VKVSS (SEQ ID NO: 2274), VQVSS (SEQ ID NO: 2275), VTVKS (SEQ ID NO: 2276), VTVQS (SEQ ID NO: 2277), VKVKS (SEQ ID NO: 2278), VKVQS (SEQ ID NO: 2279), VQVKS (SEQ ID NO: 2280), or VQVQS (SEQ ID NO: 2281). In another embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering), optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) and an extension of 1 to 5 (naturally occurring) amino acids, such as a single alanine (A) extension at the C-terminus of the single-domain antibody (e.g., VHH) (such that the C-terminus of the single-domain antibody (e.g., VHH) for example has the sequence VTVSSA (SEQ ID NO: 2282), VKVSSA (SEQ ID NO: 2283), or VQVSSA (SEQ ID NO: 2284)). In further embodiments, single-domain antibodies (e.g., VHH) of the present disclosure are modified by changes in carboxy-terminal region, for example to a terminal sequence having the sequence GQGTLVTVKPGG (SEQ ID NO: 2285) or GQGTLVTVEPGG (SEQ ID NO: 2286) or modification thereof. Additional modification to reduce binding by pre-existing antibodies in human serum can be found in e.g., WO2012 / 175741; WO2015 / 173325; WO2016 / 150845; WO2011 / 003622; WO2013 / 024059; US 11,426,468; US 10,526,397, which are incorporated herein by reference in their entireties.

[0235] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 47) or VPAG (SEQ ID NO: 2331).

[0236] In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VAGG (SEQ ID NO: 47). In one embodiment, a single-domain antibody (e.g., VHH) of the present disclosure comprises at the carboxy-terminus starting from position 111 according to Chothia the amino acid sequence VPAG (SEQ ID NO: 2331).

[0237] Additional carboxy-terminus modifications to introduce to a single-domain antibody (e.g., VHH) of the present disclosure include those described in in e.g., WO2024 / 238790, which is incorporated herein by reference in its entirety.

[0238] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs 25-40 and 1169-1904, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 74 315736224v1Attorney Docket No: 260525.000077 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid sequence at the carboxy-terminus starting from position 111 according to Chothia comprises VAGG (SEQ ID NO: 47) or VPAG (SEQ ID NO: 2331).

[0239] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs: 2381-2384, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0240] In some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure are modified to enhance binding to staphylococcal protein A (SpA) or streptococcal protein G (SpG). Binding of SpA and SpG to antibodies or antibody fragments can be useful in the manufacturing process of the antibodies or antibody fragments. The high-affinity interaction of the IgG Fc region with SpA and SpG has been extensively exploited and became the gold standard for monoclonal antibody purification (Björck and Kronvall, 1984). Other non-Fc containing antibody fragments, such as VHHs and Fabs do not have the capacity to bind to SpA or SpG via their Fc regions. However, sequence-dependent interaction with SpA has been demonstrated for these non- Fc containing antibody fragments(Graille et al., 2000; Henry et al., 2016). This characteristic circumvents potential use of affinity tags fused to the drug candidate for affinity chromatography that have the disadvantage as being regarded as a sequence liability, as it may impact protein immunogenicity as well as protein structure and stability and could compromise functionality. The interaction of the single-domain antibodies (e.g., VHH) to SpA relies on an alternative binding mode, with a 1-5 µM affinity, which is comparable to the 0.2 -3 µM measured for VH-SpA interactions (To et al., JBC, 2005; Henry et al., Plos One, 2016).

[0241] In some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure have, or are modified to have a SpA-binding motif. For example, The VHH-SpA interface has been mapped to thirteen residues, which cluster within the framework at the back side of the V- body, distant to the CDRs (Graille et al., 2000, Henry et al., 2016). In the absence of a VHH-SpA co-structure, superposition of a SpA-Fab crystal structure and a VHH allows for visualizing the binding mode. Based on a structural and functional analysis, the thirteen residues of the VHH-SpA interface have been characterized to be intolerant to substitutions (residues Gly15, Arg19, Tyr59, 75 315736224v1Attorney Docket No: 260525.000077 Gly65, and Arg66), tolerant to specific substitutions (residues Thr / Lys / Arg57, Thr68, Gln81, Asn82a, and Ser82b) or generally tolerant to a variety of substitutions (residues Ser17, Lys64, and Ser70) (all residue positions refer to Kabat numbering) (Henry et al., Plos One, 2016). Thus, a SpA-binding motif included in a single-domain antibody (e.g., VHH) of the present disclosure may include one or more, or all of the thirteen residues.

[0242] In some embodiments, single-domain antibodies (e.g., VHH) of the present disclosure comprise one or more modifications at N-terminus to prevent formation of a pyroglutamate and product heterogeneity. In one embodiment, the amino acid residue Glu at the first position of a described single-domain antibody (e.g., VHH) is replaced with Asp (E1D). In one embodiment, the amino acid residue Gln at the first position of a described single-domain antibody (e.g., VHH) is replaced with Asp (Q1D).

[0243] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID NOs 25-40 and 1169-1904, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, wherein the amino acid residue Glu at the first position of the single-domain antibody (e.g., VHH) is replaced with Asp (E1D).

[0244] In some embodiments, a single-domain antibody (e.g., VHH) of the present disclosure comprises an amino acid sequence selected from any one of SEQ ID Nos 2381-2384, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. Alternative protein scaffolds

[0245] In some embodiments, serum albumin-binding proteins of the present disclosure can adopt an alternative protein scaffold. Such alternative protein scaffold may be a single chain polypeptidic framework, optionally with a reduced size (e.g., less than about 200 amino acids), that contains a highly structured core associated with variable domains of high conformational tolerance allowing insertions, deletions, or other substitutions. Such antigen-binding proteins may be generated by grafting CDRs or variable regions described herein onto a suitable protein 76 315736224v1Attorney Docket No: 260525.000077 scaffold. The structure of alternative scaffolds may vary, but preferably are of human origin for those developed as therapeutics.

[0246] Alternative protein scaffolds of the present disclosure can be based either on a conventional immunoglobulin (Ig) backbone or are derived from a completely unrelated protein. These variable domains can be modified to create novel binding interfaces toward any targeted antigen. In some embodiments, an alternative protein scaffold of the present disclosure can be derived from Protein A, e.g., the Z-domain thereof (affibodies), ImmE7 (immunity proteins), BPTI / APPI (Kunitz domains), Ras-binding protein AF-6 (PDZ-domains), charybdotoxin (Scorpion toxin), CTLA-4, Min-23 (knottins), lipocalins (anticalins), neokarzinostatin, a fibronectin domain (used in “adnectin”), an ankyrin repeat (AR) domain (used in “DARPins”), avidity multimers (also known as “avimers”), or thioredoxin (Skerra, A., Curr. Opin. Biotechnol. 18:295-304 (2005); Hosse et al., Protein Sci.15:14-27 (2006); Nicaise et al., Protein Sci.13:1882- 1891 (2004); Nygren and Uhlen, Curr. Opin. Struc. Biol.7:463-469 (1997), all of which are hereby incorporated by reference in their entirety).

[0247] Anticalins are a suitable type of non-Ig based alternative scaffolds for use in the antigen-binding molecules of the present disclosure. Anticalins are a class of engineered ligand- binding proteins that are based on the lipocalin scaffold. Lipocalins are a family of proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. Lipocalins have limited sequence homology but share a common tertiary structure architecture based on eight antiparallel β-barrels. Lipocalins contain four exposed loops built on the rigid β-barrel structure. Exemplary anticalin proteins that are commonly used are about a size of about 180 amino acids and a mass of about 20 kDa.

[0248] DARPins are another suitable non-Ig based alternative scaffold that can be used in the antigen-binding molecules of the present disclosure. DARPins are genetically engineered antibody mimetic proteins typically exhibiting highly specific and high-affinity target protein binding. They are derived from natural ankyrin repeat (AR) proteins, which usually contain a 33 amino acid protein motif consisting of two α-helices separated by loops, which repeats mediate protein-protein interactions. DARPins can be generated using combinatorial AR libraries constructed based on the 33 amino acid AR motif with seven randomized positions. DARPin libraries can be screened using ribosome display, and library members typically are well produced in Escherichia coli, do not aggregate, and display high thermodynamic stability. Preferably, DARPins contain two to four of 77 315736224v1Attorney Docket No: 260525.000077 these motifs flanked by N- and C-terminal capping motifs to shield hydrophobic regions and allow increased solubility.

[0249] The avimer structure can also be used as a protein backbone to generate a suitable non- Ig based alternative scaffold. Avimers typically consist of two or more peptide sequences of 30 to 35 amino acids each, connected by peptide linker. The individual sequences are derived from A- domains of various membrane receptors and have a rigid structure, stabilized by disulfide bridges and calcium. Each A-domain can bind to a certain epitope of the target protein. The combination of domains binding to different epitopes of the same protein increases affinity to this protein, an effect known as avidity.

[0250] Proteins derived from fibronectin III (FN3) domains can also be used to generate a suitable non-Ig based alternative scaffold (also known as “monobody”). For example, the tenth fibronectin type III domain (FN10) of human fibronectin corresponds to a β-sandwich with seven β-strands and three connecting loops showing structural homologies to Ig domains without disulfide bridges. In some cases, the connecting loops of FN10, each about 15 to 21 amino acids in length, can be randomized and the domains displayed on both phage and yeast to select for a scaffold with the desirable properties. Adnectins™ is an exemplary scaffold generated using 10thFN3 domains randomized and displayed in this way. Another exemplary scaffold comprising FN3 domains is a Centyrin™. Centryrins™ contain the consensus sequence of FN3 domains of human Tenascin C (TNC), which is found in the extracellular matrix of various tissues. Centyrin™ scaffolds have loops that have structural homology to antibody variable domains (i.e., CDR1, CDR2, and CDR3), and are small (about 10 kDa), simple, and highly stable single domain proteins that do not contain cysteine, disulfides, or glycosylated residues. Centyrin™ possess excellent biophysical properties such as stability to heat, pH, denaturant and organic solvents, reversible unfolding and monodispersity. Another recent exemplary FN3-based scaffold that can be used in the present disclosure is fluctuation-regulated affinity proteins (FLAPs), as described in See et al., 2020. Biotechnology Journal 15(12):e2000078, which is incorporated herein by reference in its entirety. Fusion Proteins and Conjugates

[0251] In one aspect, provided herein are fusion proteins and conjugates comprising at least one serum albumin-binding protein (e.g., antibody such as a single-domain antibody) linked, 78 315736224v1Attorney Docket No: 260525.000077 directly or indirectly, to one or more additional domains or moieties. The at least one serum albumin-binding protein can specifically bind serum albumin. In some embodiments, the fusion protein or conjugate of the present disclosure comprises a single polypeptide. In other embodiments, the fusion protein or conjugate of the present disclosure comprises more than one polypeptide. For example, the fusion protein or conjugate of the present disclosure can comprise two polypeptides.

[0252] In some embodiments, the fusion protein or conjugate of the present disclosure comprises at least one serum albumin-binding protein (e.g., antibody such as a single-domain antibody) described herein. In some embodiments, the fusion protein or conjugate is multivalent. For example, the fusion protein or conjugate of the present disclosure may be at least bivalent, but can also be e.g., trivalent, tetravalent, pentavalent, hexavalent, etc. The terms “bivalent”, “trivalent”, “tetravalent”, “pentavalent”, or “hexavalent” all fall under the term “multivalent” and indicate the presence of two, three, four, five, or six binding units (e.g., VHHs), respectively.

[0253] In certain embodiments, the fusion protein or conjugate is multispecific. For example, in some cases, the one or more additional domain or moieties may be one or more additional binding domain that binds to one or more further antigen or protein. The fusion protein or conjugate of the present disclosure may be, for example, bispecific, trispecific, tetraspecific, pentaspecific, etc. The terms “bispecific”, “trispecific”, “tetraspecific”, “pentaspecific”, etc., all fall under the term “multispecific” and refer to binding to two, three, four, five, etc., different target molecules, respectively.

[0254] In some embodiments, a fusion protein or conjugate of the present disclosure comprises one or more of the serum albumin-binding proteins described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises two or more of the serum albumin- binding proteins described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises three or more of the serum albumin-binding proteins described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises four or more of the serum albumin-binding proteins described herein. In some embodiments, a fusion protein or conjugate of the present disclosure comprises one, two, three, four, five, six, seven, eight, nine, or ten, or more of the serum albumin-binding proteins described herein. 79 315736224v1Attorney Docket No: 260525.000077

[0255] In some embodiments, the one or more antigen-binding proteins can bind to the same epitope on serum albumin. In some embodiments, the one or more antigen-binding proteins can bind to different epitopes on serum albumin.

[0256] In various embodiments, the one or more antigen-binding proteins can be one or more single-domain antibodies disclosed herein, for example, one or more VHHs disclosed herein.

[0257] When two or more serum albumin-binding proteins are included in a fusion protein or conjugate, the two or more serum albumin-binding proteins may comprise the same sequence or may comprise different sequences. In such embodiments, the two or more serum albumin-binding proteins may bind to the same epitope on serum albumin or different epitopes on serum albumin. For example, a fusion protein or conjugate of the present disclosure may be biparatopic, e.g., if two VHHs bind two different epitopes on serum albumin. Fusion or Conjugation to Fc regions

[0258] In some embodiments, a fusion protein or conjugate of the present disclosure comprises at least one serum albumin-binding protein (e.g., antibody such as a single-domain antibody) provided herein operably linked to an immunoglobulin Fc region. An immunoglobulin Fc region may be linked indirectly or directly to the at least one serum albumin-binding protein (e.g., antibody such as a single-domain antibody). In some embodiments, a fusion protein or conjugate of the present disclosure comprises one, two, three, four, five, six, or more serum albumin-binding proteins provided herein operably linked to an Fc region.

[0259] In some embodiments, the immunoglobulin Fc region described herein may be linked indirectly or directly to one or more antigen-binding proteins that bind to a different target.

[0260] A “Fc region” as used herein refers to a portion of a heavy chain constant region comprising CH2 and CH3. In some embodiments, an Fc region comprises a hinge, CH2, and CH3. In various embodiments, when an Fc region comprises a hinge, the hinge can mediate dimerization between two Fc-containing polypeptides. An Fc region included in a fusion protein or conjugate may be an Fc region from any species, or derived from any species, including, but not limited to, human, mouse, rat, monkey (e.g., cyno), camel, llama, shark, goat, rabbit, and / or bovine. In various embodiments, an Fc region included in a fusion protein or conjugate of the present disclosure is a human immunoglobulin Fc region or is derived from a human immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is of IgG, IgE, IgM, IgD, IgA, or IgY isotype. In 80 315736224v1Attorney Docket No: 260525.000077 some embodiments, the immunoglobulin Fc region is an IgG isotype, such as IgG1, IgG2, IgG3, or IgG4 subclass. The immunoglobulin Fc region may comprise a variant or fragment of a native IgG Fc region.

