WO2024254562A2 - Domaines lourds variables d'immunoglobuline modifiés ayant une immunogénicité réduite - Google Patents

Domaines lourds variables d'immunoglobuline modifiés ayant une immunogénicité réduite Download PDF

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WO2024254562A2
WO2024254562A2 PCT/US2024/033155 US2024033155W WO2024254562A2 WO 2024254562 A2 WO2024254562 A2 WO 2024254562A2 US 2024033155 W US2024033155 W US 2024033155W WO 2024254562 A2 WO2024254562 A2 WO 2024254562A2
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polypeptide
amino acid
cytokine receptor
group
vhh
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WO2024254562A3 (fr
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Patrick L. LUPARDUS
Deepti ROKKAM
Michael TOTAGRANDE
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Synthekine Inc
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Synthekine Inc
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Priority to KR1020267000338A priority Critical patent/KR20260021032A/ko
Priority to IL324785A priority patent/IL324785A/en
Priority to AU2024285577A priority patent/AU2024285577A1/en
Publication of WO2024254562A2 publication Critical patent/WO2024254562A2/fr
Publication of WO2024254562A3 publication Critical patent/WO2024254562A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • the present disclosure generally relates to single domain antibodies (sdAbs), such as VH, VHH, and scFv polypeptide fragments, comprising C-terminal modifications that have reduced or eliminated binding to endogenous pre-existing antibodies, isolated polypeptides comprising such modifications, nucleotides encoding such polypeptides, and methods of using such antibody polypeptides.
  • sdAbs single domain antibodies
  • the exposed C-terminal VTVSS (SEQ ID NO: 1) amino acid sequence of camelid- derived single-domain antibody fragments, such as a VHH and scFv fragments, is recognized by circulating pre-existing antibodies of the immune system, resulting in an immunogenic response, and limiting the efficacy of therapeutic VHH and scFv drug therapeutics.
  • Prior efforts have been made to reduce this pre-existing antibody immune response by modifying the C- terminal amino acid sequence of single-domain antibodies having an exposed C-terminal VTVSS (SEQ ID NO: 1) amino acid sequence. For example, Nieba et al.
  • WO 11/07586 discloses mutating positions 99, 101, and/or 148 of a VL domain or positions 12, 97, 98, 99, 103, and/or 144 of a VH domain (corresponding to amino acid positions 11, 83, 84, 85, 89 and 103 according to Kabat). Not all such efforts have been sufficiently effective to eliminate the pre-existing antibody immune response. Accordingly, there is a significant need to develop sdAbs that eliminate or further reduce this pre-existing antibody immune response.
  • the present disclosure relates to modifications to the newly exposed C- terminal VTVSS (SEQ ID NO: 1) amino acid sequence to eliminate or reduce recognition by and an immune response due to pre-existing antibodies.
  • such amino acid sequence modifications to the C-terminal VTVSS (SEQ ID NOT) amino acid sequence alters the neoepitope resulting from the newly exposed C-terminal VTVSS (SEQ ID NOT) amino acid sequence on single-chain antibodies, such as a sdAb, VHH, multi-domain antibody comprising fusions of IgGs or HS A with another single-chain antibody, including bispecific antibodies comprising a single chain, an scFv, sdAb, Fab. diabody, cFab. or any other antigen binding domain or Fc- fusion protein that will expose a normally unexposed N- or C-terminal sequence.
  • single-chain antibodies such as a sdAb, VHH, multi-domain antibody comprising fusions of IgGs or HS A with another single-chain antibody, including bispecific antibodies comprising a single chain, an scFv, sdAb, Fab. diabody, cFab. or any other antigen binding domain or Fc-
  • the disclosure relates to an isolated single-domain antibody comprising a C-terminal modification, wherein the C-terminal modification comprises the substitution, addition, or deletion of at least one amino acid residue such that the modification to the singledomain antibody eliminates the interaction of at least one pre-existing antibody with the singledomain antibody without interfering with the binding of the single domain antibody with its target.
  • the C-terminal amino acid sequence of the single-domain antibody is exposed such that the exposed C-terminal is available for interaction with the pre-existing antibody.
  • the present disclosure provides a polypeptide comprising a single domain antibody (sdAb) comprising a modified C-terminal amino acid sequence aligning to the endogenous sdAb amino acid residues (T/Lios)Vio9TiioVinSii2Sii3 (numbered according to the Kabat numbering scheme for human VH carboxy -terminal amino acid residues).
  • sdAb single domain antibody
  • the modified amino acid sequence comprises the formula X108X109X110V111X112 X113Y, wherein:
  • X108 is selected from the group consisting of L, T, and Q;
  • X109 is selected from the group consisting of V, G, N, and L;
  • X110 is selected from the group consisting of T and Q;
  • X112 is selected from the group consisting of S, C. T, A. and G. or optionally absent;
  • X113 is selected from the group consisting of S, C, A, G, and T, or optionally absent; with the provisos that:
  • X112 and X113 are not both C;
  • Y comprises a polypeptide comprising from 1-5 amino acids and wherein such amino acids are independently selected from the group consisting of A, G, S, T, L, and V, or is optionally absent.
  • the sdAb is further modified to comprise an amino acid substitution selected from the group consisting of LI IS, LI IQ, LUG, and P14A, numbered in accordance with the Kabat numbering scheme.
  • Xios is L; X109 is selected from the group consisting of V, G. and L; X110 is T; X112 is selected from the group consisting of S, T. and C; X113 is selected from the group consisting of S, T. C, and A; Y comprises a polypeptide comprising 1-5 amino acids independently selected from the group consisting of A, G, S, T, L, and V; and wherein Y is optionally absent.
  • the amino acid sequence X109X110V111X112 X113 is V109T110V111S112 Ans and Y is AA.
  • the sdAb comprises the amino acid sequence X109X110V111X112 X113 is selected from the group consisting of GTVSS (SEQ ID NO: 3), LTVSS EQ ID NO: 27), NTVSS (SEQ ID NO: 22), VTVCS (SEQ ID NO: 8), VTVSC (SEQ ID NO: 10), VTVTS (SEQ ID NO: 23), VTVTT (SEQ ID NO: 24).
  • polypeptides of the disclosure exhibit reduced binding to pre-existing antibodies.
  • single domain antibody is a VHH. .
  • the disclosure relates to a polypeptide of the formula:
  • VHH1-Ln-VHH2 wherein VHH1 is a first VHH, L is a polypeptide linker comprising from 1-50 amino acids, n is 0 or 1, and VHH2 is a second VHH, which may be the same as or different from VHH1.
  • VHH1 and VHH2 each independently bind to the extracellular domain of a cytokine receptor.
  • the cytokine receptors to which VHH1 and VHH2 bind is selected from the group consisting of IL2Ra, IL2RP, IL2Ry, ILlORa, IL10RP, IL12RP1, IL12RP2, IL18Ra, IL18RP, IL22R1, IL27Ra, gp!30, IL23R, IL28Ra, IFNRyl, IFNR/2, IL21R.
  • VHH1 and VHH2 selectively bind to a pair of cytokine receptors selected from the following pairs: ILlORa/ILlORp. IL27Ra/gpl30, IFNyRl/IFNyR2, IL10R(3/IL28Ra, IL2Rp/IL2Ry, IL18Ra/IL18Rp, IL22R1/IL10RP, IL10Ra/IL2Ry, IL2Rp/IL2Ry, ILlORl/IFNRy, IFNRy/IL28Ra, IL12Rpi/IL12Rp2, IL12RP1/IL23R. and IL 10Ra/IL2Ry.
  • the linker molecule L may be a GS linker.
  • GS linker Various suitable GS linkers are described and exemplified herein.
  • the polypeptide is PEGylated. In some embodiments, the polypeptide is conjugated to an Fc domain.
  • the single-domain antibodies and antigen-binding fragments thereof can be conjugated to various other chemical entities, including antibody-drug conjugates (ADCs).
  • ADCs antibody-drug conjugates
  • the single-domain antibodies or antigen-binding fragments described herein may also be used for diagnostic purposes.
  • modified polypeptides of the present disclosure show reduced immunogenicity resulting from pre-existing antibodies when administered to a human subject, relative to a single-domain polypeptide having an endogenous heavy chain C-terminal amino acid sequence VTVSS (SEQ ID NO:1).
  • the present disclosure also includes methods of treating a mammalian subject suffering from a disease, disorder or condition, the method comprising the step of administering to the mammalian subject of a therapeutically effective amount of polypeptide of the present disclosure.
  • the present disclosure includes the administration of the polypeptides of the present disclosure, by the administration of an agent comprising a nucleic acid sequence encoding a polypeptide of the present disclosure with modifications to the C- terminus.
  • the present disclosure provides a method of reducing an immune response to single-domain antibodies.
  • the present disclosure includes methods of reducing the immunogenicity of single-domain antibodies resulting from pre-existing antibodies (an innate immune response) by use of a modified C-terminal amino acid sequence disclosed herein or reducing immunogenicity to B-cell derived antibodies (via acquired immunity), pharmaceutically acceptable formulations of the modified sdAbs and polypeptides described herein, nucleic acids encoding such sdAbs and polypeptides, vectors comprising such nucleic acids, and host cells comprising such vectors.
  • XI 08 is selected from the group consisting of L, T, and Q
  • X109 is selected from the group consisting of V, G, N
  • LLXHO is selected from the group consisting of T and Q
  • Xm is V
  • X112 is selected from the group consisting of S, C. T, A.
  • X113 is selected from the group consisting of S, C, A, G, and T, or optionally absent; with the provisos that: (a) if X109 is V, then XI 12 and XI 13 are not both S; and (b) X112 and X113 are not both C; and Y comprises a polypeptide comprising from 1-5 amino acids independently selected from the group consisting of A, G, S. T, L. and V, or Y is optionally absent; and wherein the polypeptide is optionally further modified to comprise an amino acid substitution selected from the group consisting of LI IS, LI IQ, LUG, and P14A, numbered in accordance with the Kabat numbering scheme.
  • the amino acid sequence of the formula X108V109T110V111S112S113Y of VHH2 is selected from the group consisting of SEQ ID NOs:2- 24, and optionally further comprises an amino acid substitution selected from the group consisting of LI IS, LI IQ, LUG, and P14A, amino acid residues numbered in accordance with the Kabat numbering scheme.
  • the cytokine receptor binding polypeptide exhibits reduced immunogenicity 7 relative to the polypeptide absent the amino acid sequence of the formula X108V109T110V111S112S113Y in VHH2.
  • the cytokine receptor binding polypeptide are modified to extend half-life in vivo. In some embodiments, the cytokine receptor binding polypeptide is PEGylated.
  • the cytokine receptor is the IL 10 receptor
  • the first cytokine receptor subunit is ILlORa
  • the second cytokine receptor subunit is ILlORb.
  • the cytokine receptor binding polypeptide comprises an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-72.
  • the cytokine receptor binding polypeptide exhibits reduced immunogenicity relative to DR2485aa (SEQ ID NO:49).
  • the cytokine receptor binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-72.
  • the cytokine receptor binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 50-72.
  • the cytokine receptor is the IL18 receptor, the first cytokine receptor subunit is IL 18Ra and the second cytokine receptor subunit is IL18Rb.
  • the cytokine receptor binding polypeptide comprises an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 111-134. In some embodiments, the cytokine receptor binding polypeptide exhibits reduced immunogenicity relative to R3905aa (SEQ ID NO: 110). In some embodiments, the cytokine receptor binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 111-134. In some embodiments, the cytokine receptor binding protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 111-134.
  • the cytokine receptor is the IL2 receptor, the first cytokine receptor subunit is IL2Rb (CD122) and the second cytokine receptor subunit is IL2Rg (CD 132).
  • the cytokine receptor is the IL 18 receptor, the first cytokine receptor subunit is IL18Ra and the second cytokine receptor subunit is IL18Rb.
  • the cytokine receptor is the IL27 receptor, the first cytokine receptor subunit is IL27Ra and the second cytokine receptor subunit is gp!30.
  • the cytokine receptor is the IL22 receptor, the first cytokine receptor subunit is IL22Ra and the second cytokine receptor subunit is IL12Rb.
  • the cytokine receptor is the IL4 receptor, the first cytokine receptor subunit is IL4Ra and the second cytokine receptor subunit is IL2Rg (CD 132).
  • the cy tokine receptor is the IL7 receptor, the first cytokine receptor subunit is IL7Ra and the second cytokine receptor subunit is IL2Rg (CD 132).
  • the cytokine receptor is the IL9 receptor
  • the first cytokine receptor subunit is IL9Ra
  • the second cytokine receptor subunit is IL2Rg
  • the cytokine receptor is the IL 12 receptor
  • the first cytokine receptor subunit is IL12Ra
  • the second cytokine receptor subunit is IL12Rb.
  • a bivalent IL6R/HSA binding polypeptide of the formula VHH1- Ln-VHH2, wherein one of VHH1 or VHH2 is a VHH that selectively binds to the extracellular domain of IL6Ra and the other of VHH1 or VHH2 is a VHH that selectively binds human serum albumin, L is a polypeptide linker, n 0 (absent) or (1) present, and wherein the VHH2 comprises an amino acid sequence of the formula X108V109T110V111S112S113Y, the amino acids numbered according to the Kabat numbering scheme, wherein Xios is selected from the group consisting of L, T, and Q; X109 is selected from the group consisting of V, G.
  • Xno is selected from the group consisting of T and Q;
  • X111 is V;
  • X112 is selected from the group consisting of S, C, T, A, and G, or optionally absent;
  • X113 is selected from the group consisting of S, C, A, G, and T, or optionally absent; with the provisos that: (a) if X109 is V, then X112 and X113 are not both S; and (b) X112 and X113 are not both C; and
  • Y comprises a polypeptide comprising from 1-5 amino acids independently selected from the group consisting of A.
  • G, S, T, L, and V, or Y is optionally absent; and wherein the polypeptide is optionally further modified to comprise an amino acid substitution selected from the group consisting of LI IS, LI IQ, LI 1G, and P14A, numbered in accordance with the Kabat numbering scheme.
  • the amino acid sequence of the formula X108V109T110V 111S112S113Y ofVHH2 is selected from the group consisting of SEQ ID NOs:2-24, and optionally further comprises an amino acid substitution selected from the group consisting of LHS, LHQ, LUG, and P14A, amino acid residues numbered in accordance with the Kabat numbering scheme.
  • the bivalent IL6R/HSA binding polypeptide exhibits reduced immunogenicity relative to the polypeptide absent the amino acid sequence of the formula X108V109T110V111S112S113Y in VHH2.
  • the bivalent IL6R/HSA binding polypeptide is modified to extend half-life in vivo.
  • the bivalent IL6R/HSA binding polypeptide is PEGylated.
  • the bivalent IL6R/HSA binding polypeptide comprises an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 85-109.
  • the bivalent IL6R/HSA binding polypeptide exhibits immunogenicity relative to DR2514aa (SEQ ID NO:84).
  • the bivalent IL6R/HSA binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 85-109.
  • an anti-HSA (human serum albumin) VHH that selectively binds human serum albumin
  • the a anti-HSA VHH comprises an amino acid sequence of the formula X108V109T110V111S112S113Y, the amino acids numbered according to the Kabat numbering scheme, wherein Xios is selected from the group consisting of L, T, and Q; X109 is selected from the group consisting of V, G, N, and L;Xno is selected from the group consisting of T and Q; X111 is V; X112 is selected from the group consisting of S, C, T, A, and G, or optionally absent; X113 is selected from the group consisting of S, C, A, G, and T.
  • Y comprises a polypeptide comprising from 1-5 amino acids independently selected from the group consisting of A. G, S, T, L, and V. or Y is optionally absent; and wherein the polypeptide is optionally further modified to comprise an amino acid substitution selected from the group consisting of LI IS, LI IQ, LUG, and P14A, numbered in accordance with the Kabat numbering scheme.
  • the amino acid sequence X108V109T110V111S112S113Y is selected from the group consisting of SEQ ID NOs:2-24, and optionally further comprises an amino acid substitution selected from the group consisting of LI IS, LI IQ, LI 1G, and P14A, amino acid residues numbered in accordance with the Kabat numbering schemein some embodiments, the anti-HSA VHH exhibits reduced immunogenicity relative to the polypeptide absent the amino acid sequence of the formula X108V109T110V111S112S113Y. In some embodiments, the anti-HSA is modified to extend halflife in vivo. In some embodiments, the anti-HSA is PEGylated.
  • the anti-HSA comprises an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 160-194.
  • the anti-HSA VHH exhibits reduced immunogenicity relative to a polypeptide of SEQ ID NO: 159.
  • the anti-HSA comprises an amino acid sequence an amino acid sequence selected from the group consisting of SEQ ID NOs: 160-194.
  • Exemplary anti-HSA VHHs are provided as SEQ ID NOS: 160-194 in Table 9 derived from the reference sequence DR2830aa (SEQ ID NOSEQ ID NOS: 160-194 in Table 9 derived from the reference sequence DR2830aa (SEQ ID NOS: 160-194 in Table 9 derived from the reference sequence DR2830aa (SEQ ID NOS: 160-194 in Table 9 derived from the reference sequence DR2830aa (
  • FIG. 1 is a scater plot graph comparing normalized percent binding activity (and presumed immunogenicity) of various VHH constructs of the present disclosure compared to the wild-type sdAb C-terminal amino acid motif VTVSS (SEQ ID NO: 1) and other amino acid VTVSS (SEQ ID NO: 1) variants described in the scientific literature.
  • FIG. 2 is a scatter plot graph comparing normalized binding activity (and expected immunogenicity) in terms of measured bioluminescence (relative light units, or RLU) of the VHH construct C-terminal amino acid modifications of the present disclosure relative to the wild-type sdAb C-terminal amino acid motif VTVSS (SEQ ID NO:1) and other amino acid VTVSS (SEQ ID NO: 1) variants described in the scientific literature as more fully described in Example 1 herein.
  • FIG. 3 is a graph showing the data of Table 12 in a violin-type scater plot format.
  • FIG.4 a scater plot graph comparing normalized binding activity (and expected immunogenicity) in terms of measured bioluminescence (relative light units, or RLU) of the aIL6R-aHSA VHH dimers comprising amino acid modifications of the present disclosure relative to the the parent reference molecule as more fully described in Example 2 herein.
  • FIG.5 a scater plot graph comparing normalized binding activity (and expected immunogenicity) in terms of measured bioluminescence (relative light units, or RLU) of the aIL18Ra-aIL18Rp VHH dimers comprising amino acid modifications of the present disclosure relative to the the parent reference molecule as more fully described in Example 3 herein.
  • FIG.6 a scater plot graph comparing normalized binding activity (and expected immunogenicity) in terms of measured bioluminescence (relative light units, or RLU) of - aHSA VHH dimers molecules comprising amino acid modifications of the present disclosure relative to the parent reference molecule as more fully described in Example 4 herein.
  • the present disclosure provides engineered sdAbs that comprise one or more amino acid substitution, deletion, and/or addition that results in improved reduction of immunogenicity from pre-existing endogenous human antibodies relative to the parent sdAb from which the engineered sdAb is derived.
  • the engineered sdAbs also exhibit binding affinity, specificity, stability, or expression efficiency comparable to the parent sdAb from which the engineered sdAb is derived.
  • the sdAbs of the present disclosure comprise one or more amino acid substitutions, deletions, and/or additions in a complementarity -determining region (CDR) (also referred to as a hypervariable region (HVR)) of the parent antibody from which the sdAb is derived.
  • CDR complementarity -determining region
  • HVR hypervariable region
  • the modified immunoglobulin heavy’ chain single-domain antibodies (sdAb) and fragments of the present disclosure can be derived from various sources, including, but not limited to, VHH, VNAR, engineered VH, or VK domains.
  • VHHs can be generated from camelid heavy-chain-only antibodies and libraries thereof.
  • VNARS can be generated from cartilaginous fish heavy-chain-only antibodies and libraries thereof.
  • Various methods have been implemented to create monomeric sdAbs from conventionally dimeric Vn and VK domains, including interface engineering and selecting specific germline families.
  • the modified sdAbs of the present disclosure are human or humanized.
  • ADAs endogenous human anti-drug antibodies
  • a sdAb has an exposed carboxy terminus.
  • the present disclosure provides mutations within the sdAb carboxy -terminal regions that prevent or reduce ADA recognition.
  • the sdAb derived from a non-human source e.g., camelids
  • the sdAb is referred to as being “humanized,’’ as discussed in greater detail hereinbelow.
  • the sdAb of the present disclosure may also comprise one or more amino acid substitutions or deletions in other regions of the sdAb.
  • one or more amino acid substitutions or deletions may be made within the framework region 1 (FW1).
  • the sdAb comprises one or more amino acid substitutions or deletions within the framework region 2 (FW2).
  • the sdAb comprises one or more amino acid substitutions or deletions within framework region 3 (FW3).
  • the sdAb comprises one or more amino acid substitution or deletion within framework region 4 (FW4).
  • the sdAb is modified within a single region.
  • the sdAb comprises one or more amino acid substitutions or deletions in one or more of FW1, FW2, FW3, and FW4.
  • a sdAb may be modified within FW1 and FW4.
  • Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as commonly accomplished in the art or as described herein.
  • the preceding techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present disclosure.
  • the nomenclature utilized in connection with, and the laboratory' procedures and techniques of, analytical chemistry, synthetic organic chemistry', and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard methods are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.
  • the terms “specifically bind, 7 ’ “immunoreactive with,’’ or “directed against” mean that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd>I O 6 ).
  • Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab' and F(ab')2 fragments. Fv, scFvs, Fab expression library, and single domain antibody (sdAb) fragments, for example, VHH, VNAR, engineered Vu or VK.
  • antigen binding site refers to the part of the immunoglobulin molecule that participates in or influences antigen binding.
  • the antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“FI”) and light (“L”) chains.
  • V N-terminal variable
  • FI heavy
  • L light
  • hypervariable regions Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking regions known as “framework regions,” or “FRs,” which are amino acid sequences that are naturally found between, and adjacent to, hypervariable regions in immunoglobulins.
  • the three hypervariable regions of alight chain and the three hypervariable regions of a heavy chain are located relative to each other in three- dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is complementary to and binds to the three-dimensional surface of a bound antigen via non- covalent chemical interactions.
  • amino acid residues are numbered as follows (amino acid residue numbers in parentheses): FR1 (1-30), CDR1 (31-35), FR2 (36-49), CDR2 (50-65), FR3 (66-94), CDR3 (95-102), and FR4 (103- 113).
  • the total number of amino acid residues in each of the CDR's may vary and may not correspond exactly to the total number of amino acid residues indicated by the standard Kabat numbering scheme (that is, 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).
  • the numbering according to the standard Kabat numbering scheme may or may not correspond to the actual numbering of the amino acid residues in the actual sequence used by Kabat for sdAbs.
  • position 1 according to the Kabat numbering corresponds to the start of FR1 and vice versa
  • position 36 according to the Kabat numbering corresponds to the start of FR2 and vice versa
  • position 66 according to the Kabat numbering corresponds to the start of FR3 and vice versa
  • position 103 according to the Kabat numbering corresponds to the start of FR4 and vice versa.
  • epitope includes any protein domain involved in specific binding to/by an immunoglobulin or fragment thereof or a T-cell receptor.
  • Epitope domains usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains. Usually, they have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An antibody is said to “specifically bind” an antigen when the dissociation constant is ⁇ 1 pM; for example, ⁇ 100 nM, or alternatively ⁇ 10 nM, or alternatively ⁇ 1 nM.
  • immunological binding refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength or affinity of immunological binding interactions can be expressed as the interaction dissociation constant (Ka), wherein a smaller K ⁇ i represents a greater affinity’.
  • Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method measures the rates of antigen-binding site/antigen complex formation and dissociation, wherein the rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that influence the rate in both directions.
  • both the “on rate constant” (k on ) and the “off rate constant” (k o ff) can be determined by calculation of the concentrations and the actual rates of association and dissociation, as described in Nature 361 : 186-87. 1993.
  • the ratio of koff/kon enables the cancellation of all parameters not related to the affinity and is equal to the dissociation constant Rd as generally described, for example, in Davies et al. 1990. Annual Rev Biochem. 59:439-473.
  • An antibody of the present disclosure is said to “specifically bind” to an antigen when the equilibrium binding constant (Kd) is ⁇ 1 pM, or alternatively ⁇ 100 nM, or alternatively ⁇ 10 nM, or alternatively ⁇ 100 pM to about 1 pM, as measured by assays such as radioligand binding assays, surface plasmon resonance (SPR), flow cytometry’ binding assay, or similar assays known to those skilled in the art.