[0261] In some embodiments, an Fc region included in a fusion protein or conjugate described herein may be a murine (e.g., a mouse or a rat) immunoglobulin Fc region, or derived from a murine immunoglobulin Fc region. In some embodiments, an Fc region included in a fusion protein or conjugate described herein may be a cyno immunoglobulin Fc region, or derived from a cyno immunoglobulin Fc region.

[0262] A native Fc region typically possesses an effector function, including but not limited to, Fc receptor binding, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (for example B-cell receptor), and B-cell activation, etc. Such effector functions generally require the Fc region to be combined with a binding domain (for example, an antibody variable domain) and can be assessed using various assays.

[0263] In some embodiments, a fusion protein or conjugate of the present disclosure can comprise a dimer of Fc regions. In some embodiments, an Fc region mediates dimerization of the serum albumin-binding units at physiological conditions, such as when expressed from a cell, such that a dimer is formed that doubles the number of serum albumin binding units. For example, a fusion polypeptide comprising one VHH domain that binds serum albumin and an Fc region is monovalent as a monomer, but the Fc region can mediate dimerization; as a result, the fusion protein is bivalent (i.e., having two anti-serum albumin VHH domains per molecule). Similarly, in some embodiments, two anti-serum albumin VHH domains (2x) are fused to an IgG Fc region and as a result of dimerization, the fusion protein is tetravalent (i.e., having four anti-serum albumin VHH domains per molecule). In some embodiments, three anti-serum albumin VHH domain (3×) are fused to an IgG Fc region and as a result of dimerization, the fusion protein is hexavalent (i.e., having six anti-serum albumin VHH domains per molecule).

[0264] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure: (anti- serum albumin VHH)n-Linker-Fc, wherein n can be any integral number (e.g., 1, 2, 3, 4, 5, etc). When n≥2, each anti-serum albumin VHH may be optionally operably linked to another anti-serum albumin VHH via a linker. 81 315736224v1Attorney Docket No: 260525.000077

[0265] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise two polypeptide chains, each polypeptide chain having the following structure: (anti- serum albumin VHH)n-Linker-Fc-(anti-serum albumin VHH)m, wherein n and m can independently be any integral number (e.g., 1, 2, 3, 4, 5, etc). When n≥2 or m≥2, each anti-serum albumin VHH may be optionally operably linked to another anti-serum albumin VHH via a linker.

[0266] In some embodiments, a fusion protein or conjugate of the present disclosure is bivalent. In some embodiments, the bivalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti- serum albumin VHH)-Linker-Fc.

[0267] In some embodiments, a fusion protein or conjugate of the present disclosure is tetravalent. In some embodiments, the tetravalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti- serum albumin VHH)-Linker-(anti-serum albumin VHH)-Linker-Fc. In some embodiments, the tetravalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-serum albumin VHH)-Linker-Fc-Linker- (anti-serum albumin VHH). The multiple linkers used in the fusion protein are not necessarily the same.

[0268] In some embodiments, a fusion protein or conjugate of the disclosure is hexavalent. In some embodiments, the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-serum albumin VHH)-Linker-(anti-serum albumin VHH)-Linker-(anti-serum albumin VHH)-Linker-Fc. In some embodiments, the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-serum albumin VHH)-Linker-(anti-serum albumin VHH)-Linker-Fc-linker-(anti-serum albumin VHH). In some embodiments, the hexavalent fusion protein or conjugate of the disclosure comprises two polypeptide chains, each polypeptide chain having the following structure: (anti-serum albumin VHH)-Linker-Fc-Linker-(anti-serum albumin VHH)-Linker-(anti-serum albumin VHH). The multiple linkers used in the fusion protein are not necessarily the same.

[0269] In some embodiments, the CH3 domain of the Fc region can be used as homodimerization domain, such that the resulting fusion protein may be formed from two identical polypeptides. In other cases, the CH3 dimer interface region of the Fc region can be mutated to 82 315736224v1Attorney Docket No: 260525.000077 enable heterodimerization. For example, a heterodimerization domain can be incorporated into the fusion protein such that the construct is a heterodimeric fusion protein.

[0270] When a dimer of Fc regions is used in a fusion protein or conjugate of the present disclosure, the first and second Fc regions may be of the same IgG isotype such as, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second Fc regions may be of different IgG isotypes such as, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0271] In some embodiments, the Fc region included in a fusion protein or conjugate of the present disclosure can be mutated or modified. In some embodiments, the mutations include one or more amino acid substitutions to reduce an effector function of the Fc region. Various examples of mutations to Fc regions to alter, such as reduce an effector function, are known, including any as described below. In general, the numbering of the residues in an immunoglobulin heavy chain or portion thereof, such as an Fc region, is according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0272] In some embodiments, the human IgG Fc region is modified to alter antibody- dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Non- limiting examples of amino acid modifications that can alter ADCC and / or CDC are described in Alegre et al, 1992 J Immunol, 148: 3461-3468; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Shields et al., 2001 JBC, 276(9): 6591-6604; Lazar et al., 2006 PNAS, 103(11): 4005-4010; Stavenhagen et al., 2007 Cancer Res, 67(18): 8882-8890; Natsume et al., 2008 Cancer Res, 68(10): 3863-72; Stavenhagen et al., 2008 Advan. Enzyme Regul., 48: 152-164; Moore et al., 2010 mAbs, 2(2): 181-189; and Kaneko and Niwa, 2011 Biodrugs, 25(1):1-11, each of which is incorporated herein by reference in its entirety.

[0273] In some embodiments, an Fc region included in a fusion protein or conjugate of the present disclosure exhibits reduced effector functions (such as CDC and ADCC). Various in vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the fusion protein construct and / or cleaved components thereof lack FcγR binding (hence likely lacking ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC are NK cells which express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest 83 315736224v1Attorney Docket No: 260525.000077 are described in e.g., US 5,500,362; US 5,821,337; Hellstrom. et al., Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986); and Hellstrom et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); Bruggemann. et al., J. Exp. Med.166:1351-1361 (1987). Alternatively, non-radioactive assay methods may be employed, such as ACTI™ non-radioactive cytotoxicity assay for flow cytometry or CytoTox96™ non-radioactive cytotoxicity assay. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the fusion protein construct or cleaved components thereof is unable to bind C1q and hence lacks CDC activity (see, e.g., C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402). To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int’l. Immunol.18(12):1759-1769 (2006)).

[0274] Examples of mutations that enhance ADCC include modification at Ser239 and Ile332, for example Ser239Asp and Ile332Glu (S239D, I332E). Examples of mutations that enhance CDC include modifications at Lys326 and Glu333. In some embodiments, the Fc region is modified at one or both of these positions, for example Lys326Ala and / or Glu333Ala (K326A and E333A) using the Kabat numbering system.

[0275] In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce Fc receptor binding: 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), according to EU numbering . In some embodiments, modifications within the Fc region reduce binding to Fc-receptor-gamma receptors (FcγRs) while have minimal impact on binding to the neonatal Fc receptor (FcRn). 84 315736224v1Attorney Docket No: 260525.000077

[0276] In some embodiments, the Fc region of the fusion protein comprises at least one amino acid mutation that reduces or abolishes binding of the Fc region to the neonatal Fc receptor (FcRn). In some embodiments, modifications within the Fc region of the fusion protein reduce its binding to FcRn, while having minimal or no impact on the binding of FcRn to serum albumin. In some embodiments, the Fc region of the fusion protein is altered at one or more of the following positions to reduce FcRn binding: Ile253Ala (I253A), His310Ala (H310A), His435Ala (H435A), and / or His435Gln (H435Q), according to EU numbering. In some embodiments, the Fc region of the fusion protein is altered at one or more positions to reduce FcRn binding as described in for example and without limitation, US8,618,252; US20180251565; WO2024 / 023271; and Immunology, Volume 287, Issue 27, P22927-22937, June 2012; the disclosure of each of which is incorporated herein by reference in its entirety.

[0277] In some embodiments, the human IgG1 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent glycosylation of the fusion protein, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu235 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E) or Leu235Ala (L235A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu234Ala (L234A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Leu234 (Kabat Numbering) to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 234 and 235, e.g., Leu234Ala and Leu235Ala (L234A / L235A) or Leu234Val and Leu235Ala (L234V / L235A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 297, e.g., Leu234Ala, Leu235Ala, Asn297Ala (L234A / L235A / N297A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro239Ala (L234A / L235A / P329A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Asp265 (Kabat Numbering) to alter Fc receptor interactions, e.g., Asp265Ala (D265A). In some embodiments, the Fc region of the fusion protein is modified at amino acid Pro329 (Kabat Numbering) to alter Fc receptor interactions, e.g., Pro329Ala (P329A) or Pro329Gly (P329G). In some embodiments, the Fc region of the fusion protein is altered at both amino acids 265 and 329, e.g., Asp265Ala and Pro329Ala (D265A / P329A) or Asp265Ala and Pro329Gly (D265A / P329G). In some embodiments, the Fc region of the fusion protein is altered 85 315736224v1Attorney Docket No: 260525.000077 at amino acids at 234, 235, and 265, e.g., Leu234Ala, Leu235Ala, Asp265Ala (L234A / L235A / D265A). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Ala, Leu235Ala, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, 265 and 329, e.g., Leu234Ala, Leu235Ala, Asp265Ala, Pro329Gly (L234A / L235A / D265A / P329G).

[0278] In some embodiments, the Fc region of the fusion protein is altered at Gly235 to reduce Fc receptor binding. For example, wherein Gly235 is deleted from the fusion protein. In some embodiments, the human IgG1 Fc region is modified at amino acid Gly236 to enhance the interaction with CD32A, e.g., Gly236Ala (G236A). In some embodiments, the human IgG1 Fc region lacks Lys447 (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[0279] In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Gly, Leu235Ser, Gly236Arg (L234G / L235S / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Thr, Gly236Arg (L234S / L235T / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Ser, Leu235Val, Gly236Arg (L234S / L235V / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Gln, Gly236Arg (L234T / L235Q / G236R). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 234, 235, and 236, e.g., Leu234Thr, Leu235Thr, Gly236Arg (L234T / L235T / G236R). In some embodiments, the Fc region of the fusion protein fusion protein is altered at amino acids at 234, 235, and 329, e.g., Leu234Thr, Leu235Thr, Pro329Gly (L234A / L235A / P329G). In some embodiments, the Fc region of the fusion protein is altered at amino acids at 252, 254, and 256, e.g., Met252Tyr, Ser254Thr, Thr256Glu (M252Y / S254T / T256E).

[0280] In some embodiments, the Fc region of the fusion protein is lacking an amino acid at one or more of the following positions to reduce Fc receptor binding: Glu233 (E233), Leu234 (L234), or Leu235 (L235). In some embodiments, the Fc region of the fusion protein is lacking an amino acid at one or more of the following positions Glu233 (E233), Leu234 (L234), or Leu235 (L235) and is modified at one or more of the Asp265 (D265), Asn297 (N297), or Pro329 (P329) 86 315736224v1Attorney Docket No: 260525.000077 to reduce Fc receptor binding. For example, an Fc region included in a serum albumin binding polypeptide is derived from a human Fc domain, and comprises a three amino acid deletion in the lower hinge corresponding to IgG1 E233, L234, and L235. In some embodiments, such Fc polypeptides do not engage FcγRs and thus are referred to as “effector silent” or “effector null.” For example, Fc deletion of these three amino acids reduces the complement protein C1q binding. In some embodiments, a polypeptide with an Fc region with Fc deletion of these three amino acids retains binding to FcRn and therefore has extended half-life and transcytosis associated with FcRn mediated recycling.

[0281] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234A, L235A (also known as “LALA” variant) (mutations bolded in the sequence below) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2287)

[0282] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234A, L235A, and P329A (also known as “LALAPA” variant) (mutations bolded in the sequence below) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2288)

[0283] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 D265A, N297A, and P329A (also known as “DANAPA” variant) (mutations bolded in the sequence below) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP 87 315736224v1Attorney Docket No: 260525.000077 VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2289)

[0284] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234A, L235A, and G237A (also known as “LALAGA” variant) (mutations bolded in the sequence below) DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2290)

[0285] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234G / L235S / G236R (mutations bolded in the sequence below) DKTHTCPPCPAPEGSRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2291)

[0286] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234S / L235T / G236R (mutations bolded in the sequence below) DKTHTCPPCPAPESTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2292)

[0287] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234S / L235V / G236R (mutations bolded in the sequence below) DKTHTCPPCPAPESVRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS 88 315736224v1Attorney Docket No: 260525.000077 KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2293)

[0288] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234T / L235Q / G236R (mutations bolded in the sequence below) DKTHTCPPCPAPETQRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2294)

[0289] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234T / L235T / G236R (mutations bolded in the sequence below) DKTHTCPPCPAPETTRGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2295)

[0290] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 L234A / L235A / P329G (mutations bolded in the sequence below) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2296)

[0291] In one embodiment, the immunoglobulin Fc region of the multispecific antigen-binding protein is a variant of human IgG1 Fc region, having an amino acid sequence: IgG1 M252Y / S254T / T256E (mutations bolded in the sequence below) 89 315736224v1Attorney Docket No: 260525.000077 DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 2297)

[0292] In some embodiments, the human IgG Fc region is modified to enhance FcRn binding. Examples of Fc mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S254T, T256E, respectively) (Kabat numbering, Dall’Acqua et al 2006, J. Biol Chem Vol. 281(33) 23514-23524), Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159), Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L, respectively) (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest), Asn434Ala (N434A), Asn434Trp (N434W), Thr256Asp, Thr307Gln (T256D / T307Q), Thr256Asp, Thr307Trp (T256D / T307W), Met252Tyr, Thr256Asp (M252Y / T256D), Thr307Gln, Gln311Val, Ala378Val (T307Q / Q311V / A378V), Thr256Asp, His286Asp, Thr307Arg, Gln311Val, Ala378Val (T256D / H286D / T307R / Q311V / A378V), or Leu309Asp, Gln311His, Asn434Ser (L309D / Q311H / N434S) (see, Ko et al., BioDrugs (2021) 35:147–157).

[0293] In some embodiments, the Fc region lacks or has reduced fucose attached to the N- linked glycan-chain at N297. There are numerous ways to prevent fucosylation, including but not limited to production in a FUT8 deficient cell line; addition inhibitors to the mammalian cell culture media, for example Castanospermine; and metabolic engineering of the production cell line.

[0294] In some embodiments, the Fc domain included in a fusion protein or conjugate of the present disclosure is derived from a human Fc domain and comprises mutations M252Y and M428V. In some embodiments, the mutated or modified Fc polypeptide includes the following mutations: M252Y and M428L using the Kabat numbering system. In some embodiments, such mutations enhance binding to FcRn at the acidic pH of the endosome (near 6.5), while losing detectable binding at neutral pH (about 7.2), allowing for enhanced FcRn mediated recycling and extended half-life.

[0295] In some embodiments, the Fc domain included in a fusion protein or conjugate is derived from a human Fc domain and comprises mutations to induce heterodimerization. In some embodiments, such mutations include those referred to as “knob” and “hole” mutations. For 90 315736224v1Attorney Docket No: 260525.000077 example, having an amino acid modification within the CH3 domain at Thr366, which when replaced with a bulkier amino acid, e.g., Try (T366W), is able to preferentially pair with a second CH3 domain having amino acid modifications to less bulky amino acids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala and Val, respectively (T366S / L368A / Y407V). In some embodiments, the “knob” Fc domain comprises the mutation T366W. In some embodiments, the “hole” Fc domain comprises mutations T366S, L368A, and Y407V. Heterodimerization via CH3 modifications can be further stabilized by the introduction of a disulfide bond, for example by changing Ser354 to Cys (S354C) and Y349 to Cys (Y349C) on opposite CH3 domains (Reviewed in Carter, 2001 Journal of Immunological Methods, 248: 7-15). In some embodiments, Fc domains used for heterodimerization comprise additional mutations, such as the mutation S354C on a first member of a heterodimeric Fc pair that forms an asymmetric disulfide with a corresponding mutation Y349C on the second member of a heterodimeric Fc pair. In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A binding while maintaining FcRn binding. In some embodiments, one member of a heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435. In various embodiments, the hole Fc domain comprises the modification H435R or H435K (referred to as “hole-R” in some instances when the modification is H435R), while the knob Fc domain does not. In some instances, the hole-R mutation improves purification of the heterodimer over homodimeric hole Fc domains that may be present.

[0296] In some embodiments, the human IgG Fc region is modified to prevent dimerization. In these embodiments, the fusion proteins of the present disclosure are monomeric. For example, modification at residue Thr366 to a charged residue, e.g., Thr366Lys, Thr366Arg, Thr366Asp, or Thr366Glu (T366K, T366R, T366D, or T366E, respectively), prevents CH3-CH3 dimerization.

[0297] In some embodiments, the immunoglobulin Fc region of the fusion protein is of human IgG3 isotype, or a variant thereof. In one embodiment, the IgG3 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human 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 of Kabat et al 1991). 91 315736224v1Attorney Docket No: 260525.000077

[0298] In some embodiments, the immunoglobulin Fc region of the fusion protein is of human IgG4 isotype, or a variant thereof.

[0299] As a non-limiting example, an immunoglobulin Fc region of human IgG4 isotype may have an amino acid sequence: ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2298)

[0300] In some embodiments, the immunoglobulin Fc region of human IgG4 isotype may comprise the amino acid sequence set forth in SEQ ID NO: 2298, or a similar sequence thereof having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0301] In some embodiments, the human IgG4 Fc region comprises one or more mutations selected from, e.g., Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and / or Pro329Gly (P329G) according to EU numbering.

[0302] In some embodiments, the IgG4 Fc region of the fusion protein is altered at amino acids at 228 and 235, e.g., Ser228Pro, Leu235Glu, or Leu235Ala (S228P / L235E or S228P / L235A). In some embodiments, the IgG4 Fc region of the fusion protein is altered at amino acids at 228, 234 and 235, e.g., Ser228Pro, Phe234Ala, Leu235Glu or Leu235Ala (S228P / F234A / L235E or S228P / F234A / L235A). In some embodiments, the IgG4 Fc region of the fusion protein is altered at amino acids at 228, 235, and 329, e.g., Ser228Pro, Leu235Glu, and P329G (S228P / L235E / P329G).

[0303] In one embodiment, the human IgG4 Fc region is modified at amino acid 235 to alter Fc receptor interactions, e.g., Leu235Glu (L235E). In some embodiments, the human IgG4 Fc region is modified at amino acid Asn297 (Kabat Numbering) to prevent to glycosylation of the antibody, e.g., Asn297Ala (N297A) or Asn297Asp (N297D). In some embodiments, the human IgG4 Fc region lacks Lys447 (EU index of Kabat et al 1991).

[0304] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, L235E (mutations bolded in the sequence below) 92 315736224v1Attorney Docket No: 260525.000077 ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2299)

[0305] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, L235A (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2300)

[0306] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, F234A, L235E (mutations bolded in the sequence below) ESKYGPPCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2301)

[0307] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG4 Fc region, having an amino acid sequence: IgG4 S228P, F234A, L235A (mutations bolded in the sequence below) ESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2302) 93 315736224v1Attorney Docket No: 260525.000077

[0308] In one embodiment, the immunoglobulin Fc region of the fusion protein is a variant of human IgG4 Fc region, having an amino acid sequence: IgG4 P329G, S228P, L235E (mutations bolded in the sequence below) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLGSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 2303)

[0309] Additional IgG4 heavy chain modifications suitable for use in the fusion proteins or conjugates of the present disclosure include those described in Tables 1 and 2 of Dumet et al., mAbs, 11:8, 1341-1350, which is incorporated herein by reference in its entirety.

[0310] In some embodiments, the fusion protein or conjugate contains an immunoglobulin hinge region. In some embodiments, the hinge region serves as a linker to connect one or more serum albumin binding units (e.g., VHHs) to the Fc region. In other embodiments, the fusion protein can comprise a linker in addition to the hinge region to connect the one or more serum albumin binding units (e.g., VHHs) to the Fc region. The hinge region can be selected from any of the human IgG subclasses. For example, the fusion protein may contain a modified IgG1 hinge having the sequence of EPKSSDKTHTCPPC (SEQ ID NO: 2304), wherein the Cys220 that typically forms a disulfide bond with the C-terminal cysteine of the light chain is mutated to serine, e.g., Cys220Ser (C220S). In other embodiments, the fusion protein contains a truncated hinge having a sequence DKTHTCPPC (SEQ ID NO: 2305).

[0311] In some embodiments, the fusion protein or conjugate has a modified hinge from IgG4, which is modified to prevent or reduce strand exchange, e.g., Ser228Pro (S228P), having the sequence ESKYGPPCPPC (SEQ ID NO: 2306).

[0312] In alternative embodiments, a fusion protein or conjugate of the present disclosure may comprise sequences other than an Fc region to achieve multimerization (e.g., dimerization). For example, an amino acid sequence containing at least one cysteine residue may be included to facilitate dimerization of two polypeptides by formation of a disulfide bond between the two polypeptides. In some embodiments, such multimerizing domain may comprise one or more cysteine residues, or a short cysteine-containing peptide. Other multimerizing domains include 94 315736224v1Attorney Docket No: 260525.000077 peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0313] Fc mutations suitable for use in the fusion proteins disclosed herein are also discussed in, e.g., Wilkinson et al., Fc-engineered antibodies with immune effector functions completely abolished. PLoS One. 2021; WO2021234402A2; US 8,969,526; EP3692065B1; and US 7,083,784, each of which is incorporated herein by reference. Fusion or Conjugation to Half-Life Extension Moieties

[0314] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise one or more other moieties which provide the fusion protein or conjugate with further increased in vivo half-life when administered in a mammal, such as a human subject.

[0315] Non-limiting examples of half-life extension moieties suitable for use in the present disclosure include polyethylene glycol (PEG) molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.

[0316] In some embodiments, a fusion protein or conjugate of the present disclosure may comprise another binding moiety that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG. In one embodiment, a fusion protein or conjugate of the present disclosure may comprise another binding moiety that can also bind to human serum albumin. In one embodiment, the binding moiety is a single-domain antibody (e.g., VHH).

[0317] For example and without limitation, other albumin binders that are described in, e.g., WO 2004 / 041865, WO 2006 / 122787, WO2012 / 175400, WO 2012 / 175741, WO2015 / 173325, WO2017 / 080850, WO2017 / 085172, WO2018 / 104444, WO2018 / 134235, WO2018 / 134234, each of which is incorporated herein by reference is its entirety, can be used in the fusion protein or conjugate of the present disclosure. Fusion or Conjugation to Other Moieties

[0318] Serum albumin-binding proteins (e.g., antibodies such as single-domain antibodies) provided herein may be operably linked, directly or indirectly, to a second moiety, such as but not limited to, a detectable label, a drug, a toxin, a radionuclide, an enzyme, an immunomodulatory agent, a cytokine, a cytotoxic agent, a small molecule drug, a chemotherapeutic agent, a therapeutic agent, a diagnostic agent, or a combination thereof. 95 315736224v1Attorney Docket No: 260525.000077

[0319] In some embodiments, a conjugate of the present disclosure comprises a label, which can generate a detectable signal. Such conjugates can be used for research or diagnostic purposes, such as for the in vivo detection of cancer. Preferably, the label is capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope (such as 3H, 14C, 32P, 35S, 123I, 125I, 131I); a fluorescent (fluorophore) or chemiluminescent (chromophore) compound (such as fluorescein isothiocyanate, rhodamine or luciferin); an enzyme (such as β-galactosidase, alkaline phosphatase, or horseradish peroxidase); an imaging agent; or a metal ion. In some embodiments, the label is a radioactive atom for scintigraphic studies, for example 99Tc or 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. Zirconium-89 may also be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011 / 056983).

[0320] Serum albumin-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure may be conjugated to another moiety, such as an epitope tag, e.g., for the purpose of purification or detection. Examples of such molecules that are useful in protein purification include those that present structural epitopes capable of being recognized by a second molecule. This is commonly employed in protein purification by affinity chromatography, in which a molecule is immobilized on a solid support and exposed to a heterogeneous mixture containing a target protein conjugated to a molecule capable of binding the immobilized compound. Non-limiting examples of epitope tag molecules that can be conjugated to serum albumin-binding proteins (e.g., antibodies such as single-domain antibodies) of the present disclosure, e.g., for the purposes of molecular recognition include a poly-histidine tag (His-tag), a myc-tag, human influenza hemagglutinin (HA) tag, a FLAG-tag, maltose-binding protein, glutathione-S-transferase, biotin, and streptavidin. Conjugates containing the epitopes presented by these molecules are capable of being recognized by complementary molecules such as maltose, glutathione, a nickel-containing complex, an anti-FLAG antibody, an anti-myc antibody, an anti- HA antibody, streptavidin, or biotin, respectively. For example, one can purify a serum albumin- binding protein of the present disclosure that has been conjugated to an epitope tag from a complex mixture of other proteins and biomolecules (e.g., DNA, RNA, carbohydrates, phospholipids, etc) by treating the mixture with a solid phase resin containing a complementary molecule that can 96 315736224v1Attorney Docket No: 260525.000077 selectively recognize and bind the epitope tag of the serum albumin antibody or fragment thereof. Examples of solid phase resins include agarose beads, which are compatible with purifications in aqueous solution.

[0321] In some embodiments, a conjugate of the present disclosure may comprise one or more anti-serum albumin VHH domains described herein conjugated to a therapeutic agent, which can be cytotoxic, cytostatic, or otherwise provides some therapeutic benefit. In some embodiments, the cytotoxic agent is a drug, a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate). Such conjugates may be applicable to, e.g., the treatment or prevention of a cancer. In some embodiments, antibody drug conjugates described herein may allow targeted delivery of a drug moiety to a target tissue (e.g., tumors).

[0322] In some embodiments, a conjugate of the present disclosure comprises a toxin. In some embodiments, the toxin includes, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem.13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res.58:2928 (1998); Hinman et al., Cancer Res.53:3336-3342 (1993)). The toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Examples of other therapeutic agents that can be conjugated to a serum albumin-binding protein of the present disclosure are described elsewhere herein.

[0323] In some embodiments, serum albumin-binding proteins (e.g., antibodies such as single- domain antibodies) of the present disclosure may be fused or conjugated to one or more moieties that facilitate delivery to the central nervous system (CNS) / brain. The moiety that can facilitate delivery of a serum albumin-binding protein to the central nervous system (CNS) / brain can be for example, a peptide, a polypeptide, small molecule, a lipid, or a synthetic polymer. Various approaches to deliver single-domain antibodies into the brain are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.

[0324] As a non-limiting example, a serum albumin-binding protein (e.g., antibody such as single-domain antibody) of the present disclosure may be fused or conjugated to a moiety (e.g., an antibody) that binds to the transferrin receptor (TfR) or insulin receptor. The transferrin receptor 97 315736224v1Attorney Docket No: 260525.000077 (TfR) is highly expressed by brain capillary endothelial cells (BCECs) forming the blood-brain barrier (BBB) and has been utilized as a target for brain drug delivery. Monoclonal antibodies binding to the TfR, such as clone Ri7, have been shown to internalize into BCECs in vivo. As another example, a serum albumin-binding protein (e.g., antibody such as single-domain antibody) of the present disclosure may be conjugated to hydrophobic fatty acid moieties, such as C18 fatty acid (stearic acid), C16 fatty acid (palmitic acid) or C8 fatty acid (octanoic acid) moieties; or amphiphilic block copolymer moieties, such as poly(ethylene oxide)-poly(propylene oxide)- poly(ethylene oxide) (pluronics or poloxamers) or poly(2-oxasolines). Various fatty acid moieties and block copolymer moieties that can be utilized for brain delivery of proteins are described in, e.g., Yi and Kabanov, J Drug Target. 2013; 21(10): 940–955, which is incorporated herein by reference in its entirety.

[0325] Example methods for attaching a moiety, such as a label, to a binding protein include those described in Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel and Meares, Elsevier, N.Y. (1983); and Colcher et al., Meth. Enzymol., 121 :802-16 (1986). Additional suitable methods for preparing the conjugates of the present disclosure include those described in, e.g., WO 2009 / 067800, WO 2011 / 133886, and US2014322129, incorporated by reference herein in their entirety.

[0326] In some embodiments, the attachment between a serum albumin-binding protein and a second moiety can be covalent or non-covalent, e.g., via a biotin-streptavidin non-covalent interaction. In some embodiments, a second moiety can be attached to a serum albumin-binding protein using any of various molecular biological or chemical conjugation and linkage methods known in the art and described below. In some embodiments, linkers such as peptide linkers, cleavable linkers, non-cleavable linkers, or linkers that aid in the conjugation reaction, can be used to link, or conjugate a second moiety to a serum albumin-binding protein described herein.

[0327] In some embodiments, a serum albumin-binding protein (e.g., antibody such as single- domain antibody) is conjugated to one or more second moieties, e.g., about 1 to about 20 moieties per molecule, optionally via a linker. In some embodiments, the one or more second moieties can be the same or different. The linker may be composed of one or more linker components. For covalent attachment of an antibody and the second moiety, the linker typically has two reactive functional groups, i.e., bivalency in a reactive sense. Bivalent linker reagents which are useful to 98 315736224v1Attorney Docket No: 260525.000077 attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups have been described in, e.g., Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234-242.

[0328] In some embodiments, a linker used in a conjugate of the present disclosure may include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit”), a alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2- pyridylthio)pentanoate (“SPP”), N-Succinimidyl 4-(N-maleimidomethyl)cyclohexane-I carboxylate (“SMCC”), or N-Succinimidyl(4-iodo-acetyl)aminobenzoate (“STAB”), or a combination thereof.

[0329] In some embodiments, a linker used in a conjugate of the present disclosure may comprise amino acid residues. Exemplary amino acid linker components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine- citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues used in an amino acid linker component may include naturally occurring amino acids, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized in their selectivity for enzymatic cleavage by particular enzymes, for example, a tumor-associated protease, cathepsin B, C and D, or a plasmin protease.