  • Kd equilibrium binding constant
  • the term “substantial identity” means that two peptide sequences, when optimally aligned, using standard default gap weights of the GAP software program, share at least 80 percent sequence identity, or alternatively at least 90 percent sequence identity, or alternatively at least 95 percent sequence identity, or alternatively at least 99 percent sequence identity.
  • amino acid sequences of antibodies or immunoglobulin molecules are considered encompassed by modifications involving “conservative amino acid substitutions” of alternative amino acids having similar properties.
  • Conservative amino acid substitutions typically involve the substitution of amino acid residues with another amino acid residue having a side chain with similar chemical properties, and that is interchangeable with the amino acid residue being replaced without altering or significantly altering the activity or binding properties of the antibody.
  • Conservative amino acid substitutions are contemplated as being within the scope of the present disclosure.
  • conserve ative amino acid substitutions occur within a family of amino acids with side chains having similar chemical properties.
  • Natural amino acids are generally divided into the following families of amino acids having similar chemical properties: (1) acidic amino acids, which include aspartate and glutamate; (2) basic amino acids, which include lysine, arginine, and histidine; (3) nonpolar amino acids, which include alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids, which include glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.
  • Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine.
  • Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine.
  • ammo acids having similar chemical properties include the following: (i) serine and threonine, which are in the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are in the amide-containing family; (iii) alanine, valine, leucine, and isoleucine, which are in the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are in the aromatic family.
  • the aminotermini and carboxy -termini of fragments or analogs occur near the boundaries of functional domains.
  • Structural and functional domains can be identified by comparison of the nucleotide and amino acid sequence data to public or proprietary sequence databases.
  • computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and function. Methods for identifying protein sequences that fold into a known three-dimensional structure are known, for example, as described by Bowie et al. 1991. Science 253: 164.
  • amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, or (4) confer or modify other physicochemical or functional properties of such analogs.
  • Analogs may include various muteins of a sequence other than the naturally occurring peptide sequence.
  • single or multiple amino acid substitutions (such as conservative amino acid substitutions) may be made in the naturally occurring sequence, such as in the portion of the polypeptide outside the domain(s) forming intermolecular contacts.
  • a conservative amino acid substitution should not substantially or materially change the structural characteristics of the parent sequence.
  • a replacement amino acid should not tend to break a helix that occurs in the parent sequence or disrupt other types of secondary structures of the parent sequence.
  • Examples of art-recognized polypeptide secondary and tertiary structures are described, for example, by Creighton et al. 1984. Proteins, Structures and Molecular Principles; Branden et al. 1991. Introduction to Protein Structure (Garland Publishing. New York, N.Y.); and Thornton et al. 1991. Nature 354: 105.
  • polypeptide fragment as used herein means a polypeptide having one or more amino-terminal or carboxy-terminal deletion, but where the remaining amino acid sequence is identical or similar to the corresponding positions in the naturally occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments typically have a length of at least 5, 6, 8, or 10 amino acids; in some embodiments, at least 14 amino acids; in some embodiments, at least 20 amino acids; and in some embodiments, at least 50 amino acids, and in some embodiments at least 70 amino acids.
  • the terms comprises, “comprising,” “containing,” “having,” “includes,” and “including,” and the like are terms of art having “open-ended” construction ascribed to them by U.S. patent law, allowing additional elements not expressly recited to be added and still form a construct within the scope of the claims.
  • the terms “consisting essentially of’ or “consists essentially” likewise have the meaning ascribed by U.S. patent law and are also “open-ended,” allowing additional elements that are not expressly recited, provided the basic or novel characteristics of the expressly recited elements are not materially changed by the presence of the additional recited elements, but exclude prior art embodiments.
  • fragment means a portion of a polypeptide or nucleic acid molecule. This portion contains, in some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80. 90. or 100. 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • single-domain antibody refers to an antibody having a single (only one) monomeric variable antibody domain, which may comprise one variable domain of a heavy-chain antibody (VH) or of a common IgG molecule.
  • VH heavy-chain antibody
  • a sdAb can bind selectively to a specific antigen.
  • single-domain antibody (sdAb) also refers to VHH polypeptides, which are the variable domain of a heavy chain-only polypeptide molecule.
  • Single-domain antibodies can be obtained by immunization of dromedaries, camels, llamas, alpacas or sharks with the desired antigen and subsequent isolation of the mRNA coding for the variable region (VNAR and VHH) of heavy-chain antibodies.
  • sdAbs can be made from common murine, rabbit, or human IgG with four chains. Humans can also produce sdAbs by the random creation of a stop codon in the light chain.
  • the term “single-domain antibody” or “sdAb” also refers to single-chain variable fragments (scFv), which are fusion proteins of the variable regions of a variable heavy chain fragment (VH) and a variable light chain fragment (VL) of an immunoglobulin molecule, generally connected with a short linker peptide of from about 10 to about 25 amino acids.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine amino acids for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL (i.e., having the amino acid sequence structure VL-linker-VH, in which the VH region has a C-terminal VTVSS (SEQ ID NO: 1) motif), or vice versa (i.e., having the amino acid sequence structure VH-linker-VL, in which the VL region has a C- terminal sequence that may be the same as or similar to the C-terminal VTVSS (SEQ ID NO: 1) motif of the VH.
  • scFv proteins retain the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. With respect to the modifications of the VTVSS (SEQ ID NO: 1) motif disclosed herein,
  • VHH means a single monomeric variable domain derived from a single heavy chain polypeptide (i.e., a “heavy chain only” variable domain).
  • Heavy chain antibodies are functional antibodies that have two heavy chains that associate with each other, and no light chains. Such antibodies can be found in or produced from camelid mammals (e.g., camels, llamas) which are naturally devoid of light chains.
  • VHHs can be obtained by immunizing camelids (including camels, llamas, and alpacas (see, e g., Hamers-Casterman, et al.
  • VHH antibodies were originally described as the antigen-binding immunoglobulin (variable) domain of "heavy chain antibodies” (i.e., of "antibodies devoid of light chains”; Hamers-Casterman et al. 1993. Nature 363:446- 448).
  • VHH domain is used to distinguish these variable domains from the heavy chain variable domains that are present in conventional four-chain antibodies (which are referred to herein as “VH domains” or a “VH”) and from the light chain variable domains that are present in conventional four-chain antibodies (which are referred to herein as "VL domains” or a “VL”). VHHs are described in more detail in Muyldermans. 2001.
  • VHH domains derived from camelids can be “humanized” or made “humanlike” by being engineered, for example, by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence 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 antibody from a human being.
  • a humanized VHH domain can contain one or more fully human framework region sequences, and, in an even more specific embodiment, can contain human framework region sequences derived from DP-29, DP-47, DP-51, or parts thereof, optionally combined with JH sequences, such as JH5.
  • VHH CDRs can be grafted into multiple ty pes of binding proteins (e.g., antibodies) and the CDRs retain binding. When a VHH CDR is grafted to a framework, it may be engineered so as to have desirable binding behavior.
  • the VHH can be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. See Bannas et al. 2017. Front Immunol. 8: 1603.
  • the binding protein is selected from: a single-chain variable fragment (scFv); a recombinant camelid heavy -chain-only antibody (VHH); a shark heavy-chain-only antibody (VNAR); a microprotein; a darpin; an anticalin; an adnectin; an aptamer; a Sac7d derivative (affitins, e.g., NANOFITINS, see 2008. Journal of Molecular Biology 383(5):1058-68, the contents of which are hereby incorporated by reference), an Fv; an Fab; an Fab'; and an F(ab')2.
  • the binding protein is heterodimeric, for example, the binding protein has greater potency than each individual monomer.
  • the heteromultimeric neutralizing binding protein is multimeric and the multimeric components are associated non-covalently or covalently.
  • VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy-chain antibodies. VHH technology' is based on fully functional antibodies from camelids that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). The cloned and isolated VHH domain is a stable polypeptide harboring the antigen-binding capacity of the original heavy-chain antibody. See Castorman et al., U.S. Pat. No. 5,840,526 issuedNov. 24, 1998; and Castorman et al., U.S. Pat. No.
  • VHHs are commercially available from Ablynx Inc. (Ghent, Belgium) under the trademark of NANOBODIESTM. Suitable methods of producing or isolating antibody fragments having the requisite binding specificity and affinity are described herein and include for example, methods which select recombinant antibodies from a library, by PCR (See Ladner, U.S. Pat. No. 5,455,030 issued Oct. 3, 1995, and Devy et al., U.S. Pat No. 7,745,587 issued Jun. 29, 2010, each of which is incorporated by reference herein in its entirety).
  • a VHH may be a bispecific VHH 2 binding molecule comprised of two separate VHH molecules, which may be the same or different, or bind to the same epitope or different epitopes.
  • VHH 2 binding molecules described herein may, in some embodiments, bind to a receptor (e.g., the first receptor or the second receptor of the natural or non-natural receptor pairs) if the equilibrium dissociation constant between the VHH and the receptor is greater than about 10 6 M, alternatively greater than about 10 8 M, alternatively greater than about 10 10 M, alternatively greater than about 10 11 M, alternatively greater than about 10 10 M, greater than about 10 12 M as determined by, e.g., Scatchard analysis (Munsen et al.
  • VHH described herein can be humanized to contain human framework regions.
  • Examples of human germlines that could be used to create humanized VHHs include, but are not limited to, VH3-23 (e.g., UniProt ID: P01764), VH3-74 (e.g., UniProt ID: A0A0B4J1X5), VH3-66 (e.g., UniProt ID: A0A0C4DH42), VH3-30 (e.g., UniProt ID: P01768).
  • VH3-1 1 e.g., UniProt ID: P01762).
  • VH3-9 e.g., UniProt ID: P01782).
  • VHH 2 As used herein, the term “VHH 2 ” and “bispecific VHH 2 ” and “VHH dimer” refers to are used interchangeably to refer to a subtype of the single-domain antibody binding molecules of the present disclosure wherein the first and second sdAbs are both VHHs and first VHH binding to a first receptor, or domain or subunit thereof, and a second VHH binding to a second receptor, or domain or subunit thereof.
  • the first VHH binding domain may be the same or different from the second VHH binding domain.
  • a VHH 2 may be covalently linked via a linker or may be directly covalently linked at the C-terminal amino acid of the first VHH (VHH1) and the N-terminal amino acid of the second VHH (VHH2).
  • the dimeric variable domains can be split from a common immunoglobulin G (IgG) from humans or mice into monomers.
  • Single-domain antibodies also include sdAbs derived from light chains which specifically bind to the target epitope.
  • a “range” provided herein include all values within and inclusive of the designated values of the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. 16. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. 30. 31. 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the term “about” means within a range of standard tolerance in the art, for example, within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”
  • amino acid residues of a VHH described herein are numbered according to the general numbering scheme for VH domains developed by Kabat et al., as applied to VHH domains from Camelids in Riechmann et al. 2000. J Immunol Methods 23:240 and (1-2): 185- 195, or as otherwise referred to herein. Generally, according to this numbering system.
  • FR1 of a VHH comprises the amino acid residues at positions 1-30
  • CDR1 of a VHH comprises the amino acid residues at positions 31-35B (where 35B is an amino acid residue insertion)
  • FR2 of a VHH comprises the amino acids at positions 36-49
  • CDR2 of a VHH comprises the amino acid residues at positions 50-65
  • FR3 of a VHH comprises the amino acid residues at positions 66-92
  • CDR3 of a VHH comprises the amino acid residues at positions 93-102
  • FR4 of a VHH comprises the amino acid residues at positions 103-113.
  • the C-terminal amino acid sequence (LZT)VTVSS is numbered as L/T108V109T110V111S112S113.
  • the number of amino acid residues in each of the CDRs of a VH or VHH antibody may vary and may not correspond precisely to the total number of amino acid residues indicated by the Kabat numbering scheme and one or more positions according to the Kabat numbering scheme may not be occupied in the sequence, or the sequence may contain more amino acid residues than the number predicted by the Kabat numbering.
  • pre-existing or endogenous pre-existing antibodies can bind to the C-terminal end of sdAbs, which, in full- sized conventional 4-chain monoclonal antibodies, as well as in the ‘"heavy-chain only” antibodies that are found, for example, in Camelidae, are linked to the rest of the antibody, for example, to the CHI region in conventional monoclonal antibodies and to the hinge region in Camelidae heavy chain antibodies, respectively, and that such full-sized antibodies may be shielded from such protein interference.