[0330] Conjugates of a serum albumin-binding protein (e.g., antibody such as single-domain antibody) and second moiety (e.g., cytotoxic agent) can be made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl substrate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0331] Conjugates of the present disclosure can be prepared by a variety of methods. For example, the conjugation method may include: (1) reaction of a nucleophilic group of a VHH domain with a bivalent linker reagent, to form VHH-Linker, via a covalent bond, followed by reaction with a drug moiety; or (2) reaction of a nucleophilic group of a drug moiety with a bivalent 99 315736224v1Attorney Docket No: 260525.000077 linker reagent, to form drug-linker, via a covalent bond, followed by reaction with the nucleophilic group of a VHH domain.

[0332] Nucleophilic groups on proteins including antibodies (e.g., VHH domains), include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups (e.g., lysine), (iii) side chain thiol groups (e.g., cysteine), and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Additional nucleophilic groups can be introduced into proteins (e.g., antibodies such as VHH domains) through the reaction of lysines with 2-iminothiolane (Traut’s reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into a protein (e.g., antibody such as a VHH domain) by introducing one, two, three, four, or more cysteine residues.

[0333] Conjugates, such as antibody drug conjugates, may also be produced by modification of an antibody, such as a VHH domain, to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may lead with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid. Such aldehyde can be reacted with a drug moiety or linker nucleophile.

[0334] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBi esters, 100 315736224v1Attorney Docket No: 260525.000077 haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[0335] Alternatively, a fusion protein containing a VHH domain and cytotoxic agent may be made, e.g., by recombinant DNA techniques or peptide synthesis. A DNA sequence may be engineered to comprise respective regions encoding the two portions of the fusion protein either adjacent to one another or separated by a region encoding a linker peptide which does not impair the desired properties of the fusion protein. The DNA sequence can be then transfected into a host cell that expresses the fusion protein. The fusion protein can be recovered from the cell culture and purified using techniques known in the art. Linkers

[0336] In some embodiments, the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via peptide linkers. A peptide linker can range from 2 amino acids to 60 or more amino acids, and in certain aspects a peptide linker ranges from 3 amino acids to 50 amino acids, from 4 to 30 amino acids, from 5 to 25 amino acids, from 10 to 25 amino acids, 10 amino acids to 60 amino acids, from 12 amino acids to 20 amino acids, from 20 amino acids to 50 amino acids, or from 25 amino acids to 35 amino acids in length.

[0337] In some embodiments, a peptide linker, e.g., a peptide linker separating two VHH domains or an VHH domain and a heavy chain constant region, is at least 5 amino acids, at least 6 amino acids or at least 7 amino acids in length and optionally is up to 30 amino acids, up to 40 amino acids, up to 50 amino acids, or up to 60 amino acids in length.

[0338] In some embodiments, the linker ranges from 5 amino acids to 50 amino acids in length, e.g., ranges from 5 to 50, from 5 to 45, from 5 to 40, from 5 to 35, from 5 to 30, from 5 to 25, or from 5 to 20 amino acids in length. In other embodiments of the foregoing, the linker ranges from 6 amino acids to 50 amino acids in length, e.g., ranges from 6 to 50, from 6 to 45, from 6 to 40, from 6 to 35, from 6 to 30, from 6 to 25, or from 6 to 20 amino acids in length. In yet other embodiments of the foregoing, the linker ranges from 7 amino acids to 50 amino acids in length, e.g., ranges from 7 to 50, from 7 to 45, from 7 to 40, from 7 to 35, from 7 to 30, from 7 to 25, or from 7 to 20 amino acids in length.

[0339] In some embodiments, charged (e.g., charged hydrophilic linkers) and / or flexible linkers are used. Examples of flexible linkers that can be used in the fusion proteins of the 101 315736224v1Attorney Docket No: 260525.000077 disclosure include those disclosed by Chen et ai, 2013, Adv Drug Deliv Rev.65(10): 1357-1369 and Klein et a / ., 2014, Protein Engineering, Design & Selection 27(10): 325-330. Particularly useful flexible linkers are or comprise repeats of glycines and serines (termed “GS-linker” herein), e.g., a monomer or multimer of GnS (SEQ ID NO: 2307) or SGn (SEQ ID NO: 2308), where n is an integer from 1 to 10, e.g., 12, 3, 4, 5, 6, or 7, 8, 9, or 10. In one embodiment, the linker is or comprises a monomer or multimer of repeat of G4S (SEQ ID NO: 2309), e.g., (GGGGS)n (SEQ ID NO: 2310).

[0340] Polyglycine linkers can suitably be used in the fusion proteins of the disclosure. In some embodiments, a peptide linker used herein comprises two consecutive glycines (2Gly), three consecutive glycines (3Gly), four consecutive glycines (4Gly) (SEQ ID NO: 2311), five consecutive glycines (5Gly) (SEQ ID NO: 2312), six consecutive glycines (6Gly) (SEQ ID NO: 2313), seven consecutive glycines (7Gly) (SEQ ID NO: 2314), eight consecutive glycines (8Gly) (SEQ ID NO: 2315), or nine consecutive glycines (9Gly) (SEQ ID NO: 2316).

[0341] In some embodiments, a GS-linker used herein comprises an amino acid sequence selected from GGSGGS, i.e., (GGS)2 (SEQ ID NO: 2317); GGSGGSGGS, i.e., (GGS)3(SEQ ID NO: 2318); GGSGGSGGSGGS, i.e., (GGS)4(SEQ ID NO: 2319); and GGSGGSGGSGGSGGS, i.e., (GGS)5(SEQ ID NO: 2320). In some embodiments, the fusion proteins can include a combination of a GS-linker and a glycine linker.

[0342] In one embodiment, two or more VHHs are linked via a GGGGSGGGGSGGGGS (SEQ ID NO: 2321) linker. In one embodiment, two or more VHHs are linked via a GGGGSGGGGS (SEQ ID NO: 2322) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGSESKYGPPCPSCP (SEQ ID NO: 2323) linker. In one embodiment, a VHH and an Fc region are linked via a GGGGS (SEQ ID NO: 2309) linker.

[0343] In some embodiments, the one or more polypeptides of the fusion proteins of the present disclosure are operably linked via a “rigid” peptide linker. Such peptidic linker may comprise a proline-rich peptide. In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAP (SEQ ID NO: 2325). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 2326). In one embodiment, a rigid peptide linker comprises PAPAPAPAPAPAPAPAPGGGGS (SEQ ID NO: 2327). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGGS (SEQ 102 315736224v1Attorney Docket No: 260525.000077 ID NO: 2328). In one embodiment, a rigid peptide linker comprises GGGGSPAPAPAPAPAPAPAPAPGGGS (SEQ ID NO: 2329).

[0344] In one embodiment, a rigid peptide linker comprises A(EAAAK)nA (SEQ ID NO: 2330), where n is any integer, e.g., 12, 3, 4, 5, 6, 7, 8, 9, or 10.

[0345] Other exemplary peptide linkers that can be used in the fusion proteins described herein are shown in Table 2. Table 2. Exemplary Peptide Linker Sequences. Linker Amino acid sequence SEQ ID NO G4S GGGGS 2309103 315736224v1Attorney Docket No: 260525.000077 Linker Amino acid sequence SEQ ID NO Linker 32 IEGRMD 2361[ ] s o e un ers oo a n em o men s w ere e us on pro e ns escr e erein contain non-humanized VHH amino acid sequences, such non-humanized VHH amino acid sequences can be replaced with any of the humanized VHH amino acid sequences described herein (e.g., in Tables 1-1 and 1-2).

[0347] In some embodiments, the fusion protein described herein may further comprise a signal sequence at its N-terminus. Signal sequences may be present in the precursor molecule of the fusion protein and may be removed after the protein is secreted from the host cell during production. In some embodiments, the signal sequence is MAVMAPRTLVLLLSGALALTQTWA (SEQ ID NO: 2375) or a fragment or variant thereof. In some embodiments, the signal sequence is MYRMQLLSCIALSLALVTNS (SEQ ID NO: 2376), or a fragment or variant thereof. Polynucleotide Molecules

[0348] In another aspect, provided herein are polynucleotide molecules encoding the serum albumin-binding proteins (e.g., antibodies including single-domain antibodies) or fusion proteins described herein. Polynucleotide molecules encoding polypeptide portion(s) of a conjugate of the present disclosure are also encompassed within the present disclosure. 104 315736224v1Attorney Docket No: 260525.000077

[0349] In some embodiments, a polynucleotide molecule of the present disclosure encodes an anti-serum albumin VHH amino acid sequence selected from SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, and 1169-1536, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0350] In some embodiments, a polynucleotide molecule of the present disclosure encoding an anti-serum albumin VHH comprises the nucleotide sequence of any one of SEQ ID NOs: 1905- 2272, or a similar sequence thereof having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0351] In some embodiments, a polynucleotide molecule of the present disclosure encoding an anti-serum albumin VHH comprises the nucleotide sequence of any one of SEQ ID NOs: 1905, 1931, 1960, 2049, 2110, 2152, 2156, 2157, 2185, 2221, 2229, 2230, or a nucleotide sequence having at least 70% identity thereto.

[0352] In an embodiment provided herein, a polynucleotide molecule of the present disclosure encodes a humanized VHH amino acid sequence selected from SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, and 1537-1904, or a similar sequence thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

[0353] A polynucleotide molecule may be used to transform / transfect a host cell or host organism, e.g., for expression and / or production of a polypeptide. Suitable hosts or host cells for production of an anti-serum albumin polypeptides described herein include any suitable fungal, prokaryotic, or eukaryotic cell or cell line or any suitable fungal, prokaryotic, or eukaryotic organism. A host or host cell comprising a polynucleotide molecule encoding a serum albumin- binding protein or fusion protein described herein is also encompassed by the present disclosure.

[0354] A polynucleotide molecule may be for example DNA, RNA, or a hybrid thereof, and may also comprise (e.g., chemically) modified nucleotides, like locked nucleic acids (LNA) or peptide nucleic acids (PNA). In some embodiments, the polynucleotide is single-stranded. In some embodiments, the polynucleotide is double-stranded. In one embodiment, the polynucleotide is in the form of double-stranded DNA (e.g., plasmid). In some embodiments, the polynucleotide is in the form of a single-stranded RNA (e.g., mRNA). 105 315736224v1Attorney Docket No: 260525.000077

[0355] Techniques for generating polynucleotides may include, for example but not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and / or regions that may easily be digested and / or ligated using suitable restriction enzymes), and / or the introduction of mutations by means of a PCR reaction using one or more “mismatched” primers. Alternatively, polynucleotides of the present disclosure may be isolated from a suitable natural source. Polynucleotide sequences encoding naturally occurring (poly)peptides can for example be subjected to site-directed mutagenesis, to generate a polynucleotide molecule encoding polypeptide with sequence variation. Vectors

[0356] Also provided herein are vectors comprising the polynucleotide molecules encoding the serum albumin-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or other relevant polypeptides of the present disclosure. A “vector” as used herein is a vehicle suitable for carrying genetic material into a host cell. A vector can include a nucleic acid vector, such as a plasmid or mRNA, or nucleic acids embedded into a bigger structure, such as a liposome or viral vector.

[0357] A vector can include one or more of the following elements: an origin of replication, one or more regulatory sequences (e.g., promoters, enhancers, terminators) that regulate the expression of a polypeptide of interest, and / or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, β-galactosidase). For DNA-based vectors, this usually includes the presence of elements for transcription (e.g., a promoter and a polyA signal) and translation (e.g., Kozak sequence). In some embodiments, the vector is an expression vector, i.e. a vector suitable for expressing an encoded polypeptide or construct under suitable conditions in a host cell.

[0358] To express a serum albumin-binding protein or fusion protein (or fragments thereof) of the present disclosure, polynucleotides encoding partial or full-length polypeptide chains, e.g., obtained as described above (e.g., VHH, VHH-Fc), can be inserted into expression vectors such that the genes are operatively linked to one or more transcriptional and translational control sequences. The expression vector and expression control sequences are chosen to be compatible 106 315736224v1Attorney Docket No: 260525.000077 with the expression host cell used. Polynucleotides encoding the two or more polypeptide chains (when present and differ from one another) of a serum albumin-binding protein or fusion protein of the present disclosure can be inserted into separate vectors, or, optionally, incorporated into the same expression vector.

[0359] In addition to polynucleotides encoding the polypeptide chain(s) of a serum albumin- binding protein or fusion protein, the recombinant expression vectors of the invention may include regulatory sequences that control the expression of genes encoding the polypeptide chain(s) in a host cell. The design of the expression vector, including the selection of regulatory sequences, may depend on the choice of the host cell to be transformed and / or the desired level of protein expression. For example, suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. Additional examples of viral regulatory elements, and sequences thereof, include those described in, e.g., U.S. Pat. Nos.5, 168,062; 4,510,245; and 4,968,615; the disclosures of each of which are incorporated herein by reference.

[0360] Recombinant expression vectors of the present disclosure may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. A selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., US4,399,216; US 4,634,665; and US 5,179,017; the disclosure of each of which is incorporated herein by reference in its entirety). For example, typically the selectable marker gene confers resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin, or cytotoxic drugs, such as G418, puromycin, blasticidin, hygromycin or methotrexate, to a host cell into which the vector has been introduced. Suitable selectable marker genes can include the dihydrofolate reductase (DHFR) gene (for use in DHFR deficient host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0361] Vectors of the present disclosure may further include sequence elements that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5’ and 3’ untranslated 107 315736224v1Attorney Docket No: 260525.000077 regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.

[0362] Viral vectors can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral vectors are particularly useful for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents to induce gene integration. Examples of suitable viral vectors include a retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses (AAV) such as AAV2, AAV8, AAV9), negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpes virus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), baculovirus, coronavirus, and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding polypeptides of the present disclosure include, for example Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include, but are not limited to, avian leukosissarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M.1996. Fundamental Virology, DMKDN Fields, PM Howley, ed. (Philadelphia, Lippincott-Raven Publishers): 763-843., the disclosure of which is incorporated herein by reference). Other examples of viral genomes useful in the compositions and methods of the present disclosure include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline sarcoma virus, feline leukemia virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentiviruses. Host Cells

[0363] In one aspect, the present disclosure also provides host cells or host organisms that comprise the polynucleotides or vectors encoding the serum albumin-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or other relevant polypeptides 108 315736224v1Attorney Docket No: 260525.000077 described herein. Suitable host cells or host organisms can be any suitable fungal, prokaryotic, or eukaryotic cell or cell line or any suitable fungal, prokaryotic, or eukaryotic organism. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells can also include cells transfected in vivo with a polynucleotide(s) or vector provided herein.

[0364] Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris); plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER.C6®cells (Crucell), COS cells, SP2 / 0 cells, and 293 and CHO cells, and their derivatives, such as 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. Exemplary prokaryotic cells include bacterial cells such as Escherichia coli. Preparation Methods

[0365] The present disclosure also provides methods of producing the serum albumin-binding proteins (e.g., antibodies including single-domain antibodies), fusion proteins, or conjugates described herein.

[0366] In some embodiments, a method may comprise transforming / transfecting a host cell or host organism with a polynucleotide encoding a serum albumin-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or other relevant polypeptide(s) described herein, expressing the serum albumin-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or other relevant polypeptide(s) in the host, optionally followed by one or more isolation and / or purification steps.

[0367] When recombinant expression vectors encoding one or more polypeptide(s) of an anti- serum albumin antigen binding protein (e.g., antibody such as single-domain antibody), fusion protein, or conjugate of the present disclosure are introduced into mammalian host cells, the host cells are cultured for a period of time sufficient to allow for expression of the protein(s) or polypeptide(s) in the host cells or secretion of the protein(s) or polypeptide(s) into the culture medium in which the host cells are grown. Protein(s) or polypeptide(s) can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as VHH domains. 109 315736224v1Attorney Docket No: 260525.000077

[0368] Once a protein or polypeptide of the present disclosure has been produced by recombinant expression, it can be purified by any method known in the art for purification of a protein or polypeptide, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for serum albumin after Protein A or Protein G selection, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the proteins or polypeptides of the present disclosure can be fused to heterologous polypeptide sequences described herein (e.g., His-tag) or otherwise known in the art to facilitate purification or to produce therapeutic conjugates below). Once isolated, a protein or polypeptide of the present disclosure can, if desired, be further purified, e.g., by high performance liquid chromatography, or by gel filtration chromatography, such as on a Superdex™ column. Compositions and Formulations

[0369] The present disclosure also provides a composition comprising serum albumin-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or conjugate of the present technology, at least one polynucleotide molecule encoding the same, at least one vector comprising such a polynucleotide molecule, or at least one host cell comprising the polynucleotide molecule or vector. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent, or excipient and / or adjuvant, and optionally comprise one or more further pharmaceutically active polypeptides and / or compounds.

[0370] As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions.

[0371] Examples of suitable formulations include, but are not limited to, solutions, suspensions, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) 110 315736224v1Attorney Docket No: 260525.000077 containing vesicles (such as LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations” PDA (1998) J Phdomain Sci Technol 52:238-311.

[0372] A pharmaceutical composition of the present disclosure may be formulated according to its intended route of administration. Examples of suitable routes of administration include, e.g., intravenous, subcutaneous, intratumoral, oral (e.g., buccal, sublingual), intranasal, inhalation, intraocular, intramuscular, intradermal, transdermal (i.e., topical), intraperitoneal, transmucosal, vaginal, and rectal administration, or injection to the CNS / brain (e.g., intraspinal, intracerebral, or intrathecal administration). Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; fixed oils; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as phosphates, acetates, or citrates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of plastic or glass.

[0373] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include, for example, physiological saline, bacteriostatic water, Cremophor EL®, or phosphate buffered saline (PBS). The composition is preferably sterile and has a proper fluidity. In most embodiments, the composition is stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the contamination by microorganisms can be achieved by the 111 315736224v1Attorney Docket No: 260525.000077 inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0374] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and / or freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0375] Oral compositions may include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, capsules, or liquid forms. Formulation in tablet and liquid forms may be used for protease insensitive VHHs. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0376] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. 112 315736224v1Attorney Docket No: 260525.000077

[0377] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0378] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0379] For brain delivery, compounds of the present disclosure may be formulated to facilitate crossing of the blood-brain barrier. For example, serum albumin-binding proteins (e.g., antibody such as single-domain antibody), fusion proteins, or conjugates of the present disclosure may be encapsulated into brain targeted liposomes, lipid nanoparticles, lipid microparticles, or lipid microcapsules for brain delivery. Example liposomes delivery systems are described in Pothin et al., Pharmaceutics 2020, 12(10), 937, which is incorporated herein by reference in its entirety.

[0380] In some embodiments, the active compounds are prepared with carriers that can protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US 4,522,811, which is incorporated herein by reference in its entirety.

[0381] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure is dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. 113 315736224v1Attorney Docket No: 260525.000077

[0382] The pharmaceutical compositions (or components thereof) can be included in a kit, container, pack, or dispenser together with instructions for administration. These pharmaceutical compositions can be included in diagnostic kits with instructions for use.

[0383] Pharmaceutical compositions are administered in an amount effective for treatment or prophylaxis of the specific indication. The therapeutically effective amount is typically dependent on the weight of the subject being treated, the physical or health condition of the subject, the extensiveness of the condition to be treated, or the age of the subject being treated. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 50 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 100 μg / kg body weight to about 50 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. Depending on the severity of the condition, the frequency and the duration of the treatment can be adjusted. Effective dosages and schedules for administering a pharmaceutical composition of the present disclosure may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Phdomainaceut. Res.8:1351).

[0384] In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 200 mg per dose.

[0385] In some embodiments wherein the antigen-binding proteins of the present disclosure are administered as a viral vector (e.g., an AAV), dose ranges and frequency of administration of the viral vector described herein can vary depending on the nature of the viral vector, and the medical condition, as well as parameters of a specific patient and the route of administration used. 114 315736224v1Attorney Docket No: 260525.000077 In some embodiments, viral vector compositions can be administered to a subject at a dose ranging from about 1×105plaque forming units (pfu) to about 1×1015pfu, depending on mode of administration, the route of administration, the nature of the disease and condition of the subject. In some cases, the viral vector compositions can be administered at a dose ranging from about 1×108pfu to about 1×1015pfu, or from about 1×1010pfu to about 1×1015pfu, or from about 1×108pfu to about 1×1012pfu. A more accurate dose can also depend on the subject in which it is being administered. For example, a lower dose may be required if the subject is juvenile, and a higher dose may be required if the subject is an adult human subject. In certain embodiments, a more accurate dose can depend on the weight of the subject. In certain embodiments, for example, a juvenile human subject can receive from about 1×108pfu to about 1×1010pfu, while an adult human subject can receive a dose from about 1×1010pfu to about 1×1012pfu.

[0386] Various delivery systems are known and can be used to administer the pharmaceutical composition of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor mediated endocytosis (see, e.g., Wu et al., 1987, J. Biol. Chem.262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intraocular, epidural, intraspinal, intracerebral, intrathecal, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.

[0387] A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical 115 315736224v1Attorney Docket No: 260525.000077 composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0388] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a controlled release system can be placed in proximity of the composition’s target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol.2, pp.115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0389] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, intramuscular, intratumoral, intraperitoneal, intraspinal, intracerebral, and intrathecal injections, drip infusions, etc. In one embodiment, the injectable preparations may be prepared, e.g., by dissolving, suspending, or emulsifying the antibody or its salt described above in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0390] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antigen-binding proteins described herein may be about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, the antigen-binding proteins described herein may be contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms. 116 315736224v1Attorney Docket No: 260525.000077

[0391] The pharmaceutical composition may be administered as needed to a subject. In some embodiments, an effective dose of the pharmaceutical composition is administered to a subject one or more times. In various embodiments, an effective dose of the pharmaceutical composition is administered to the subject once a month, less than once a month, such as, for example, every two months, every three months, or every six months. In other embodiments, an effective dose of the pharmaceutical composition is administered more than once a month, such as, for example, every two weeks, every week, twice per week, three times per week, daily, or multiple times per day. An effective dose of the pharmaceutical composition is administered to the subject at least once. In some embodiments, the effective dose of the pharmaceutical composition may be administered multiple times, including for periods of at least a month, at least six months, or at least a year. In some embodiments, the pharmaceutical composition is administered to a subject as needed to alleviate one or more symptoms of a condition.

[0392] Pharmaceutical compositions of the present disclosure may optionally include more than one active agent. For example, compositions of the present disclosure may contain a serum albumin-binding protein conjugated to, admixed with, or administered separately from another pharmaceutically active molecule to treat a disease or disorder. Methods of Use and Treatment Methods

[0393] In one aspect, provided herein is a method of using serum albumin-binding proteins, fusion proteins, and / or conjugates of the present disclosure to increase the serum half-life of a biologic molecule in a subject. In some embodiments, the method for increasing the serum half- life of a biological molecule comprises attaching a serum albumin-binding protein, fusion protein, and / or conjugate of the present disclosure to the biological molecule, and optionally, administering said biological molecule to the subject.

[0394] In some embodiments, the biological molecule is a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, or combination thereof. In some embodiments, the biological molecule when administered, binds to a target molecule in a subject.

[0395] In another aspect, provided herein is a method of removing a target molecule from serum of a subject, comprising administering the antigen-binding protein, fusion protein, and / or conjugate of the present disclosure to the subject, wherein the antigen-binding protein, fusion 117 315736224v1Attorney Docket No: 260525.000077 protein and / or conjugate is fused or conjugated to a biological molecule that binds to the target molecule.

[0396] The half-life of a biological molecule or serum half-life of a biological molecule can be calculated as the time taken for the concentration of a biological molecule to reduce to half of maximum concentration (Cmax) in the blood. An increase in half-life can be an increase in the area under the curve (AUC), t1 / 2-alpha, t1 / 2-beta, or a combination thereof.

[0397] In some embodiments, the serum half-life of a biologic molecule that is attached to a serum albumin-binding protein, the fusion protein, and / or the conjugate of the present disclosure is increased by from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 10% to about 50%, from about 10% to about 60%, from about 10% to about 70%, from about 10% to about 80%, from about 10% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%, as compared to the serum half-life of a biological molecule not attached to a serum albumin-binding protein, the fusion protein, and / or the conjugate of the present disclosure. The serum half-life of a biologic molecule that is attached to a serum albumin-binding protein, the fusion protein, and / or the conjugate described herein can be increased by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100%

[0398] In one aspect, provided herein is a method of using serum albumin-binding proteins, fusion proteins, and / or conjugates of the present disclosure to reduce the clearance of a biologic molecule in a subject. In some embodiments, the method for reducing the clearance of a biological molecule comprising attaching the serum albumin-binding protein, fusion protein, and / or conjugate to the biological molecule and optionally, administering said biological molecule to the subject. In some embodiments, the method for reducing the clearance of a biologic molecule in a subject comprising administrating the serum albumin-binding protein, fusion protein, and / or conjugate described herein and the biological molecule to the subject. In some embodiments, the 118 315736224v1Attorney Docket No: 260525.000077 serum albumin-binding protein, fusion protein, and / or conjugate and the biological molecule are administered simultaneously or in a sequential manner. In some embodiments, the serum albumin- binding protein, fusion protein, and / or conjugate is administered after the administration of the biological molecule to the subject.

[0399] In some embodiments, the clearance of a biologic molecule that is attached to a serum albumin-binding protein, the fusion protein, and / or the conjugate of the present disclosure is reduced by from about 10% to about 20%, from about 10% to about 30%, from about 10% to about 40%, from about 10% to about 50%, from about 10% to about 60%, from about 10% to about 70%, from about 10% to about 80%, from about 10% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99% as compared to the clearance of a biological molecule not attached to a serum albumin- binding protein, the fusion protein, and / or the conjugate of the present disclosure. In some embodiments, the clearance of a biologic molecule that is attached to a serum albumin-binding protein, the fusion protein, and / or the conjugate described herein can be reduced by about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100%.

[0400] In one aspect, the serum albumin-binding protein, fusion protein, and / or conjugate improves pharmacokinetic properties of the biological molecule. The improvements in pharmacokinetic properties may include an increase of the serum half-life, a reduction of the rate of the clearance or and an enhancement of the desired effect of the biological molecule.

[0401] In some embodiments, the subject may be a mammal (e.g., a human).

[0402] In some embodiments, serum albumin-binding proteins of the present disclosure are capable of interacting with and promoting / inhibiting signal transduction events mediated by serum albumin. Serum albumin-binding proteins of the present disclosure may be able to induce conformational changes within serum albumin. 119 315736224v1Attorney Docket No: 260525.000077

[0403] In another aspect, serum albumin-binding proteins (e.g., antibody such as single- domain antibody), fusion proteins, conjugates, polynucleotide molecules, vectors, and / or host cells described herein, or pharmaceutical compositions thereof, are useful for the (prophylactic or therapeutic) treatment of a wide array of diseases or disorders, wherein the serum albumin-binding protein is fused or conjugated to a biological molecule. Accordingly, the present technology provides the described antigen-binding proteins (e.g., antibody such as single-domain antibody), fusion proteins, conjugates, polynucleotide molecules, vectors, or host cells for use as a medicament. Also provided is a (prophylactic and / or therapeutic) method of treating a disease or disorder, comprising administering to a subject in need thereof a pharmaceutically active amount of a serum albumin-binding protein (e.g., antibody such as single-domain antibody), a fusion protein, a conjugate, a polynucleotide molecule, a vector, or a host cell described herein, wherein the antigen-binding protein is fused or conjugated to the biological molecule.

[0404] In one aspect, provided herein is a method of delivering therapeutic or imaging agent(s) to a tumor in a subject, wherein the therapeutic or imaging agent(s) are conjugated to a serum albumin-binding protein (e.g., antibody such as single-domain antibody), fusion protein, or conjugate, described herein. HSA has the ability to accumulate and retain in tumors due to the enhanced permeability and retention (EPR) effect (see, e.g., Hilbrig et al., Cancers 2023, 15, 1126 and Li et al., Nanotheranostics 2017; 1(4): 346-357, both of which are incorporated herein by reference in their entirety). By taking advantage of this effect, the serum albumin-binding proteins (e.g., antibody such as single-domain antibody), fusion proteins, or conjugates described herein could be used to concentrate a therapeutic or imaging agent in the tumor micro-environment. In some embodiments, the method of delivering therapeutic agent(s) described herein is used to treat and / or prevent a cancer, or for cancer imaging.

[0405] In some embodiments, the method of delivering therapeutic or imaging agent(s) to a tumor in a subject comprises administering to the subject the therapeutic or imaging agent(s) which are conjugated to a serum albumin-binding protein (e.g., antibody such as single-domain antibody), fusion protein, conjugate described herein. In some embodiments, the therapeutic or imaging agents conjugated serum albumin-binding proteins (e.g., antibody such as single-domain antibody), fusion proteins, conjugates described herein are formulated in the form of nanoparticles. In some embodiments, the therapeutic agent is a cytotoxic agent, cytostatic agent, or otherwise provide a therapeutic benefit. In some embodiments, the cytotoxic agent is a drug, a 120 315736224v1Attorney Docket No: 260525.000077 chemotherapeutic agent, a growth inhibitory agent, a toxin, or a radioactive isotope. In some embodiments, the toxin is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof. Examples of other toxins that can be conjugated to a serum albumin-binding protein of the present disclosure are described elsewhere herein.

[0406] Examples of chemotherapeutic agents include, but are not limited to, bleomycin, carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide, daunorubicin, doxorubicin or liposomal doxorubicin, mitomycin C, actinomycin, diethylstilbestrol, daunorubicin, etoposide, 5- fluorouracil, floxuridine, melphalan, methotrexate, mitomycin, 6-mercaptopurine, teniposide, 6- thioguanine, vincristine and vinblastine, leflunomide, actinomycin, tamoxifen, interferon α-2b, glutamic acid, plicamycin, 6-mercaptopurine, 6-thioguanine, carmustine, BCNU, limousine, CCNU, cytosine arabinoside, estramustine, hydroxyurea, procarbazine, busulfan, medroxyprogesterone, estramustine phosphate sodium, ethenyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, testolactone, melphalan, chlorambucil, mechlorethamine, thiourea, betamethasone sodium phosphate, dicarbazine, asparagine, mitotane, vincristine sulfate, vinblastine sulfate, FOLFOX (folinic acid, 5-fluorouracil and oxaliplatin) or FOLFIRI (folinic acid, 5-fluorouracil, and irinotecan), and a combination thereof.