  • a single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain that binds selectively to a specific antigen, as described, for example, by Hamers-Casterman et al. 1993. Naturally Occurring Antibodies Devoid of Light Chains. Nature 363: 446-448; Nguyen et al., 2000. Camel Heavy-chain Antibodies: Diverse Germline V(H)H and Specific Mechanisms Enlarge the Antigen-binding Repertoire. EMBO, 19: 921-30; Achour et al. 2008. Tetrameric and Homodimeric Camelid IgGs Originate from the Same IgH Locus. J Immunol.
  • Single-domain antibodies typically have a molecular weight of about 12-15 kDa, much smaller than common antibodies having a molecular weight of about 150-160 kDa and two heavy protein chains and two light chains.
  • Single-domain antibodies are also smaller than Fab fragments having a molecular weight of about 50 kDa and having one light chain and half of a heavy chain, and smaller than single-chain variable fragments (scFvs) having a molecular weight of about kDa and have two variable domains, one from a light chain and one from a heavy chain.
  • scFvs single-chain variable fragments
  • Single-domain antibodies have complementary' determining regions that are part of a single-domain polypeptide. Examples 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, and single-domain scaffolds other than those derived from antibodies. Single-domain antibodies may be derived from any species including, but not limited to, mouse, human, camel, llama, goat, rabbit, and bovine. In some embodiments, a single-domain antibody is a naturally occurring single-domain antibody, known as a heavy chain antibody devoid of light chains (a VH).
  • VH heavy chain antibody devoid of light chains
  • variable domain derived from a heavy chain antibody naturally devoid of a light chain is referred to herein as a VHH to distinguish it from the conventional VH of four-chain immunoglobulins.
  • VHH molecules can be derived from antibodies raised in Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco. Species other than Camelidae may also produce heavy chain antibodies naturally devoid of light chains.
  • a single-domain antibody can be obtained by immunizing dromedaries, camels, llamas, alpacas, or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies.
  • a gene library’ of single-domain antibodies containing several million clones is produced by reverse transcription and polymerase chain reaction. Screening techniques, such as phage display and yeast surface display, are commonly used to identify clones that bind the antigen of interest, as described, for example, by Ghahroudi et al. (cited above) and Desmyter et al. 1997. Selection and Identification of Single Domain Antibody Fragments from Camel Heavy-chain Antibodies. FEBS Letters 414(3):521-526.
  • naive libraries Other methods of generating sdAbs use gene libraries from animals that have not been preimmunized, referred to as “naive libraries.” Such naive libraries typically contain only antibodies with low affinity to the desired antigen, making it necessary’ to apply affinity maturation by random mutagenesis as an additional step, as described by Saerens et al. 2008. Single-domain Antibodies as Building Blocks for Novel Therapeutics. Current Opinions in Pharmacology 8(5):600-608. The most potent clones are identified, and their DNA sequence is optimized, for example, to improve their stability towards enzy mes. Another common approach is to humanize the sdAb to prevent immunological reactions of the human organism against the antibody.
  • Humanization is generally unproblematic because of the high degree of homology between camelid VHH and human VH fragments, as described, for example, by Saerens et al. 2008.
  • the humanized sdAb is generated by translating the optimized singledomain antibody DNA in E. coli, S. cerevisiae. or other suitable organisms.
  • sdAb fragments can also be derived from conventional antibodies.
  • the sdAbs of the disclosure can be made from common murine or human IgG with four chains, as described, for example, by Holt et al. 2003. Domain Antibodies: Proteins for Therapy. Trends in Biotechnology 21(l l):484-490.
  • This process typically utilizes gene libraries from immunized or naive donors and antigen display techniques to identify the most specific antigens.
  • this approach results in a common IgG consisting of two domains (VH and VL) that constitute the binding region. Because these two IgG domains are lipophilic, they dimerize or aggregate, requiring the additional step of monomerization by replacing lipophilic amino acids with hydrophilic amino acids. This often results in a loss of affinity’ to the antigen, as described, for example, by Borrebaeck et al. 2002. Antibody Evolution Beyond Nature. Nature Biotechnology ⁇ 20(12):! 189-90. If affinity can be retained, single-domain antibodies can be produced in E. coli, S. cerevisiae, or other organisms. The modifications within a human or humanized single-domain antibody fragment described herein are useful with any sdAb fragment, regardless of the production method.
  • the present disclosure describes modifications of sdAbs to reduce or eliminate interference by ADAs.
  • modifications involve replacing, eliminating, or adding specific amino acid residues to the C-terminal end of the variable domain of a sdAb, such as a VH or VHH antibody or antibody fragment.
  • V arious methods have been implemented to generate monomeric sdAbs from conventionally heterodimeric VH and VK. domains, including interface engineering and selecting specific germline families.
  • the modified sdAb of the present disclosure are human or humanized VH or VHH antibodies or fragments thereof. Endogenous ADAs, particularly anti-human single domain antibody (sdAb) antibodies, are primarily observed in sdAbs having exposed C-terminal amino acids.
  • the present disclosure provides mutations within a camelid, human, or humanized sdAb C-terminal region that reduce or eliminate immunogenicity caused by ADAs.
  • the camelid, human or humanized sdAb C-terminal region mutations comprise amino acid substitutions of endogenous or wild-type amino acids.
  • the human or humanized sdAb C-terminal region mutations comprise amino acid additions to existing amino acids.
  • the modifications within a camelid, human, or humanized sdAb C-terminal region include amino acid deletions to existing amino acids.
  • the mutations within a human or humanized sdAb C-terminal region comprise one or more amino acid substitutions, additions, or deletions to existing amino acids.
  • Such sdAb modifications of the present disclosure prevent recognition by ADAs and an immunogenic response without substantially decreasing binding affinity, specificity, expression, or stability of the protein.
  • sdAb C-terminal substitutions, deletions, and additions to a sdAb are described herein.
  • the mutations described herein may also be applied to constructs that are multivalent, multispecific (such as bispecific), or multiparatopic (such as biparatopic) antibody constructs that contain two or more sdAbs directly linked or linked via one or more suitable linkers forming the C-terminal part of such a construct having a solvent-exposed variable domain, and in some embodiments having a variable domain at their C-terminus, such as a sdAb and scFv having a heavy chain variable domain at the C-terminus.
  • Such constructs may, for example, be entirely comprised of VH domains (such as VHH domains, humanized VHH domains, or camelized VH domains), linked directly or via one or more suitable linkers.
  • VH domains such as VHH domains, humanized VHH domains, or camelized VH domains
  • a sdAb is generally defined as an amino acid sequence that comprises an immunoglobulin fold or, under suitable physiological conditions, can form an immunoglobulin fold to form an immunoglobulin variable domain (for example, a VH. VL, or VHH domain).
  • an immunoglobulin variable domain for example, a VH. VL, or VHH domain.
  • Such sdAbs form or can create an immunoglobulin variable domain that comprises a functional antigen binding site without requiring interaction with another immunoglobulin variable domain (such as a VH-VL interaction).
  • an immunoglobulin single variable domain currently known in the art include, for example, a single-chain Fv (scFv) domain, a single domain antibody (sdAb), a variable heavy-chain (VH) antibody, and a variable heavy -chain-only (VHH) antibody (i.e., a single variable domain located on a single heavy chain antibody), commonly, but not exclusively, derived from a camelid heavy-chain variable domain.
  • scFv single-chain Fv
  • sdAb single domain antibody
  • VH variable heavy-chain
  • VHH variable heavy -chain-only antibody
  • Embodiments of the disclosure described herein are intended and suitable to be applied to single domain antibodies (sdAbs) that comprise, are based on, or have been derived from heavy chain variable domains, such as VH domains (including human VH domains), VHH domains, including humanized and sequence optimized VHH domains, or camelized VH domains.
  • VH domains including human VH domains
  • VHH domains including humanized and sequence optimized VHH domains
  • camelized VH domains camelized VH domains.
  • Such sdAbs may be synthetic, for example, obtained starting from a synthetic library or based on a fixed framework region, semi-synthetic, for example, humanized, camelized, or sequence-optimized, or obtained by affinity maturation or CDR grafting, such as by starting from a natural VH or VHH domain or fully naturally occurring VH or VHH domains.
  • Embodiments of the disclosure are described and illustrated herein with reference primarily to, but not limited to, sdAbs that are, are based on, or have been derived from or similar to VH or VFIH domains and retain at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the disclosure provides a single-domain antibody (sbAb) or antigen-binding fragments thereof that comprise at least one or more mutation that reduces or prevents recognition of the sdAb by an antibody that specifically recognizes single-domain antibodies.
  • the mutation is at least one or more mutations in a framework region, such as the C-terminal region of framework region 4 of the sdAb.
  • the mutation is a substitution of an amino acid in the carboxyterminal region of the framework 4 (FW4) region.
  • the mutation is or includes a carboxy -terminal extension in the FW4 region.
  • the mutation is a combination of substitutions and/or extensions in the C-terminus of the FW4 region.
  • the carboxy -terminal amino acid modifications comprise a C-terminal amino acid sequence modification comprising an amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs:2-24.
  • the sdAbs. VHHs. sdAb-based biologicals. or VHH-based biologicals may also be functionalized to increase their half-life by including in the construct a moiety or binding unit that increases the half-life of the construct.
  • Examples of such functionalization, moieties, or binding units will be apparent to the skilled person and may, for example be as described herein and may include, for example. PEGylation, fusion to serum albumin, or fusion to a peptide or binding unit that can bind to a serum protein such as serum albumin.
  • Such a serum-albumin binding peptide or binding domain may be any suitable serum-albumin binding peptide or binding domain capable of increasing the half-life of the construct (compared to the same construct without the serum-albumin binding peptide or binding domain) and may, in particular be serum albumin binding peptides as described in WO 2008/068280 and WO 2009/127691. or a serum-albumin binding sdAb, such as a serum-albumin binding VEH, for example, Alb-1 or a humanized version of Alb-1 such as Alb-8, as disclosed in WO 06/122787.
  • a serum-albumin binding sdAb such as a serum-albumin binding VEH, for example, Alb-1 or a humanized version of Alb-1 such as Alb-8, as disclosed in WO 06/122787.
  • In vitro assays may be used to evaluate the immunogenic properties of the modified sdAbs of the present disclosure.
  • such assays may include the ELISA, MSD, Biacore or other similar techniques, such as described in Examples 1 and 2 below.
  • the blood or serum or other biological fluids such as those mentioned herein of specific individuals or groups of individuals, may contain certain pre-existing or endogenous proteins that, under certain circumstances, may non-specifically bind to sdAbs, leading to interference in signals in specific assays used to analyze blood or serum samples obtained from such individuals.
  • the present disclosure also addresses the problem of non-specific protein interference when assaying samples that have been obtained from subjects to which a sdAb has previously been administered, as well as when assaying samples that have been obtained from subjects to whom a sdAb was administered.
  • the modified sdAbs of the present disclosure can also be used to reduce or avoid protein interference or signals due to non-specific binding in immunoassays performed on biological samples, such as blood or serum samples, obtained from a subject to whom a sdAb- based biological drug has been administered. Such samples are referred to herein as a “test sample 7 ’ or “assay sample.” Examples of immunoassays that can be used for detecting the presence of or characterizing ADAs or determining ADA-induced immunogenicity before administering a sdAb-based drug, including those described in the Guideline on the Clinical Investigation of the Pharmacokinetics of Therapeutic Proteins (document CHMP/EWP/89249/2004 dated Jan.
  • the modified sdAbs can be used to predict, eliminate, or reduce such protein interference in "‘anti-drug antibody” or “ADA” assays that are performed on test samples of biological fluids taken from subjects to whom sdAbs (such as VHHs, or sdAb-based biological therapeutic drug or VHH-based biological therapeutic drug, as further defined herein) are intended to be or have been administered.
  • sdAbs such as VHHs, or sdAb-based biological therapeutic drug or VHH-based biological therapeutic drug, as further defined herein
  • the compounds and methods of the present disclosure can be used to predict, avoid or reduce such protein interference or aspecific signals usually associated with the same in biological test samples obtained from a subject to whom one or more such sdAbs or VHHs or a sdAb-based biological therapeutic drug or VHH-based biological therapeutic drug, as further defined herein, may be or have been administered, wherein said samples as suitable for or intended for use in an immunological assay, such as an ADA assay.
  • an immunological assay such as an ADA assay.
  • a biological sample may be whole blood, serum or plasma, ocular fluid, bronchoalveolar fluid/BALF, cerebrospinal fluid, or any other suitable biological fluid or sample suitable for use in an immunoassay, and in particular, an ADA assay.
  • variable domains as previously disclosed in the art, to reduce, eliminate, substantially eliminate, or essentially eliminate such protein interference.