[0407] In some embodiments, the tumor can be a solid tumor. In some embodiments, the tumor is adenoid cystic carcinoma, adrenal gland tumor, amyloidosis, anal cancer, appendix cancer, astrocytoma, ataxia-telangiectasia, Beckwith-Wiedemann syndrome, bile duct cancer (cholangiocarcinoma), Birt-Hogg-Dubé syndrome, bladder cancer, bone cancer (sarcoma of bone), brain stem glioma, brain tumor, breast cancer, inflammatory breast cancer, metastatic breast cancer, male breast cancer, Carney complex, central nervous system tumors (brain and spinal cord), cervical cancer, childhood cancer, colorectal cancer, Cowden syndrome, craniopharyngioma, desmoid tumor, desmoplastic infantile ganglioglioma, childhood tumor, ependymoma, esophageal cancer, Ewing sarcoma, eye cancer, eyelid cancer, familial adenomatous polyposis, gastrointestinal stromal tumor (GIST), familial GIST, familial malignant melanoma, familial pancreatic cancer, gallbladder cancer, germ cell tumor, gestational trophoblastic disease, head and neck cancer, hereditary breast and ovarian cancer, hereditary diffuse gastric cancer, hereditary leiomyomatosis and renal cell cancer, hereditary mixed polyposis syndrome, hereditary pancreatitis, hereditary papillary renal carcinoma, HIV / AIDS-related cancer, juvenile polyposis 121 315736224v1Attorney Docket No: 260525.000077 syndrome, kidney cancer, lacrimal gland tumor, laryngeal and hypopharyngeal cancer, Li- Fraumeni syndrome, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lynch syndrome, mastocytosis, medulloblastoma, melanoma, meningioma, mesothelioma, multiple endocrine neoplasia type 1, multiple endocrine neoplasia type 2, multiple myeloma, MUTYH (or MYH)-associated polyposis, myelodysplastic syndromes (MDS), nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine tumor of the gastrointestinal tract, neuroendocrine tumor of the lung, neuroendocrine tumor of the pancreas, neuroendocrine tumors, neurofibromatosis type 1, neurofibromatosis type 2, nevoid basal cell carcinoma syndrome, oral and oropharyngeal cancer, osteosarcoma, ovarian, fallopian tube, and peritoneal cancer, pancreatic cancer, parathyroid cancer, penile cancer, Peutz-Jeghers syndrome, pheochromocytoma and paraganglioma, pituitary gland tumor, pleuropulmonary blastoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Kaposi sarcoma, soft tissue sarcomas, skin cancer (non-melanoma), small bowel cancer, stomach cancer, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, tuberous sclerosis complex, uterine cancer, vaginal cancer, Von Hippel-Lindau syndrome, vulvar cancer, Werner syndrome, Wilms tumor, or xeroderma pigmentosum.

[0408] In some embodiments, serum albumin-binding proteins, fusion proteins, or conjugates of the present disclosure may be admixed, conjugated, or administered with, or administered separately from, another therapeutic agent.

[0409] In one aspect, provided herein is a method of using serum albumin-binding proteins, fusion proteins, and / or conjugates of the present disclosure to isolate albumin, a derivative, or a fragment thereof. In some embodiments, the method of isolating albumin, or a derivative or fragment thereof comprising contacting a sample comprising albumin, or a derivative, or a fragment thereof with a solid or semi-solid support attached to serum albumin-binding proteins, fusion proteins, and / or conjugates of the present disclosure.

[0410] In one aspect, provided herein is a method of isolating serum albumin-binding proteins, fusion proteins, and / or conjugates of the present disclosure. In some embodiments, the method of isolating serum albumin-binding proteins, fusion proteins, and / or conjugates of the present disclosure comprising contacting a sample comprising serum albumin-binding proteins, fusion proteins, and / or conjugates of the present disclosure to a solid or semi solid support. In some 122 315736224v1Attorney Docket No: 260525.000077 embodiments, the solid or semi solid support is a resin, a column, a medium, a hydrogel, a cell, a tissue, or a combination thereof. EXAMPLES

[0411] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments. Example 1. Camelid immunization

[0412] Two juvenile alpacas (Vicugna pacos) were immunized by six subcutaneous injections with human albumin (isolated from serum, Merck Millipore Sigma, cat. nr. 126654), porcine albumin (isolated from serum, abbexa, cat. nr. 065007), canine albumin (isolated from serum, abcam, cat. nr. ab119814) and rat albumin (isolated from serum, Merck Millipore Sigma, cat. nr. A6272) using two different adjuvants (complete / incomplete Freund’s adjuvant (CFA / IFA); or Gerbu FAMA) using standard protocols to elicit a humoral immune response that includes the generation of antigen-specific conventional and heavy-chain only antibodies. The immune response was determined by ELISA using sera from day 0 (before immunization), day 28 and day 43. In brief, MaxiSorp plates were coated overnight at 4 ˚C with 50 µl of antigen at 2 µg / ml in PBS. The next day, the antigen was removed, plates were washed with PBS and then blocked with 4% milk powder in PBS (MPBS). Next, the wells were incubated with serially diluted sera for 1 hour at room temperature. Bound antibodies in the sera were detected with rabbit-anti-VHH, followed by donkey-anti-rabbitHRP. In between incubations, plates were washed with PBS with 0.05% Tween-20 (PBST) or PBS. Bound HRP-coupled antibodies were detected using o- phenylenediamine dihydrochloride (OPD) with H2O2as substrate and converted substrate is measured at 490nm wavelength. Example 2. Phage library construction

[0413] Four to ten days after the fourth injection blood samples were collected, and four to six days after the fourth injection a bone marrow sample was aspirated. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood or bone marrow following density gradient purification with Ficoll-PaqueTM Plus. Total RNA was extracted from freshly isolated PBMCs. 123 315736224v1Attorney Docket No: 260525.000077

[0414] To generate VHH immune libraries, total RNA was reversely transcribed to cDNA using random hexamer primers. Conventional and heavy chain IGH cDNA fragments were amplified by PCR using primers annealing to the IGH leader sequence region and the CH2 region. The resulting amplicons represent the VHH and VH cDNAs, respectively. The VHH fragment was isolated and used as template for a nested PCR to introduce appropriate endonuclease recognition sites for cloning into the pQ81 phagemid in frame with geneIII. Libraries were transformed into electrocompetent E. coli TG1 cells. In total, four libraries were built with 96% to 100% VHH insert frequency and maximum library sizes between 2x108and 4x108. Phage for phage display was prepared following standard protocols.

[0415] Binders to serum albumin were enriched from VHH immune libraries by two rounds of phage display. The panning strategy was illustrated in the Figure 1 using human serum albumin (ACROBiosystems, cat. nrs. HSA-H5220 and HSA-H82E3), rat serum albumin (Albumin Bioscience, cat. nr.4001) or mouse serum albumin (ACROBiosystems, cat. nrs. MSA-52H8 and MSA-M82E4). For the 1stpanning round, libraries originating from the blood and the bone marrow samples of the same animal had been pooled in equal parts (at the phage level), resulting in 2 pooled input libraries. Each pooled library was panned under 6 conditions (panning substrates from 3 species and 2 ways of antigen immobilization) at pH 7.4 resulting in 12 panning reactions. For the 2ndround of panning, the 12 output samples (enriched libraries) from the first round served as input libraries. Pannings of the 2ndround were at the same concentrations of the panning substrates and pH 7.4 or pH 5.8.

[0416] Phages were produced according to QVQ’s standard operating procedures and phage titers were determined to ensure at least 10-fold excess over the maximum diversity of the libraries. Panning substrates were commercially purchased. The panning substrates were immobilized either by direct coating on ELISA plates or by binding of biotinylated antigen on neutravidin-coated ELISA plate. For the panning, the albumin-free SuperBlock blocking buffer (Thermo Fisher Scientific, cat. Nr.37515) was used. Glycerol stocks were prepared from all outputs and are stored at -80°C.

[0417] Panning outputs were analyzed by random clone picking, ELISAs with periplasmic extracts PE-ELISA and Sanger sequencing (QVQ), and NGS (Genewiz / PipeBio).

[0418] For random colony picking, rescued outputs of the 2ndpanning rounds were plated out and 1760 random single anti-albumin clones (equal numbers of colonies from each condition) were 124 315736224v1Attorney Docket No: 260525.000077 picked to create masterplates (96-well format). From the masterplates, expression cultures in deep- well plates were inoculated to produce periplasmic extracts containing monoclonal VHH. Periplasmic extracts were used to determine binding of individual VHHs to human antigen by ELISA. Positive clones were rearrayed into new masterplates and sequenced by the Sanger method. Following sequencing, unique clones were rearrayed into new masterplates. Periplasmic extracts were then generated and used to determine binding of individual VHHs to cynomolgus, mouse, and rat antigen.

[0419] For NGS analysis, minipreps from input libraries and outputs after the 1stand 2ndrounds of panning were prepared, amplified by PCR, and sequenced by NGS. Example 3. Next-generation sequencing

[0420] Following two rounds of panning with the previously mentioned conditions, phages were eluted and corresponding phagemid DNA was extracted. Identification of initial V-body candidates was performed in a parallelized fashion, employing a random colony picking as well as a next generation sequencing (NGS) approach as orthogonal techniques to yield a particularly diverse set of initial candidates. Prior to next generation sequencing techniques, random colony picking was the prevalent method for initial hit identification, which involves transformation of a phagemid pool (from a panning elution) and picking of individual bacterial colonies to isolate single clones. Following this approach, 1760 single colonies were randomly picked from the 40 samples of panning round 2 for HSA (Figure 1). Then, individual clones were expressed and subjected to ELISA screening against the target antigen to select for antigen binding V-bodies, which were further functionally characterized. To mitigate the risk of losing rare binders, all panning elutions were also sequenced using next generation sequencing.

[0421] In brief, the entire VHH region first needed to be PCR-amplified from isolated phagemid pools by primers annealing to universal phagemid sequences 5’ and 3’ of the VHH-encoding region. In a second step, the generated amplicons were fused to sequencing-compatible and sample-specific barcodes. By fusing unique barcodes, it was possible to multiplex hundreds of different samples. An Illumina NovaSeq 6000 with an SP flowcell was employed for sequencing, yielding 250 bp reads from each direction and a total of ~600 million reads. To account for differences in the number of expected unique sequences in the library, and both panning rounds, each library was sequenced with a total of 20 million reads, compared to the first and second round 125 315736224v1Attorney Docket No: 260525.000077 of panning with 5 million reads each. This strategy allowed for covering sufficient sequence space in the libraries as well as in the panning elutions, which were expected to have drastically fewer unique sequences compared to the initial libraries. Moreover, spike-in of 30% of a standard PhiX reference genome control into the sequencing reaction helped to provide a technical quality control for assessing sequencing accuracy. The NGS raw data contained multiplexed sequencing reads, which are de-multiplexed based on the sample-specific barcodes. The de-multiplexed data, containing unmerged sequencing reads were then processed by the following strategy depicted in Figure 2, employing an NGS analysis platform of an external service provider.

[0422] Based on CDR3 identity, V-body sequences were clustered, allowing for a detailed analysis of V-body enrichment during phage display, sequence diversity, CDR3 length distribution and cluster abundance. Based on these analyses, up to ~500 candidates were selected for DNA synthesis by Twist and further characterization. Among which, 368 V-bodies can be classified into eight distinct clusters, as follows: H-001 (Group A), H-005 (Group B), H-006 (Group C), H-007 (Group D), H-009 (Group E), H-010 (Group F), H-011 (Group G), and H-012 (Group H). The following Table 3-1 to Table 3-24 display the amino acid frequency distribution at each amino acid (AA) position (IMGT) for the complementarity determining regions (CDR1, CDR2 and CDR3) of V-bodies for the eight clusters. Table 4 provides the sequence identifiers of amino acid sequences of the complementarity determining regions (CDR1, CDR2 and CDR3), amino acid and DNA sequences of the full-length VHH domain for the 368 identified V-bodies. Table 3-1. CDR1 amino acid frequency distribution for cluster H-001 (Group A) Position A i 1 2 3 4 5 6 7 8 1315736224v1Attorney Docket No: 260525.000077 Position Amino 1 2 3 4 5 6 7 8 acid3 6 Table. y Group A) Position Amino 1 2 3 4 5 6 7 8 2 8127 315736224v1Attorney Docket No: 260525.000077 Table 3-3. CDR3 amino acid frequency distribution for cluster H-001 (Group A) Position Amino 1 2 3 4 5 6 7 8 9 10 11 12 13 14 acid A39 56 51 44Table 3-4. CDR1 amino acid frequency distribution for cluster H-005 (Group B) Position Amino 1 2 3 4 5 6 7 8128 315736224v1Attorney Docket No: 260525.000077 Position Amino 1 2 3 4 5 6 7 8 acid) Position Amino Acid 1 2 3 4 5 6 7 8 0 0Table 3-6. CDR3 amino acid frequency distribution for cluster H-005 (Group B) Position129 315736224v1Attorney Docket No: 260525.000077 Position Amino Acid 1 2 3 4 5 6 7 8 9 5a e . amno ac requency sr u on or cuser roup C) Position Amino Acid 1 2 3 4 5 6 7 8 3 7130 315736224v1Attorney Docket No: 260525.000077 Table 3-8. CDR2 amino acid frequency distribution for cluster H-006 (Group C) Position Amino 1 2 3 4 5 6 7 8 3 7Table 3-9. CDR3 amino acid frequency distribution for cluster H-006 (Group C) Position Amino 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0 0 0 0 0 0 0 0 0 0 0 0 0315736224v1Attorney Docket No: 260525.000077 Position Amino 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Acid Q0 0 0 0 0 0 00. Position Amino Acid 1 2 3 4 5 6 7 8Table 3-11. CDR2 amino acid frequency distribution for cluster H-007 (Group D) Position315736224v1Attorney Docket No: 260525.000077 Position Amino Acid 1 2 3 4 5 6 7 8 G0 Tabe . amno ac requency sr u on or cuser roup D) Position Amino 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0133 315736224v1Attorney Docket No: 260525.000077 Table 3-13. CDR1 amino acid frequency distribution for cluster H-009 (Group E) Position Amino 1 2 3 4 5 6 7 8 9 4 4 7Table 3-14. CDR2 amino acid frequency distribution for cluster H-009 (Group E) Position A i 1 2 3 4 5 6 7 8134 315736224v1Attorney Docket No: 260525.000077 Position Amino 1 2 3 4 5 6 7 8 Acid3 1 Table. y Group E) Position Amino 1 2 3 4 5 6 7 8 9 10 11 12 13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00Table 3-16. CDR1 amino acid frequency distribution for cluster H-010 (Group F) Position 7315736224v1Attorney Docket No: 260525.000077 Position Amino Acid 1 2 3 4 5 6 7 8 C3 Table 3-17. CDR2 amino acid frequency distribution for cluster H-010 (Group F) Position Amino 1 2 3 4 5 6 7 8 6 8136 315736224v1Attorney Docket No: 260525.000077 Position Amino 1 2 3 4 5 6 7 8 Acid7 TableGroup F) Position Amino 1 2 3 4 5 6 7 8 9 10 11 12 13 92 31Table 3-19. CDR1 amino acid frequency distribution for cluster H-011 (Group G) Position 7315736224v1Attorney Docket No: 260525.000077 Position Amino 1 2 3 4 5 6 7 8 Acid3 Table 3- . amno ac requency sr u on or cuser - (Group G) Position Amino 1 2 3 4 5 6 7 8 038 315736224v1Attorney Docket No: 260525.000077 Position Amino 1 2 3 4 5 6 7 8 AcidTable 3(Group G) Position Amino 1 2 3 4 5 6 7 8 0Table 3-22. CDR1 amino acid frequency distribution for cluster H-012 (Group H) Position 4139 315736224v1Attorney Docket No: 260525.000077 Position Amino 1 2 3 4 5 6 7 8 Acid1 1 Table. a o ac eque cy s u o o cuse Group H) Position Amino 1 2 3 4 5 6 7 8 4140 315736224v1Attorney Docket No: 260525.000077 Table 3-24. CDR3 amino acid frequency distribution for cluster H-012 (Group H) Position Amino 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Acid 67 26Table 4. Sequence Identifiers of V-bodies identified from panning Group CDR1 CDR1 CDR1 VHH VHH DNA Amino Amino Amino Amino Acid Sequence141 315736224v1Attorney Docket No: 260525.000077 Group CDR1 CDR1 CDR1 VHH VHH DNA Amino Amino Amino Amino Acid Sequence Ai Ai Ai142 315736224v1Attorney Docket No: 260525.000077 Group CDR1 CDR1 CDR1 VHH VHH DNA Amino Amino Amino Amino Acid Sequence Ai Ai Ai143 315736224v1Attorney Docket No: 260525.000077 Group CDR1 CDR1 CDR1 VHH VHH DNA Amino Amino Amino Am...

Claims

Attorney Docket No: 260525.000077 Claims 1. An antigen-binding protein that specifically binds serum albumin, comprising a complementarity determining region 3 (CDR3) comprising an amino acid sequence selected from a) (Q / L)AYRQ(K / S)GS(N / K / Q / A)(A / S)PLI(V / I) (SEQ ID NO: 41); b) KAWYLTTTY (SEQ ID NO: 2); c) AAAYGAGRYRMVKQYDY (SEQ ID NO: 3); d) AADLWAVCPTGASGRGSDYDW (SEQ ID NO: 4); e) NAF(T / V)SWPLEDY(D / N)Y (SEQ ID NO: 45); f) NAGNS(G / W)G(L / W)G(P / T)FDY (SEQ ID NO: 46); g) APTSSWYL (SEQ ID NO: 7); and h) HAYR(A / Q)KTSSGKL(H / I)I (SEQ ID NO: 48).

2. The antigen-binding protein of claim 1, wherein the CDR3 comprises an amino acid sequence selected from LAYRQKGSNSPLIV (SEQ ID NO: 1), LAYRQKGSKSPLIV (SEQ ID NO: 209), LAYRQSGSNSPLIV (SEQ ID NO: 120), LAYRQKGSNSPLII (SEQ ID NO: 91), LAYRQKGSNAPLII (SEQ ID NO: 2378), LAYRQKGSQSPLII (SEQ ID NO: 2379), LAYRQKGSASPLII (SEQ ID NO: 2380), KAWYLTTTY(SEQ ID NO: 2), AAAYGAGRYRMVKQYDY(SEQ ID NO: 3), AADLWAVCPTGASGRGSDYDW (SEQ ID NO: 4), NAFVSWPLEDYNY (SEQ ID NO: 5), NAGNSWGLGTFDY(SEQ ID NO: 6), APTSSWYL (SEQ ID NO: 7), and HAYRQKTSSGKLHI (SEQ ID NO: 8).

3. The antigen-binding protein of claim 1 or 2, further comprising a CDR1 comprising an amino acid sequence selected from a) GFTF(S / T)(D / F)Y(A / S) (SEQ ID NO: 49); b) GFTFRNYV (SEQ ID NO: 10); c) G(G / R)(S / T)F(N / S)(I / T)Y(T / V); d) G(F / L)TLANYS (SEQ ID NO: 52); e) G(F / L)T(F / L)(I / S)SY(A / D) (SEQ ID NO: 53); f) GFTFS(V / Y)Y(A / R) (SEQ ID NO: 54); g) GFTFSSY(A / P) (SEQ ID NO: 55); and 164 315736224v1Attorney Docket No: 260525.000077 h) (E / G)FTFS(G / R / S)Y(A / S) (SEQ ID NO: 56).

4. The antigen-binding protein of claim 3, wherein the CDR1 comprises an amino acid sequence selected from GFTFTDYS (SEQ ID NO: 9), GFTFSFYS (SEQ ID NO: 488), GFTFRNYV (SEQ ID NO: 10), GGSFNIYT (SEQ ID NO: 11), GFTLANYS (SEQ ID NO: 12), GFTFISYD (SEQ ID NO: 13), GFTFSVYA (SEQ ID NO: 14), GFTFSSYA (SEQ ID NO: 15), and EFTFSSYA (SEQ ID NO: 16).

5. The antigen-binding protein of any one of claims 1-4, further comprising a CDR2 comprising an amino acid sequence selected from a) TRTTGG(N / S)I (SEQ ID NO: 57); b) (I / V)TSV(D / G)DST (SEQ ID NO: 58); c) (I / V)(S / T)(R / W)SG(N / R / S)(G / R / S)(L / T); d) ISRSGGST (SEQ ID NO: 20); e) I(S / T)S(T / A)GR(N / T)T (SEQ ID NO: 61); f) ITST(G / S)(G / S)(R / S)(L / T) (SEQ ID NO: 62); g) ITSGG(E / G)ST (SEQ ID NO: 63); and h) ITTT(D / G)(G / N / S)(S / T)(T / -) (SEQ ID NO: 64).

6. The antigen-binding protein of claim 5, wherein the CDR2 comprises an amino acid sequence selected from TRTTGGSI (SEQ ID NO: 17); TRTTGGNI (SEQ ID NO: 856); ITSVDDST (SEQ ID NO: 18); ITWSGSRL (SEQ ID NO: 19); ISRSGGST (SEQ ID NO: 20); ITSTGRNT (SEQ ID NO: 21); ITSTSSRL (SEQ ID NO: 22); ITSGGGST (SEQ ID NO: 23); and ITTTDNT (SEQ ID NO: 24).

7. The antigen-binding protein of claim 1, 3, or 5, comprising i. a CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR2 comprising an amino acid sequence of SEQ ID NO: 57, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 41; 165 315736224v1Attorney Docket No: 260525.000077 ii. a CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR2 comprising an amino acid sequence of SEQ ID NO: 58, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 2; iii. a CDR1 comprising the amino acid sequence of G(G / R)(S / T)F(N / S)(I / T)Y(T / V), a CDR2 comprising an amino acid sequence of (R / W)SG(N / R / S)(G / R / S)(L / T), and a CDR3 comprising an amino acid sequence of SEQ ID NO: 3; iv. a CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR2 comprising an amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 4; v. a CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a CDR2 comprising an amino acid sequence of SEQ ID NO: 61, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 45; vi. a CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a CDR2 comprising an amino acid sequence of SEQ ID NO: 62, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 46; vii. a CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR2 comprising an amino acid sequence of SEQ ID NO: 63, and a CDR3 comprising an amino acid sequence of SEQ ID NO: 7; and / or viii. a CDR1 comprising the amino acid sequence of SEQ ID NO: 56, a CDR2 comprising an amino acid sequence of SEQ ID NO: 64, and a CDR3 comprising an amino acid sequence of SEQ ID NO:

48.

8. An antigen-binding protein that specifically binds serum albumin, comprising a CDR1 comprising an amino acid sequence selected from SEQ ID NOs: 9-16 and 433-800, a CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 17-24 and 801-1168, and / or a CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 1-8, 65-432, and 2378-2380.

9. The antigen-binding protein of any one of claims 1-8, wherein the antigen-binding protein comprises 166 315736224v1Attorney Docket No: 260525.000077 a) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNSPLIV (SEQ ID NO: 1); b) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSKSPLIV (SEQ ID NO: 209); c) a CDR1 comprising an amino acid sequence of GFTFSFYS (SEQ ID NO: 488), a CDR2 comprising an amino acid sequence of TRTTGGNI (SEQ ID NO: 856), and a CDR3 comprising an amino acid sequence of LAYRQSGSNSPLIV (SEQ ID NO: 120); d) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNSPLII (SEQ ID NO: 91); e) a CDR1 comprising an amino acid sequence of GFTFRNYV (SEQ ID NO: 10), a CDR2 comprising an amino acid sequence of ITSVDDST (SEQ ID NO: 18), and a CDR3 comprising an amino acid sequence of KAWYLTTTY (SEQ ID NO: 2); f) a CDR1 comprising an amino acid sequence of GGSFNIYT (SEQ ID NO: 11), a CDR2 comprising an amino acid sequence of ITWSGSRL (SEQ ID NO: 19), and a CDR3 comprising an amino acid sequence of AAAYGAGRYRMVKQYDY (SEQ ID NO: 3); g) a CDR1 comprising an amino acid sequence of GFTLANYS (SEQ ID NO: 12), a CDR2 comprising an amino acid sequence of ISRSGGST (SEQ ID NO: 20), and a CDR3 comprising an amino acid sequence of AADLWAVCPTGASGRGSDYDW (SEQ ID NO: 4); h) a CDR1 comprising an amino acid sequence of GFTFISYD (SEQ ID NO: 13), a CDR2 comprising an amino acid sequence of ITSTGRNT (SEQ ID NO: 21), and a CDR3 comprising an amino acid sequence of NAFVSWPLEDYNY (SEQ ID NO: 5); i) a CDR1 comprising an amino acid sequence of GFTFSVYA (SEQ ID NO: 14), a CDR2 comprising an amino acid sequence of ITSTSSRL (SEQ ID NO: 22), and a CDR3 comprising an amino acid sequence of NAGNSWGLGTFDY (SEQ ID NO: 6); 167 315736224v1Attorney Docket No: 260525.000077 j) a CDR1 comprising an amino acid sequence of GFTFSSYA (SEQ ID NO: 15), a CDR2 comprising an amino acid sequence of ITSGGGST (SEQ ID NO: 23), and a CDR3 comprising an amino acid sequence of APTSSWYL (SEQ ID NO: 7); k) a CDR1 comprising an amino acid sequence of EFTFSSYA (SEQ ID NO: 16), a CDR2 comprising an amino acid sequence of ITTTDNT (SEQ ID NO: 24), and a CDR3 comprising an amino acid sequence of HAYRQKTSSGKLHI (SEQ ID NO: 8); l) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNAPLII (SEQ ID NO: 2378); m) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSQSPLII (SEQ ID NO: 2379); and / or n) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), or a CDR3 comprising an amino acid sequence of LAYRQKGSASPLII (SEQ ID NO: 2380).

10. The antigen-binding protein of claim 9, wherein the antigen-binding protein comprises a) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNSPLII (SEQ ID NO: 91); b) a CDR1 comprising an amino acid sequence of a GFTFRNYV (SEQ ID NO: 10), a CDR2 comprising an amino acid sequence of ITSVDDST (SEQ ID NO: 18), and a CDR3 comprising an amino acid sequence of KAWYLTTTY (SEQ ID NO: 2); c) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSNAPLII (SEQ ID NO: 2378); d) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSQSPLII (SEQ ID NO: 2379); and / or 168 315736224v1Attorney Docket No: 260525.000077 e) a CDR1 comprising an amino acid sequence of GFTFTDYS (SEQ ID NO: 9), a CDR2 comprising an amino acid sequence of TRTTGGSI (SEQ ID NO: 17), and a CDR3 comprising an amino acid sequence of LAYRQKGSASPLII (SEQ ID NO: 2380).

11. The antigen-binding protein of any one of claims 1-9, wherein the antigen-binding protein is a single-domain antibody.

12. The antigen-binding protein of claim 11, wherein the single-domain antibody is a VHH, a VNAR, or a VH domain.

13. The antigen-binding protein of claim 12, wherein the single-domain antibody is a VHH.

14. The antigen-binding protein of claim 13, wherein the VHH is a camelid VHH.

15. The antigen-binding protein of claim 14, wherein the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, and 1169-1536, or an amino acid sequence having at least 75% identity thereto.

16. The antigen-binding protein of claim 15, wherein the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 25, 26, 27, 28, 29, 30, 31, 32, 1195, 1224, 1313, and 1420, or an amino acid sequence having at least 75% identity thereto.

17. The antigen-binding protein of any one of claims 14-16, wherein the VHH comprises an amino acid sequence of SEQ ID NO: 26 or 1195, or an amino acid sequence having at least 75% identity thereto.

18. The antigen-binding protein of claim 13, wherein the VHH is a humanized VHH.

19. The antigen-binding protein of claim 18, wherein the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 1537- 1904, and 2381-2384, or an amino acid sequence having at least 75% identity thereto. 169 315736224v1Attorney Docket No: 260525.000077 20. The antigen-binding protein of claim 19, wherein the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 1563, 1592, 1681, 1788, and 2381-2384, or an amino acid sequence having at least 75% identity thereto.

21. The antigen-binding protein of any one of claim 18-20, wherein the humanized VHH comprises an amino acid sequence of SEQ ID NO: 34, 1563, 2381, 2382, 2383, or 2384, or an amino acid sequence having at least 75% identity thereto.

22. An antigen-binding protein that competes for binding to serum albumin with the antigen- binding protein of any one of claims 1-21.

23. An antigen-binding protein that binds to the same epitope as the antigen-binding protein of any one of claims 1-21.

24. The antigen-binding protein of any one of claims 1-23, wherein the antigen-binding protein binds to domain I and / or domain II of human serum albumin (HSA).

25. The antigen-binding protein of any one of claims 1-23, wherein the antigen-binding protein binds to domain II of human serum albumin (HSA).

26. The antigen-binding protein of any one of claims 1-23, wherein the antigen-binding protein binds to domain III of human serum albumin (HSA).

27. An antigen-binding protein that specifically binds human serum albumin (HSA), wherein the antigen-binding protein binds human serum albumin at an epitope comprising one or more amino acid residues selected from: a) Glu227, Phe228, Ala229, Glu230, Lys233, Lys240, Asp308, Glu321, Val325, and / or Met329; and / or; 170 315736224v1Attorney Docket No: 260525.000077 b) Glu542, Asp549, Asp550, Ala553, Phe554, Glu556, Lys557, Lys560, Glu570, Glu571, and / or Leu575, wherein the numbering is with respect to amino acids 25-609 of SEQ ID NO:

42.