  • this modification may include eliminating, substituting, or adding one or more amino acid residues to the C-terminal region of the variable domain in accordance with the present disclosure.
  • substituting, eliminating, or adding a single amino acid residue to the C-terminal end can reduce or eliminate ADA-induced immunogenicity of an antibody-based biotherapeutic drug.
  • the modification involves substituting a single amino acid residence.
  • the modification consists in substituting a single amino acid residence in combination with adding one or more amino acid residues to the C-terminal end of the sdAb.
  • other mutations in the C- terminal region, or in a framework region are, in combination with the above substitutions and additions to the C-terminal end of the sdAb, other mutations in the C- terminal region, or in a framework region. For example, it is well known to make mutations to amino acid residues within the C -terminus (including at those positions that are explicitly referred to by Nieba et al.
  • variable domain including, without limitation, a VHH domain
  • VH domain a variable domain
  • the modifications disclosed herein can be applied to any variable domain that is not linked to or otherwise associated with a constant domain (or with another group or peptide moiety that functions to "shield.” cover, or “bury” the C-terminal region of the variable domain) and more generally to any variable domain that has a C-terminal region that is solvent- exposed.
  • the methods, assays, and modifications may be applied to heavy chain variable domains (VH domains) and according to one specific aspect of the disclosure to VHH domains.
  • the modifications described herein can also be applied to other constructs having a solvent-exposed variable domain at their C-terminus, such as a single chain Fv (scFv) having a heavy chain variable domain at its C-terminus.
  • modifications, methods, and assays described herein can also be applied to protein constructs that contain one or more variable domains and to such constructs in which a variable domain forms the C-terminal part of the construct or in which the C-terminal region of a variable domain is solvent-exposed.
  • the methods, assays, and modifications are applied to constructs in which a VH domain (and a VHH domain) forms the C-terminal portion of the construct or is otherwise solvent-exposed.
  • VH domains including human VH domains
  • VHH domains including humanized and sequence- optimized VHH domains
  • camelized VH domains may be synthetic (such as sdAbs obtained starting from a synthetic library or based on a fixed framework regions), semisynthetic (such as sdAbs that have been humanized, camelized, sequence-optimized, or obtained by affinity maturation or CDR grafting, starting from a natural VH or VHH domain) or fully naturally occurring VH or VHH domains.
  • the disclosure will therefore be further described herein using, as non-limiting examples. sdAbs that are based on and have been derived from VH or VHH domains.
  • a sdAb of the present disclosure is modified within a single region. In other embodiments, the sdAb is modified at more than one region.
  • a sdAb may be modified within the VTVSS (SEQ ID NO: 1) canonical wildtype sequence, as well as at other positions, for example, at amino acid positions 8. 9, 10, 11, 12. or 13 of FW1, or may be modified by adding additional amino acids.
  • the modifications to the endogenous wild-type sdAb e.g..
  • VHH or scFv C-terminal amino acid motifs LVTVSS (SEQ ID NO: 20) or TVTVSS (SEQ ID NO: 21) (collectively referred to herein as “(T/L)VTVSS’ ? ) in FW4, as described herein, may be combined with modifications at amino acid positions 11 and 14 of FW1.
  • the modifications may include LI IS, LI IQ, LUG, or P14A.
  • the sdAb modifications of the present disclosure eliminate or reduce ADA-based immunogenicity resulting from pre-existing endogenous antibodies, without substantially decreasing the protein's binding affinity, specificity, expressibility. or stability.
  • the disclosure relates to a VHH, such as a humanized VHH or a camelized VH, including a camelized human polypeptide, VH or sdAb or sdAb-based drug or VHH-based drug or other sdAb at its C-terminal end that is a VHH or VH domain, such as a sdAb that is a VH domain or derived from a VH domain, or that has been based on or has been derived from the amino acid sequence of a VHH or VH domain, which sdAb or VHH comprises at its C-terminal end the amino acid sequences in which the canonical wild type C-terminal sequences (T/L)VTVSS C-terminal sequence has been modified.
  • a VHH such as a humanized VHH or a camelized VH, including a camelized human polypeptide, VH or sdAb or sdAb-based drug or VHH-based drug or other sdAb at its C-terminal
  • the disclosure relates to a single domain antibody (sdAb) comprising a modified amino acid sequence aligning to the endogenous sdAb amino acid residues (T/Lio8)Vio9TiioViiiSii2Sii3, numbered according to the Kabat numbering scheme for human VH carboxy-terminal amino acid residues, wherein the modified amino acid sequence comprises the formula X108X109X110V 111X112 XnsY (“V” representing the amino acid valine) and one or more of the amino acid substitutions for X108X109X110V111X112 X113; and/or one or more of the C-terminal amino acid additions at position Y; as set forth in the following Table 1 :
  • polypeptide modifications disclosed herein may optionally further comprise one or more amino acid substitution at positions 11, 12, 13, and 14, for example, substitutions selected from the group consisting of LI IS, LI IQ, LUG, and P14A.
  • the single letter amino acid abbreviations T, L, Q, G, N, S, C, A. represent, respectively, the amino acids threonine, lysine, glutamine, glycine, asparagine, serine, cysteine, and alanine.
  • the disclosure relates to a single domain antibody (sdAb) comprising a modified amino acid sequence aligning to the endogenous sdAb amino acid residues (T/Lio8)Vio9TiioViiiSii2Sii3, numbered according to the Kabat numbering scheme for human VH carboxy-terminal amino acid residues, wherein the modified amino acid sequence comprises the formula X108X109X110V 111X112 X113Y. wherein:
  • X108 is selected from the group consisting of L, T. and Q;
  • X109 is selected from the group consisting of V, G, N, and L;
  • X110 is selected from the group consisting of T and Q;
  • Xii2 is selected from the group consisting of S, C, T, A, and G, or optionally absent;
  • Xii3 is selected from the group consisting of S, C, A, G, and T, or optionally absent; with the provisos that:
  • Y comprises a polypeptide of from 1-5 amino acids and wherein such amino acids are independently selected from the group consisting of A, G, S, T, L, and V. or Y is optionally absent.
  • the sdAb is optionally further modified to comprise an amino acid substitution selected from the group consisting of LI IS, LI IQ, LI 1G. and P14A, numbered in accordance with the Kabat numbering scheme.
  • Xios is L; X109 is selected from the group consisting of V, G, and L; Xno is T; X112 is selected from the group consisting of S, T, and C; X113 is selected from the group consisting of S, T, C, and A; X114 comprises a polypeptide comprising 1-5 amino acids independently selected from the group consisting of A, G, S, T, L. and V ; and Y is absent or is a dipeptide of the amino acid sequence AA.
  • the sdAb having the amino acid sequence X109X110V111X112 X113 includes specific variants, including, but not limited to, any one of GTVSS (SEQ ID NO: 3), LTVSS EQ ID NO: 27), VTVCS (SEQ ID NO: 8), VTVSC (SEQ ID NO: 10), NTVSS (SEQ ID NO: 22), VTVTS (SEQ ID NO: 23), and VTVTT (SEQ ID NO: 24).
  • Y is AA.
  • the amino acid sequence X109X110V111X112 X113 is VTVSA (SEQ ID NO: 6), and Y is AA.
  • the amino acid sequence X109X110V111X112 X113 is selected from the group consisting of GTVSS (SEQ ID NO: 3), LTVSS EQ ID NO: 27), VTVCS (SEQ ID NO: 8), VTVSC (SEQ ID NO: 10), NTVSS (SEQ ID NO: 22), VTVTS (SEQ ID NO: 23), and VTVTT (SEQ ID NO: 24).
  • Y is AA.
  • the amino acid sequence X109X110V 111X112 X113 is VTVSA (SEQ ID NO: 6), and Y is AA.
  • the sdAb is a VHH.
  • the sdAb is a polypeptide of the formula VHH 1-L n -VHH2, wherein VHH1 is a VHH, L is a polypeptide linker comprising from 1-50 amino acids, n is 0 or 1, and VHH2 is a VHH of claim 6.
  • the C-terminus of the single-domain antibody of the present disclosure has an amino acid sequence that is selected from the group consisting of one of the amino acid SEQ ID NOs: 2-24 of the following Table 2
  • FR4 framework region 4
  • a sdAb such as a VHH or scF v. having the FR and CDR domain structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the C-terminus of the single-domain antibody of the present disclosure is VTVSSAA (SEQ ID NO: 2). In some embodiments, the C-terminus of the singledomain antibody of the present disclosure is GTVSS (SEQ ID NO: 3). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is LTVSS (SEQ ID NO: 4). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is LTVSSAA (SEQ ID NO: 5). In some embodiments, the C-terminus of the singledomain antibody of the present disclosure is VTVSA (SEQ ID NO: 6).
  • the C-terminus of the single-domain antibody of the present disclosure is VTVSAAA (SEQ ID NO: 7). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is VTVCS (SEQ ID NO: 8). In some embodiments, the C-terminus of the singledomain antibody of the present disclosure is VTVCS A (SEQ ID NO: 9). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is VTVSCSAA (SEQ ID NO: 10). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is VTVSC (SEQ ID NO: 11).
  • the C-terminus of the singledomain antibody of the present disclosure is VTVSC AA (SEQ ID NO: 12). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is LLTVSS (SEQ ID NO: 13). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is LLTVSSA (SEQ ID NO: 14). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is LLTVSSAA (SEQ ID NO: 15). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is LLTVSS SEQ ID NO:13) further comprising an L11S substitution.
  • the C-terminus of the single-domain antibody of the present disclosure is LLTVSSA (SEQ ID NO: 14) further comprising an LI IS substitution. In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is LLTVSSAA (SEQ ID NO: 15) further comprising an LI IS substitution. In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is LVTVTT (SEQ ID NO: 16). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is LVTVTTAA (SEQ ID NO: 17). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is (SEQ ID NO:16) further comprising an LI IS substitution.
  • the C-terminus of the single-domain antibody of the present disclosure is (SEQ ID NO: 17) further comprising an LI IS substitution.
  • the C-terminus of the single-domain antibody of the present disclosure is TLTVSS (SEQ ID NO: 18).
  • the C-terminus of the single-domain antibody of the present disclosure is TLTVSSA (SEQ ID NO: 19).
  • the C-terminus of the single-domain antibody of the present disclosure is TLTVSSAA (SEQ ID NO: 20).
  • the C-terminus of the single-domain antibody of the present disclosure is TVTVSS (SEQ ID NO: 21).
  • the C-terminus of the single-domain antibody of the present disclosure is NTVSS (SEQ ID NO: 22). In some embodiments, the C-terminus of the singledomain antibody of the present disclosure is VTVTS (SEQ ID NO: 23). In some embodiments, the C-terminus of the single-domain antibody of the present disclosure is VTVTT (SEQ ID NO: 24).
  • the present disclosure provides a VHH wherein the C-terminus of the VHH comprises an amino acid sequence selected from the group consisting amino acid SEQ ID NOs: 2-24 wherein the single domain antibody optionally wherein the single domain antibody further comprises one or more amino acid substitutions selected from the group consisting of LI IS, LI IQ, LUG. and P14A, numbered in accordance with the Kabat numbering scheme.
  • the sdAb is a polypeptide of the formula VHH1-L n -VHH2, wherein the VHH1 and VHH2 each independently bind to different molecules.
  • VHH1 binds to a molecule expressed on a first cell and VHH2 binds to a molecule expressed on a second cell.
  • th first cell and the second cell are different cell types.
  • one of VHH1 or VHH2 of the polypeptide of the formula VHH1-L n -VHH2 binds to an antigen preferentially expressed on a cancer cell and the other of of VHH1 or VHH2 binds to an antigen preferentially expressed on an immune cell, pro- inflammatory cell such as activated immune cell.
  • the antigen expressed on a cancer cell is selected from the group consisting tumor antigens for which antibody binding molecules have been identified and their clinical therapeutic targets include include CD19 (e.g., hematological malignancies e.g., ALLs, CLLs, B cell lymphomas), CD20 (e.g., refractor ⁇ ' or relapsed CD2(T B-cell lymphoma), BCMA (e.g., multiple myeloma, Carpenter, etal. (2013) Clin Cancer Res; 19(8); 2048-60), CD22 (B-cell malignancies including pediatric B cell precursor ALL as described in Pan.
  • CD19 e.g., hematological malignancies e.g., ALLs, CLLs, B cell lymphomas
  • CD20 e.g., refractor ⁇ ' or relapsed CD2(T B-cell lymphoma)
  • BCMA e.g., multiple myeloma, Carpenter, etal. (2013) Clin Cancer Res; 19(8);
  • CD30 e.g., CD30+ lymphomas including Hodgkin lymphoma; Grover. (2019) BMC Cancer 19, 203
  • CD70 e.g., acute myeloid leukemia (AML; Sauer, et al (2019) Blood 134 (Supplement 1): 1932
  • Lewis Y e.g., AML; Ritchie, et al (2013) Molecular Therapy 21(l l):2122-9
  • GD2 e.g., gliomas; Mount, el al (2016) Nal Med 24, 572-579
  • GD3 e.g., metastatic melanoma and nuroecodermal tumors; Agnes, et al.
  • mesothelin e.g., mesothelioma, lung, pancreas, breast, ovarian and other solid tumors; Beatty, et. Al., (2014) Cancer Immunol Research 2(2)), ROR-1 (e.g., chronic lymphocytic leukemia; Aghebati-Maleki, et al (2017) Biomedicine and Pharmacology 88: 814-822), CD44 (e.g., AML and multiple myeloma; Casuccia. et al (2013) Blood 122 (20): 3461-3472).
  • mesothelin e.g., mesothelioma, lung, pancreas, breast, ovarian and other solid tumors; Beatty, et. Al., (2014) Cancer Immunol Research 2(2)
  • ROR-1 e.g., chronic lymphocytic leukemia; Aghebati-Maleki, et al (2017) Biomedicine and Pharmacology 88: 8
  • CD171 e.g., neuroblastoma; Kunkele, et al (2017) Clin Cancer Research 23(2):466-477
  • EGP2 EphA2 (e.g., glioblastoma; Yi, et al (2016) Molecular Therapy: Methods & Clinical Development 9:70-80), ErbB2, ErbB3/4, FAP, FAR ILllRa, PSCA (prostate cancer), PSMA(prostate cancer), NCAM, HER2(, NY-ESO-1, MUC 1. CD123. FLT3, B7-H3.
  • CD33 IL1RAP, CLL1 (CLEC 12A)PSA, CEA, VEGF, VEGF-R2, c-Met, Glycolipid F77, FAP, EGFRvIII, MAGE A3, 5T4, WT1, K.G2D ligand, a folate receptor (FRa), and Wntl antigens.
  • CD123 Additionally, the ABD may have specificity for more than one tumor antigen (e.g. CD 19 and CD20 as described in Zah, et al (2016) Cancer Immunol Res; 4(6); 498- -508; CD19 and CD22 as described in Tu. et al (2019) Frontiers in Oncology 9: 1350).
  • one of VHH1 or VHH2 of the polypeptide of the formula VHH1- Ln-VHH2 binds to an antigen preferentially expressed on an immune cell, including but not limited to an activated immune cell or proinflammatory immune cell, including but not limited to the IL1R1 receptor, IL-i receptor accessory protein, the IL6 receptor subunit (IL6R), HLA- DR, HLA-DR a-chain, HLA-DR p-cham the TNFR1, TNFR2, CD4, CD8, F4/80, CCR2, CD169, CX3CR1, CD206. CD163 and, Lyvel.
  • an activated immune cell or proinflammatory immune cell including but not limited to the IL1R1 receptor, IL-i receptor accessory protein, the IL6 receptor subunit (IL6R), HLA- DR, HLA-DR a-chain, HLA-DR p-cham the TNFR1, TNFR2, CD4, CD8, F4/80, CCR2, CD169, CX3CR1, CD206
  • the sdAb is a polypeptide of the formula VHH1-L n -VHH2, wherein the VHH1 and VHH2 each independently bind to the extracellular domain of a cytokine receptor. In some embodiments, the sdAb is a polypeptide of the formula VHHl-Ln- VHH2.
  • the cytokine receptor is selected from the group consisting of IL2Ra, IL2RP, IL2Ry, ILlORa, IL10R
  • the sdAb is a polypeptide of the formula VHH1-L n -VHH2. wherein the VHH1 and VHH2 selectively bind to a pair of cytokine receptors selected from the following sets: IL I ORa/IL I ORp.
  • IL27Ra/gpl30 IFNyRl/IFNyR2, IL1 OR0/IL28Ra, IL2R0/IL2Ry, IL18Ra/ILl 8R0. IL22R1/IL1OR0. IL10Ra/IL2Ry. IL2R0/IL2Ry, ILlORl/IFNRy, IFNRy/IL28Ra. IL12R01/IL12RP2, IL12R01/IL23R, IL12Rp2/gpl30, and IL10Ra/IL2Ry.
  • the sdAb is a a VHH that selectively binds to the extracellular domain of a subunit of a cytokine receptor.
  • cytokine receptor is selected from the group consisting of IL2Ra, IL2Rb, IL2Rg. IL6Ra, ILlORa, ILlORb, IL12Rbl. IL12Rb2, IL18Ra, IL18Rb, IL22R1, IL27Ra, gpl30, IL23R, IL28Ra, IFNRyl, IFNRy2, and IL21R.
  • the sdAb is a polypeptide that binds to human serum albumin.
  • the sdAb is a polypeptide of the formula VHH1-L n -VHH2, wherein L is a linker comprising the amino acids G and S.
  • the sdAb is a polypeptide that is PEGylated.
  • the sdAb is a polypeptide that is conjugated to an Fc domain.
  • the polypeptide has reduced immunogenicity to pre-existing endogenous antibodies when administered to a human subject compared to a polypeptide of the corresponding native human sequence VTVSS (SEQ ID NO: 1).
  • the disclosure relates to a polypeptide of the formula VHHl-Ln- VHH2, wherein VHH1 is a VHH, L is a polypeptide linker comprising from 1 -50 amino acids, n is 0 or 1, and VHH2 is a sdAb VHH.
  • VHH1 and VHH2 each independently bind to the extracellular domain of a cytokine receptor.
  • VHH1 and VHH2 may independently bind to one or more of the cytokine receptors IL2Ra, IL2RJ3, IL2Ry, ILlORa, IL10RP, IL12RJ31, IL12RP2, IL18Ra.
  • VHH1 and VHH2 may also selectively bind to a pair of cytokine receptors such as ILlORa/ILlORP; IL27Ra/gpl30; IFNyRl/IFNyR2, IL10Rp/IL28Ra, IL2Rp/IL2Ry, IL18Ra/IL18Rp, IL22R1/IL10RP, IL10Ra/IL2Ry, IL2Rp/IL2Ry, ILlORl/IFNRy, IFNRy/IL28Ra, IL12RP1/IL12RP2. IL12RP1/IL23R. and IL 10Ra/IL2Ry.
  • IL12RP1/IL23R. IL 10Ra/IL2Ry.
  • the forward slash symbol indicates that the cytokine pair may be linked together in any order.
  • the cytokine pair “ILlORa/ILlORP” may be oriented as “ILlORa-ILlORP” or, alternatively, as “IL10RP- ILlORa.”
  • Xuo is selected from the group consisting of T and Q;
  • Xm is V;
  • X112 is selected from the group consisting of S, C, T, A, and G, or optionally absent;
  • X113 is selected from the group consisting of S, C, A, G, and T, or optionally absent; with the provisos that: (a) if X109 is V, then X112 and X113 are not both S; and (b) X112 and X113 are not both C; and
  • Y comprises a polypeptide comprising from 1-5 amino acids independently selected from the group consisting of A, G, S, T, L, and V, or Y is optionally absent; and wherein the polypeptide is optionally further modified to comprise an amino acid substitution selected from the group consisting of LI IS, LI IQ.
  • cytokine receptor binding binding polypeptide of the formula VHH1-L n -VHH2 wherein the amino acid sequence of the formula X108V109T110V111S112S113Y of VHH2 is selected from the group consisting of SEQ ID NOs:2- 24, and optionally further comprises an amino acid substitution selected from the group consisting of LI IS, LI IQ. LUG, and P14A, amino acid residues numbered in accordance with the Kabat numbering scheme.
  • the cytokine receptor binding polypeptide exhibits reduced immunogenicity relative to the polypeptide absent the amino acid sequence of the formula X108V109T110V111S112S113Y in VHH2.
  • the cytokine receptor binding polypeptide is further modified to extend halflife in vivo.
  • the cytokine receptor binding polypeptide is PEGylated.
  • the present disclosure provides a nucleic acid sequence encoding the cytokine receptor binding polypeptide.
  • the cytokine receptor is the IL 10 receptor
  • the first cytokine receptor subunit is ILlORa and the second cytokine receptor subunit is ILlORb.
  • the cytokine receptor is the IL2 receptor
  • the first cytokine receptor subunit is IL2Rb (CD 122)
  • the second cytokine receptor subunit is IL2Rg (CD132).
  • the cytokine receptor is the IL18 receptor
  • the cytokine receptor is the IL27 receptor, the first cytokine receptor subunit is IL27Ra and the second cytokine receptor subunit is gpl30.
  • the cytokine receptor is the IL22 receptor, the first cytokine receptor subunit is IL22Ra and the second cytokine receptor subunit is IL12Rb.
  • the cytokine receptor is the IL4 receptor, the first cytokine receptor subunit is IL4Ra and the second cytokine receptor subunit is IL2Rg (CD 132).
  • the cytokine receptor is the IL7 receptor, the first cytokine receptor subunit is IL7Ra and the second cytokine receptor subunit is IL2Rg (CD 132).
  • the cytokine receptor is the IL9 receptor, the first cytokine receptor subunit is IL9Ra and the second cytokine receptor subunit is IL2Rg (CD132).
  • the cytokine receptor is the IL12 receptor, the first cytokine receptor subunit is IL12Ra and the second cytokine receptor subunit is IL12Rb.
  • Xno is selected from the group consisting of T and Q;
  • X111 is V;
  • X112 is selected from the group consisting of S, C, T, A, and G, or optionally absent;
  • Xu? is selected from the group consisting of S, C, A, G, and T, or optionally absent; with the provisos that: (a) if X109 is V, then X112 and X113 are not both S; and (b) X112 and X113 are not both C; and
  • Y comprises a polypeptide comprising from 1-5 amino acids independently selected from the group consisting of A, G, S.
  • T, L, and V, or Y is optionally absent; and wherein the polypeptide is optionally further modified to comprise an amino acid substitution selected from the group consisting of LUS, L11Q, LUG, and P14A, numbered in accordance with the Kabat numbering scheme.
  • IL 10 receptor binding polypeptide of the formula VHH 1-L n -VHH2 wherein the amino acid sequence of the formula X108V109T110V111S112S113Y of VHH2 is selected from the group consisting of SEQ ID NOs:2-24, and optionally further comprises an amino acid substitution selected from the group consisting of LI IS, LI IQ, LUG, and P14A, amino acid residues numbered in accordance with the Kabat numbering scheme.
  • the IL10 receptor binding polypeptide exhibits reduced immunogenicity relative to the polypeptide absent the amino acid sequence of the formula X108V109T110V111S112S113Y in VHH2.
  • the IL 10 receptor binding polypeptide is further modified to extend halflife in vivo.
  • the IL 10 receptor binding polypeptide is PEGylated.
  • the present disclosure provides a nucleic acid sequence encoding the IL 10 receptor binding polypeptide.
  • the IL 10 receptor binding polypeptide comprises an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity' to an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-72 wherein the IL10 receptor binding polypeptide exhibits reduced immunogenicity relative to DR2485 (SEQ ID NO:49)
  • the IL10 receptor binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-72.
  • Exemplary IL10 receptor binding molecules as described herein are provided as SEQ ID NOS:50-72 in Table 4 derived from the reference sequence DR2485aa (SEQ ID NO:49). [0108] In some embodiments, the present disclosure provides nucleic acid molecules comprising a nucleic sequence encoding a polypeptide of Table 4 above as provided in Table 5 below:
  • Xno is selected from the group consisting of T and Q;
  • X111 is V;
  • X112 is selected from the group consisting of S, C, T, A, and G, or optionally absent;
  • X113 is selected from the group consisting of S, C, A, G, and T, or optionally absent; with the provisos that: (a) if X109 is V, then X112 and X113 are not both S; and (b) X112 and X113 are not both C: and
  • Y comprises a polypeptide comprising from 1-5 amino acids independently selected from the group consisting of A, G, S. T, L.
  • the bivalent IL6R/HSA binding polypeptide exhibits reduced immunogenicity relative to the polypeptide absent the amino acid sequence of the formula X108V109T110V111S112S113Y in VHH2.
  • the bivalent IL6R/HSA binding polypeptide is further modified to extend half-life in vivo.