28. The antigen-binding protein of claim 27, wherein the epitope further comprises one or more amino acid residues selected from: a) Ala226, Ser232, Thr236, Tyr263, Ser270, Asp301, Pro303, Phe309, Asn318, Ala322, Lys323, Asp324, Tyr332, Glu333, and / or Arg336; and / or; b) Lys545, Ala546, Ala552, Ala561, Asp563, Thr566, Cys567, Lys574, and / or Ala578, wherein the numbering is with respect to amino acids 25-609 of SEQ ID NO:

42.

29. An antigen-binding protein that comprises a paratope that binds human serum albumin (HSA), wherein the paratope comprises one or more amino acid residues selected from: a) TyrH37, ArgH45, ArgH52, ThrH52A, TyrH59, AspH61, TyrH95, ArgH96, GlnH97, LeuH100D, and / or TrpH103; and / or b) ValH2, GlyH26, PheH27, ThrH28, ValH31, TyrH32, SerH52A, SerH55, AsnH96, SerH97, TrpH98, GlyH100A, ThrH100B, AspH100D, and / or TyrH102, wherein the numbering is according to Chothia numbering.

30. The antigen-binding protein of claim 29 wherein the paratope further comprises one or more amino acid residues selected from: a) SerH33, GluH44, LeuH47, AlaH50, ThrH53, IleH57, ValH58, LysH64, LeuH93, GlyH99, SerH100, and / or IleH101; and / or b) SerH30, ThrH53, LeuH100, and / or PheH100C, wherein the numbering is according to Chothia numbering.

31. An antigen-binding protein that comprises a paratope that binds human serum albumin (HSA), wherein the paratope comprises a complementarity determining region 3 (CDR3) comprising an amino acid sequence a). (Q / L)AYRQ(K / S)GS(N / K / Q / A)(A / S)PLI(V / I) (SEQ ID NO: 41); or b). NAGNS(G / W)G(L / W)G(P / T)FDY (SEQ ID NO: 46). 171 315736224v1Attorney Docket No: 260525.000077 32. The antigen-binding protein of claim 31, wherein the CDR3 comprises an amino acid sequence selected from a) LAYRQKGSNSPLIV (SEQ ID NO: 1), LAYRQKGSKSPLIV (SEQ ID NO: 209), LAYRQSGSNSPLIV (SEQ ID NO: 120), LAYRQKGSNSPLII (SEQ ID NO: 91), LAYRQKGSNAPLII (SEQ ID NO: 2378), LAYRQKGSQSPLII (SEQ ID NO: 2379), or LAYRQKGSASPLII (SEQ ID NO: 2380); and b) NAGNSWGLGTFDY(SEQ ID NO: 6).

33. The antigen-binding protein of claim 31 or 32, wherein the paratope further comprises one or more amino acid residues selected from: a). TyrH37, ArgH45, ArgH52, ThrH52A, TyrH59, AspH61, and / or TrpH103; or b). ValH2, GlyH26, PheH27, ThrH28, ValH31, TyrH32, SerH52A, and / or SerH55, wherein the numbering is according to Chothia numbering.

34. The antigen-binding protein of claim 33, wherein the paratope further comprises one or more amino acid residues selected from: a). SerH33, GluH44, LeuH47, AlaH50, ThrH53, IleH57, ValH58, and / or LysH64; or b). SerH30 and / or ThrH53, wherein the numbering is according to Chothia numbering.

35. The antigen-binding protein of any one of claims 27-34, wherein the antigen-binding protein is a single-domain antibody.

36. The antigen-binding protein of claim 35, wherein the single-domain antibody is a VHH, a VNAR, or a VH domain.

37. The antigen-binding protein of claim 36, wherein the single-domain antibody is a VHH.

38. The antigen-binding protein of claim 37, wherein the VHH is a camelid VHH. 172 315736224v1Attorney Docket No: 260525.000077 39. The antigen-binding protein of claim 38, wherein the VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 25, 1313, 1224, 1195, and 30, or an amino acid sequence having at least 75% identity thereto.

40. The antigen-binding protein of claim 37, wherein the VHH is a humanized VHH.

41. The antigen-binding protein of claim 40, wherein the humanized VHH comprises an amino acid sequence selected from any one of SEQ ID NOs: 33, 1563, 1592, 1681, 38, 2381, 2382, 2383, and 2384, or an amino acid sequence having at least 75% identity thereto.

42. The antigen-binding protein of any one of claims 1-41, wherein the antigen-binding protein binds to HSA at neutral pH.

43. The antigen-binding protein of any one of claims 1-41, wherein the antigen-binding protein binds to HSA at endosomal pH.

44. The antigen-binding protein of any one of claims 1-43, wherein the antigen-binding protein binds to HSA at both neutral pH and endosomal pH.

45. The antigen-binding protein of claim 42 or claim 44, wherein the antigen-binding protein binds to HSA with a KD of less than about 3×10−7M at neutral pH.

46. The antigen-binding protein of claim 45, wherein the antigen-binding protein binds to HSA with a KD of about 1×10−9to 5×10−8M at neutral pH.

47. The antigen-binding protein of claim 43 or claim 44, wherein the antigen-binding protein binds to HSA with a KD of less than about 3×10−7M at endosomal pH.

48. The antigen-binding protein of claim 47, wherein the antigen-binding protein binds to HSA with a KDof less than about 1×10−9x 7×10−8M at endosomal pH. 173 315736224v1Attorney Docket No: 260525.000077 49. The antigen-binding protein of any one of claims 1-48, wherein the antigen-binding protein binds to cyno serum albumin at neutral pH and / or endosomal pH.

50. The antigen-binding protein of any one of claims 1-48, wherein the antigen-binding protein binds to mouse serum albumin at neutral pH and / or endosomal pH.

51. The antigen-binding protein of any one of claims 42, 44-46, 49-50, wherein the neutral pH is in the range from about pH 7.2 to about pH 7.

8.

52. The antigen-binding protein of claim51, wherein the neutral pH is about pH 7.

4.

53. The antigen-binding protein of any one of claims 43-44 and 47-50, wherein the endosomal pH is in the range from about pH 5.3 to about pH 6.

3.

54. The antigen-binding protein of claim 53, wherein the endosomal pH is about pH 5.

8.

55. The antigen-binding protein of any one of claims 1-54, wherein the antigen-binding protein binds to serum albumin without disrupting the interaction of serum albumin with neonatal Fc receptor / beta-2 microglobulin (FcRn / β2M).

56. The antigen-binding protein of any one of claims 1-54, wherein the antigen-binding protein binds to serum albumin at the FcRn / β2M binding site on serum albumin.

57. The antigen-binding protein of any one of claims 1-56, wherein the antigen-binding protein comprises one or more modifications that reduce binding of said antigen-binding protein by pre-existing antibodies found in human blood or serum.

58. The antigen-binding protein of any one of claims 35-57, wherein the single-domain antibody comprises one or more modifications at the amino-terminus and / or the carboxy-terminus. 174 315736224v1Attorney Docket No: 260525.000077 59. The antigen-binding protein of claims 58, wherein the single-domain antibody comprises the amino acid sequence VPAG (SEQ ID NO: 4698) or VAGG (SEQ ID NO: 4697) at the carboxy-terminus starting from position 111 according to Chothia.

60. The antigen-binding protein of claim 58 or 59, wherein the single-domain antibody comprises a substitution of amino acid residue Glu with Asp (E1D) at the first position of the amino-terminus.

61. The antigen-binding protein of any one of claims 58-60, wherein the single-domain antibody comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2381, 2382, 2383, and 2384, or an amino acid sequence having at least 75% identity thereto.

62. An antigen-binding protein that specifically binds serum albumin, comprising a means for binding an epitope within human serum albumin bound by an antibody selected from H-001, H-002, H-002Hu1.DVQ.VPAG, H-002Hu1.DVQ.VPAG.NA, H-002Hu1.DVQ.VPAG.QS, H-002Hu1.DVQ.VPAG.AS, H-003, H-004, H-005, H-006, H-007, H-008, H-009, H-010, H- 011, and H-012.

63. A fusion protein comprising one or more of said antigen-binding proteins of any one of claims 1-62.

64. The fusion protein of claim 63, wherein one or more of said antigen-binding proteins are operably linked to a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, or combinations thereof.

65. The fusion protein of claims 63 or 64, wherein the one or more antigen-binding proteins bind to the same epitope on serum albumin.

66. The fusion protein of claims 63 or 64, wherein the one or more antigen-binding proteins bind to different epitopes on serum albumin. 175 315736224v1Attorney Docket No: 260525.000077 67. The fusion protein of any one of claims 63-66, wherein the one or more antigen-binding proteins are one or more single-domain antibodies.

68. The fusion protein of claim 67, wherein one or more single-domain antibodies are one or more VHHs.

69. The fusion protein of any one of claims 63-68, which further comprises an immunoglobulin Fc region.

70. The fusion protein of claim 69, wherein the immunoglobulin Fc region is an Fc region of a human immunoglobulin.

71. The fusion protein of claim 70, wherein the immunoglobulin Fc region is an Fc region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof.

72. The fusion protein of claim 69, wherein the immunoglobulin Fc region is a Fc variant that has reduced or abolished binding to FcRn.

73. The fusion protein of claim 70 or 71, wherein the immunoglobulin Fc region is an Fc region of human IgG1, or a variant thereof.

74. The fusion protein of claim 73, wherein the Fc region of human IgG1 comprises one or more mutations selected from Ile253Ala (I253A), His310Ala (H310A), His435Ala (H435A), and / or His435Gln (H435Q), according to EU numbering.

75. The fusion protein of claim 73, wherein the Fc region of human IgG1 comprises one or more mutations selected from 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), 176 315736224v1Attorney Docket No: 260525.000077 Asn297Ala (N297A), Pro329Ala (P329A), Pro239Gly (P329G), Asn325Glu (N325E) ,and / or Ala327Ser (A327S) according to EU numbering.

76. The fusion protein of claim 75, wherein the Fc region of human IgG1 comprises a set of mutations selected from 1). L234A and L235A; 2). L234A, L235A, and P329A; 3). D265A, N297A and P329A; 4). L234A, L235A, and G237A; 5). L234G, L235S, and G236R; 6). L234S, L235T, and G236R; 7). L234S, L235V, and G236R; 8). L234T, L235Q, and G236R; 9). L234T, L235T, and G236R; 10). L234A, L235A, and P329G; and 11). M252Y, S254T, and T256E.

77. The fusion protein of claim 70 or 71, wherein the immunoglobulin Fc region is an Fc region of human IgG4, or a variant thereof.

78. The fusion protein of claim 77, wherein the Fc region of human IgG4 comprises one or more mutations selected from Ser228Pro (S228P), Leu235Glu (L235E), Leu235Ala (L235A), Phe234Ala (F234A), and / or Pro329Gly (P329G) according to EU numbering.

79. The fusion protein of claim 78, wherein the Fc region of human IgG4 comprises a set of mutations selected from 1). S228P and L235E; 2). S228P and L235A; 3). S228P, F234A, and L235E; 4). S228P, F234A, and L235A; and 5). P329G, S228P, and L235E. 177 315736224v1Attorney Docket No: 260525.000077 80. A conjugate comprising the antigen-binding protein of any one of claims 1-62 or the fusion protein of any one of claims 63-79, wherein the antigen-binding protein or the fusion protein is conjugated to a second moiety.

81. The conjugate of claim 80, wherein the second moiety is selected from a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a detectable label, a drug, a toxin, a radionuclide, an immunomodulatory agent, a cytokine, a cytotoxic agent, a chemotherapeutic agent, a diagnostic agent, or a combination thereof.

82. A polynucleotide molecule encoding the antigen-binding protein of any one of claims 1-62 or the fusion protein of any one of claims 63-79.

83. The polynucleotide molecule of claim 82, which comprises the nucleotide sequence of any one of SEQ ID NOs: 1905-2272, or a nucleotide sequence having at least 70% identity thereto.

84. The polynucleotide molecule of claim 83, which comprises the nucleotide sequence of any one of SEQ ID NOs: 1905, 1931, 1960, 2049, 2110, 2152, 2156, 2157, 2185, 2221, 2229, 2230, or a nucleotide sequence having at least 70% identity thereto.

85. A recombinant vector comprising the polynucleotide molecule of any one of claims 82-84.

86. A host cell comprising the polynucleotide molecule of any one of claims 82-84, or the recombinant vector of claim 85.

87. A kit comprising the antigen-binding protein of any one of claims 1-62, the fusion protein of any one of claims 63-79, the conjugate of any one of claims 80-81, the polynucleotide molecule of any one of claims 82-84, or the recombinant vector of claim 85, and optionally, instructions and / or packaging for the same. 178 315736224v1Attorney Docket No: 260525.000077 88. A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1- 62, the fusion protein of any one of claims 63-79, the conjugate of any one of claims 80-81, the polynucleotide molecule of any one of claims 82-84, or the recombinant vector of claim 85, and a pharmaceutically acceptable carrier and / or excipient.

89. A method for preparing an antigen-binding protein or a fusion protein that specifically binds serum albumin, comprising the steps of: (a) culturing the host cell of claim 86 in a culture medium under conditions suitable for expression of the antigen-binding protein or fusion protein, and (b) isolating the antigen-binding protein or fusion protein from the host cell and / or culture medium.

90. A method for increasing the serum half-life of a biological molecule comprising attaching to the biological molecule the antigen-binding protein of any one of claims 1-62, the fusion protein of any one of claims 63-79, or the conjugate of any one of claims 80-81.

91. A method of removing a target molecule from serum of a subject, comprising administering the antigen-binding protein of any one of claims 1-62, the fusion protein of any one of claims 63-79, or the conjugate of any one of claims 80-81 to the subject, wherein the antigen-binding protein is fused or conjugated to a biological molecule that binds to the target molecule.

92. The method as claimed in any one of claims 90 or 91, wherein the biological molecule is a peptide, a polypeptide, a protein, an enzyme, an antibody, an antibody fragment, a small molecule, or combination thereof.

93. The method as claimed in claim 92, wherein the biological molecule binds to a target molecule in a subject.

94. A method of delivering a therapeutic or imaging agent to a tumor in a subject in need thereof, comprising administering the antigen-binding protein of any one of claims 1-62 or the fusion 179 315736224v1Attorney Docket No: 260525.000077 protein of any one of claims 63-79 to the subject, wherein the antigen-binding protein or the fusion protein is fused or conjugated to the therapeutic or imaging agent.

95. The method as claimed in claim 94, wherein the therapeutic agent is a drug, a chemotherapeutic agent, a growth inhibitory agent, a toxin, or a radioactive isotope.

96. A solid or semi-solid support for isolating albumin, or a derivative or fragment thereof, comprising attached thereon the antigen-binding protein according to any one of claims 1-62, the fusion protein of any one of claims 63-79, or the conjugate of any one of claims 80-81.

97. A method of isolating albumin, or a derivative or fragment thereof, comprising contacting a sample comprising albumin with the solid or semi-solid support of claim 96.

98. A method of isolating the antigen-binding protein of any one of claims 1-62, the fusion protein of any one of claims 63-79, or the conjugate of any one of claims 80-81 from a sample comprising contacting the sample to a solid or semi-solid support, wherein the solid or semi- solid support comprises attached thereon albumin, or a derivative or fragment thereof. 180 315736224v1