  • the bivalent IL6R/HSA binding polypeptide is PEGylated.
  • the present disclosure provides a nucleic acid sequence encoding a bivalent IL6R/HSA binding polypeptide.
  • the bivalent IL6R/HSA binding polypeptide comprises an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 85-109
  • the bivalent IL6R/HSA binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 85- 109.
  • Exemplary bivalent IL6R/HSA binding polypeptide molecules as described herein are provided as SEQ ID NOS: 85-109 of Table 6 derived from the reference sequence DR2514aa (SEQ ID NO: 84).
  • Y comprises a polypeptide comprising from 1-5 amino acids independently selected from the group consisting of A, G, S. T, L. and V, or Y is optionally absent; and wherein the polypeptide is optionally further modified to comprise an amino acid substitution selected from the group consisting of LUS, L11Q, LUG, and P14A, numbered in accordance with the Kabat numbering scheme.
  • the IL 18 receptor binding polypeptide exhibits reduced immunogenicity relative to the polypeptide absent the amino acid sequence of the formula X108V109T110V111 S112S113Y in VHH2.
  • the IL 18 receptor binding polypeptide comprises an amino acid sequence having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 111-134 In some embodiments, the IL18 receptor binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 111-134.
  • Exemplary IL18 receptor binding molecules as described herein are provided as SEQ ID NOS: 111-134 in Table 7 derived from the reference sequence DR3905aa (SEQ ID NO: 110).
  • polypeptides described in the table above may be encoded by a DNA sequence as set forth in table 8 below:
  • the present disclosure provides an anti-HSA VHH that selectively binds human serum albumin, wherein the a HSA VHH comprises an amino acid sequence of the formula X108V109T110V111S112S113Y, the amino acids numbered according to the Kabat numbering scheme, wherein Xios is selected from the group consisting of L, T, and Q; X109 is selected from the group consisting of V, G, N, and L;Xno is selected from the group consisting of T and Q; Xm is V; X112 is selected from the group consisting of S. C, T.
  • X113 is selected from the group consisting of S, C, A, G, and T, or optionally absent; with the provisos that: (a) if X109 is V, then X112 and X113 are not both S; and (b) X112 and X113 are not both C; and Y comprises a polypeptide comprising from 1-5 amino acids independently selected from the group consisting of A, G, S, T, L. and V, or Y is optionally absent; and wherein the polypeptide is optionally further modified to comprise an amino acid substitution selected from the group consisting of Ll lS, LHQ, LUG, and P14A, numbered in accordance with the Kabat numbering scheme.
  • the amino acid sequence of the formula XiosVit ⁇ TiioViiiSinSiisY is selected from the group consisting of SEQ ID NOs:2-24. and optionally further comprises an amino acid substitution selected from the group consisting of LI IS, LI IQ, LUG, and P14A, amino acid residues numbered in accordance with the Kabat numbering scheme.
  • the anti-HSA VHH exhibits reduced immunogenicity relative to the polypeptide absent the amino acid sequence of the formula X108V109T110V111S112S113Y.
  • the anti-HSA VHH is PEGylated.
  • the present disclosure provides a nucleic acid sequence encoding the anti-HSA VHH.
  • the anti-HSA VHH comprises an amino acid sequence having at least 95%, alternatively at least 96%. alternatively at least 97%, alternatively at least 98%, alternatively at least 99%. or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 160-194 and exhibits reduced immunogenicity relative to a polypeptide of SEQ ID NO: 159.
  • the anti-HSA VHH comprises an amino acid sequence an amino acid sequence selected from the group consisting of SEQ ID NOs: 160-194. Exemplary anti-HSA VHHs are provided as SEQ ID NOS: 160-194 in Table 9 derived from the reference sequence DR2830aa (SEQ ID NO: 159).
  • the anti-HSA VHH molecules comprising amino acid substitutions that exhibit reduced immunogenicity which may be conjugated (covalently linked) to a second molecule (e.g. a therapeutic protein or antibody) to extend the in vivo half-life of a therapeutic proteinin
  • a conjugate comprising a therapeutic protein and an anti-HSA sdAb the anti-HSA sdAb polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting SEQ ID NOs: 160-194.
  • the present disclosure provides a conjugate comprising a therapeutic protein and an anti-HSA sdAb the anti-HSA sdAb polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting SEQ ID NOs: 160-194 wherein the conjugate exhibits reduced immunogenicity relative to a conjugate comprising a polypeptide of SEQ ID NO: 159.
  • the disclosure relates to a composition wherein the polypeptide is PEGylated. In other embodiments, the disclosure relates to a composition wherein the polypeptide is conjugated to an Fc domain.
  • the polypeptide has reduced immunogenicity when administered to a human subj ect compared to a polypeptide of the corresponding native human sequence VTVSS.
  • the naturally occurring C-terminal amino acid sequence VTVSS of the sdAb or sdAb-containing construct may also be substituted with a non-naturally occurring C-terminal amino acid sequence.
  • the covalent linkage of the respective sdAb dimers may further include a linker.
  • VHH1 and VHH2 may be linked together with a linker, represented as “L.”
  • Linkers are molecules selected from the group, including, but not limited to, peptide linkers and chemical linkers.
  • the linker joins the C-terminus of the first sdAb to the N-terminus of the second sdAb.
  • the linker joins the C- terminus of the second sdAb to the N-terminus of the first sdAb.
  • the linker is a peptide linker.
  • a peptide linker can include between 1 and 50 amino acids (e.g., between 2 and 50, between 5 and 50, between 10 and 50, between 15 and 50, between 20 and 50, between 25 and 50, between 30 and 50, between 35 and 50, between 40 and 50, between 45 and 50, between 2 and 45, between 2 and 40, between 2 and 35. between 2 and 30, between 2 and 25, between 2 and 20, between 2 and 15, between 2 and 10, between 2 and 5 amino acids).
  • the linker "‘L” is a GS linker. Glycine and glycine-serine polymers are relatively unstructured and may be a neutral linker between components.
  • Examples of glycine polymers include (G) n , glycine-alanine polymers, alanine-serine polymers, glycine-serine polymers (for example, (GmSo)n, (GSGGS)n, (GmSoGm)n, (GmSoGmSoGm)n, (GSGGSm)n, (GSGSmGjn and (GGGSm)n, and combinations thereof, where m, n, and o are each independently selected from an integer of at least 1 to 20 (e.g., 1-18, 2-16, 3-14, 4-12, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), and other flexible linkers.
  • Exemplary flexible peptide linkers useful in preparing the sdAb binding molecules and polypeptides of the present disclosure can include but are not limited to specific linkers shown in Table 10.
  • a chemical linker may covalently link the first and second domains.
  • the term “linker” refers to a link between two elements, such as polypeptide domains.
  • a linker can be a covalent bond or a peptide linker.
  • the bond may be a chemical bond, such as an amide bond, a disulfide bond, or any bond created from a chemical reaction or chemical conjugation.
  • the linker may also be a peptide linker comprising an amino acid or polypeptide that links two protein domains to provide space or flexibility between the two protein domains.
  • L is a ”GSA‘ linker selected from the group consisting of linkers comprising the amino acid residues G, S, and A, as described in the above table.
  • any modified proteins or polypeptides may, for example, be a construct containing two or more sdAbs (such as two or more VHHs, or a scFv comprising a VH domain linked to a VH domain), optionally linked via one or more suitable linkers.
  • a construct may be a bivalent, trivalent. tetravalent.
  • pentavalent construct such as a bivalent, trivalent, tetravalent, or pentavalent VHH construct
  • conjugation of a molecule to an antibody, including a sdAb or antibody fragment, that binds to human serum albumin is capable of extending the half life of the molecule in vivo.
  • the present disclosure provides anti-HSA VHH molecules comprising amino acid substitutions that exhibit reduced immunogenicity which may be conjugated (covalently linked) to a second molecule (e.g. a therapeutic protein or antibody) to extend the in vivo halflife of a therapeutic protein.
  • a conjugate comprising a therapeutic protein and an anti-HSA sdAb monomer, the anti-HSA sdAb monomer polypeptide having at least 95%, alternatively at least 96%, alternatively at least 97%, alternatively at least 98%, alternatively at least 99%, or alternatively 100% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 160-194.
  • the disclosure may be applied to other proteins or polypeptides (and antibody fragments such as Fab fragments or other proteins or polypeptides based on antibody fragments, such as scFvs) that have a VH-domain at their C- terminal end.
  • the disclosure relates to a protein or polypeptide (such as an scFv) with a VH domain at its C-terminal end having an amino acid sequence, as described above.
  • Antibodies such as the single-domain antibodies and antigen-binding fragments thereof, as disclosed herein, can also be conjugated to various other chemical entities, such as small drug molecules, enzymes, liposomes, polymers such as polyethylene glycol (PEG), radionuclides, antibody Fc domains, and the like.
  • Such antibodies and fragments are useful for therapeutic, diagnostic, kit or other purposes, and include antibodies coupled, for example, to dyes, radioisotopes, enzymes, or metals, such as colloidal gold (see, e.g., Le Doussal et al. 1991. J Immunol. 146: 169-175; Gibellini et al. 1998. J Immunol.
  • Such conjugated moieties may be conjugated to a single-domain antibody or fragment at any suitable position on the single-domain antibody or fragment thereof, for example, at either the N-terminal end.
  • C-terminal end either as a fusion protein or conjugated to a side chain of a residue of the antibody, for example at a sulfhydryl or SH-group of a cysteine residue, for example, at a cysteine amino acid residue at amino acid positions 112 or 113.
  • the single-domain antibody and antigen-binding fragment compounds disclosed herein may be conjugated to polymers.
  • polymers include, but are not limited to, hydrophilic polymers which include but are not limited to polyethylene glycol (PEG), for example, PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethylene glycol (rnPEG).
  • PEG polyethylene glycol
  • rnPEG dextran and monomethoxypolyethylene glycol
  • the conjugation to PEG or mPEG may increase a compound’s biological (e.g., serum) half-life.
  • the antibody or fragment typically is reacted with a reactive form of polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C 1-C 10) alkoxy - or aryloxy-polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody or fragment to be PEGylated is a non-gly cosylated antibody or fragment.
  • Methods for PEGylating proteins are well known in the art and can be applied to the antibodies of the present disclosure. See, e.g., EP 0 154 316 and EP 0 401 384, Lee et al. 1999. Bioconj Chem. 10:973- 981 (PEG conjugated single-chain antibodies), Wen et al. 2001. Bioconj Chem. 12:545-553 (antibodies conjugated with PEG attached to a radiometal chelator, diethylenetriaminpentaacetic acid (DTP A)).
  • DTP A diethylenetriaminpentaacetic acid
  • the single-domain antibodies or antigen-binding fragments thereof described herein can be conjugated to a therapeutic agent having immunostimulatory (agonist) or immunoinhibitory (antagonist) activity to form an antibody-drug conjugate (ADC) compound.
  • ADC antibody-drug conjugate
  • Suitable therapeutic agents include, for example, a cytotoxic agent (e.g., a chemotherapeutic agent), a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), and/or a radioactive isotope (i.e., a radio-conjugate).
  • Additional suitable agents include, e.g., antimetabolites, alky lating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and anti-mitotic agents.
  • ADCs with the single-domain antibodies or antigen-binding fragment thereof described herein (for example, conjugated to a cytotoxic agent) that bind to immunosuppressive cells, such as regulatory T cells, can be used to deplete immunosuppressive cells, for example, from the tumor mi croenvironment.
  • the single-domain antibodies or antigen-binding fragments thereof disclosed herein may also be conjugated with fluorescent or chemilluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o- phthaladehyde, fluorescamine, 152 Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridimium salt label, an oxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals.
  • fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothi
  • the single-domain antibodies or antigen-binding antibody fragments thereof disclosed herein may also be conjugated with radiolabels such as: "Tc, 90 Y, m In, 32 P, 14 C, 123 I, 3 H, 131 I, “C, 13 O, 13 N, 18 F, 33 S, 51 Cr, 37 To, 226 Ra, 60 Co, 39 Fe, 37 Se , 152 EU, 67 CU, 217 Ci, 21 ’At, 212 Pb, 47 Sc, 109 Pd, 234 Th, and 40 K, 157 Gd, 53 Mn, 32 Tr, and 56 Fe.
  • the radionuclides used for making radioconjugated single-domain antibodies or fragments thereof are 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.
  • ADCs may be formed with enzymatically active toxins and fragments thereof, such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin.
  • enzymatically active toxins and fragments thereof such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, mode
  • cytotoxins or cytotoxic agents include, e.g., taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D. 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5 -fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU).
  • antimetabolites e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5 -fluorouracil decarbazine
  • alkylating agents e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU).
  • cyclothosphamide busulfan, dibromomanmtol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.. vincristine and vinblastine).
  • anthracyclines e.g., daunorubicin (formerly daunomycin) and doxorubicin
  • antibiotics e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)
  • anti-mitotic agents e.g.. vincristine and vinblastine
  • the antibody and therapeutic agent preferably are conjugated via a cleavable linker such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker such as Val-Cit, Ala-Vai, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu- Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys. Lys, Cit, Ser, Glu, or others know to those skilled in the art.
  • the ADCs can be prepared as described in U.S. Pat. Nos.
  • Immunoconjugates can also be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity (e.g., lymphokines, tumor necrosis factor, IFNy, grow th factors).
  • Immunoconjugates 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 suberate), 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 tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene).
  • imidoesters such as dimethyl adipimidate HCL
  • Carbon- 14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid is an exemplary 7 chelating agent for conjugation of radionucleotide to the antibody (see, e.g., PCT publication number WO94/11026).
  • compositions that comprise a sdAb (and preferably a therapeutic sdAb) or a protein or polypeptide comprising at least one sdAb, such as at least one therapeutic sdAb. wherein the sdAb.
  • protein or polypeptide is as further described herein (i.e., a sdAb, protein or polypeptide according to one or more of the aspects described herein, and in particular according to one or more of the aspects described on the preceding pages; and more, in particular, a sdAb, protein or polypeptide that has a C-terminal end/ sequence that is according to one or more of the aspects described herein), and at least one suitable carrier, diluent or excipient suitable for pharmaceutical use, and optionally one or more further active substances.
  • compositions, carriers, diluents, or excipients can, for example, be as described in WO 08/020079 for pharmaceutical compositions that comprise a VHH or a protein or polypeptide that comprises at least one sdAb. which sdAb may be a VHH.
  • the disclosure further relates to a sdAb or a protein or polypeptide comprising at least one sdAb for use in therapy of a disease in a human being (e.g., a patient in need of such treatment), wherein said VHH, protein, or polypeptide is as further described herein, such as a VHH, protein or polypeptide according to one or more of the aspects described herein, and in some embodiments to one or more of the aspects described on the preceding pages; and in other aspects a VHH, protein or polypeptide that has a C-terminal end/sequence that is according to one or more of the aspects described herein.
  • the disclosure further relates to the use of a sdAb or a protein or polypeptide comprising at least one sdAb in the preparation of a pharmaceutical composition, wherein said sdAb, protein, or polypeptide is as further described herein, for example, a sdAb. protein, or polypeptide according to one or more of the aspects described herein, and in some embodiments according to one or more of the aspects described on the preceding pages; and in some embodiments a sdAb, protein or polypeptide that has a C-terminal end/sequence that is according to one or more of the aspects described herein.
  • the disclosure further relates to a method of treatment that comprises administering to a human subject (e.g., to a patient in need of such treatment) a sdAb or a protein or polypeptide comprising at least one sdAb in the preparation of a pharmaceutical composition, wherein said sdAb, protein or polypeptide is as further described herein (i.e., a sdAb, protein or polypeptide according to one or more of the aspects described herein, and in some embodiments according to one or more of the aspects described on the preceding pages; and in some embodiments a sdAb, protein or polypeptide that has a C-terminal end sequence that is according to one or more of the aspects described herein; or pharmaceutical compositions (as described above) that comprise at least one such sdAb, protein or polypeptide.
  • a human subject e.g., to a patient in need of such treatment
  • a sdAb or a protein or polypeptide comprising at least one sdAb in the preparation
  • the disclosure relates to the therapeutic use of the sdAbs. proteins, and polypeptides described herein, as such therapeutic use (or the clinical development of such sdAbs, proteins, and polypeptides for such therapeutic use), including the preliminary use of ADA assays using the sdAbs or polypeptides to determine whether the sdAb, protein or polypeptide is immunogenic and could give rise to an ADA when administered to a human subject.
  • a sdAb, protein, polypeptide or pharmaceutical composition as described herein is intended for treatment of a chronic disease in a human being, and/or such sdAb, protein, polypeptide as described herein is intended to be present in the circulation of the subject (for example, at pharmacologically active levels) to which it is administered (for example, at a therapeutically active dose) for at least a period of one week, preferably at least two weeks, for example, at least one or more months; and/or such sdAb, protein, polypeptide as described herein is such that it has a half-life (for example, expressed as t'A-beta) in a human subject of at least 3 days, such as at least one week, and up to 10 days or more; and/or such a sdAb protein, polypeptide or pharmaceutical composition as described herein is intended to be administered to a human being as two or more doses that are administered over a period of at least 3 days,
  • the disclosure further relates to a method for (substantially) reducing or essentially preventing the tendency of a sdAb, a VHH or a sdAb-based drug, or a VHH-based drug to give rise to immunogenic protein interference, said method comprising at least the steps of (1) optionally determining the tendency of the sdAb, VHH.
  • sdAb-based drug or VHH-based drug to give rise to protein interference, using a method that at least comprises steps (i) and (ii) as referred to herein; and (2) modifying said sdAb, VHH, sdAb-based drug or VHH-based drug by introducing one or more one or more amino acid substitutions, additions or deletions in the sdAb or VHH, or the C-terminal sdAb or VHH (if any) of the sdAb-based drug or VHH-based drug; and in some embodiments introducing one or more amino acid substitutions or additions in the C-terminal region of the sdAb or VHH, or the C-terminal region of the C-terminal sdAb or VHH (if any) of the sdAb-based drug or VHH-based drug.
  • sdAbs comprising the C-terminal modifications described herein can humanized to contain human framework regions.
  • humanized versions are prepared and evaluated for binding target receptors using binding assays, such as surface plasmon resonance spectroscopy (Biacore®).
  • VHHs When humanizing VHHs that bind to a target, one consideration for the design of the humanized VHH is the distribution of amino acids at each position of the non-human framework, which suggests amino acid residues, the modification of which may introduce substantial modifications in the secondary' and tertiary structure of the protein.
  • human germlines that could be used to create humanized VHHs include, but are not limited to, VH3-23 (e.g. UmProt ID: P01764).
  • VH3-74 e.g. UmProt ID: A0A0B4J1X5)
  • VH3-66 e.g.
  • UmProt ID: A0A0C4DH42 VH3-30 (e.g., UniProt ID: P01768), VH3-11 (e.g., UniProt ID: P01762), and VH3-9 (e.g., UniProt ID: P01782).
  • Specific framework residues of the canonical llama VHH sequence VH3-66 include positions V37, G44, L45, and W47 and vernier zone residues R94 and W103 may also be residues that are not readily susceptible to modification.
  • VHH sdAb sequences are obtained, and an internally developed R Script is used to evaluate distributions of amino acids at each position.
  • An amino acid distribution chart can identify rare residues at each position.
  • the protein sequences disclosed herein may be tested in a direct anti-single domain antibody (ADA) detection assay in determining pre-existing ADA in serum from human donors.
  • ADA direct anti-single domain antibody
  • pre-existing antibodies within human serum can be identified by an ELISA wherein the sdAb is immobilized on a plate and blocked with BSA.
  • human serum (neat or diluted in PBS+0.01% Tween-20) is then incubated with the plate-bound sdAb.
  • unbound serum IgG is washed away, and any remaining antibodies to the sdAb are detected using anti -human IgKappa and anti -human IgLamda HRP- conjugated antibodies.
  • An indirect ADA assay is useful for analyzing multiple variants of a given sdAb to measure the sdAb-specific serum antibody reactivity.
  • the indirect ADA involves immobilization of a sdAb and blocking with BSA. Human serum (neat or diluted in PBS+0.01% Tween-20) is then pre-incubated with soluble sdAb (either the same sdAb or variants of the parental sdAb with modifications of the present disclosure) and then added to the plate-bound sdAb.
  • a soluble sdAb variant to block the ADA recognition of the immobilized sdAb is indicative of the soluble sdAb being recognized by the AD As and is thus an indirect measurement of the pre-existing ADA response.
  • the inability of soluble sdAbs to prevent the ADA recognition of the immobilized sdAb is indicative of the soluble sdAb variant being non-immunogenic or otherwise not recognized by the pre-existing ADAs.
  • 8-16 assays are conducted in parallel with serum from separate donors previously determined to contain ADAs.
  • ADA antidrug antibody
  • ADA assays such as immunoassays
  • immunoassays see, for example, the review by Shankar et al., 2008. Journal of Pharmaceutical and Biomedical Analysis. 48: 1267-1281; as well as Mire-Sluis et al. 2004. J Immunol Meth. 289: 1-16; Peng et al. 2011. J Pharm. and Biomed. Analysis. 54:629-635; and Loyet et al. 2009. J Immunol Meth. 345: 17-28.
  • ADA assays and methods for performing such methods are common knowledge in the field of pharmacology and are routinely used during the clinical development of biological drug products (as well as being required by various regulatory 7 agencies worldwide).
  • ADA assay formats such as “ELISA-bridging format.” “ELISA-Direct Format.” “Indirect Format,” Radio Immuno-precipitation Assay (RIP), “Surface Plasmon Resonance,” and “Electrochemiluminescence-Bridging Format,” are known and described by Mire-Sluis et al. and Peng et al. Other formats for performing ADA immunoassays are well-known to those skilled in the art.
  • ADA assays can be used. These include but are not limited to the MSD platform (Meso Scale Diagnostics), Gyrolab (Gyros), and the octet platform (Fortebio).
  • MSD platform Meso Scale Diagnostics
  • Gyrolab Gyrolab
  • octet platform Formebio
  • Indirect ADA assays may also be used to demonstrate the impact of multiple modifications of a humanized VHH compared with humanized VHH having the native carboxy-terminal sequence, VTVSS (SEQ ID NO: 1).
  • the single-domain antibodies described herein may also be used for diagnostic purposes.
  • Such sdAbs can be conjugated to an appropriate detectable agent to form an immunoconjugate.
  • appropriate agents are detectable labels that include radioisotopes, for whole body imaging, and radioisotopes, enzy mes, fluorescent labels and other suitable antibody tags for sample testing.
  • the detectable labels can be any of the various types used currently in the field of in vitro diagnostics, including particulate labels, isotopes, chromophores, fluorescent markers, luminescent markers, metal labels (e g., for CyTOF, imaging mass cytometry), phosphorescent markers and the like, as well as enzyme labels that convert a given substrate to a detectable marker, and polynucleotide tags that are revealed following amplification such as bypolymerase chain reaction. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase and the like.
  • the label can be the enzyme alkaline phosphatase, detected by measuring the presence or formation of chemiluminescence following conversion of 1,2 dioxetane substrates such as adamantyl methoxy phosphoryl oxy phenyl dioxetane (AMPPD), disodium 3 -(4-(methoxy spiro ⁇ 1 ,2-dioxetane-3 ,2'-(5 '-chi orojtri cyclo ⁇ 3.3.
  • 1,2 dioxetane substrates such as adamantyl methoxy phosphoryl oxy phenyl dioxetane (AMPPD), disodium 3 -(4-(methoxy spiro ⁇ 1 ,2-dioxetane-3 ,2'-(5 '-chi orojtri cyclo ⁇ 3.3.
  • AMPPD adamantyl methoxy phosphoryl oxy phenyl diox
  • CSPD 3,7 ⁇ decan ⁇ -4-yl phenyl phosphate
  • CDP and CDP-Star® other luminescent substrates well-known to those in the art, for example the chelates of suitable lanthanides such as Terbium(III) and Europium(III).
  • the detection means is determined by the chosen label. Appearance of the label or its reaction products can be achieved using the naked eye, in the case where the label is particulate and accumulates at appropriate levels, or using instruments such as a spectrophotometer, a luminometer, a fluorimeter, and the like, all in accordance with standard practice.
  • conjugation methods result in linkages that are non-immunogenic or have reduced immunogenicity, e.g., peptide-(i.e. amide-), sulfide-, (sterically hindered), disulfide-, hydrazone-, and ether linkages.
  • linkages are nearly non-immunogenic and show reasonable stability within serum (see e.g., Senter. P. D., 2009. Curr Opin Chem Biol. 13:235- 244; and PCT Publication numbers WO 2009/059278 and WO 95/17886).
  • site-specific reaction and covalent coupling is based on transforming a natural amino acid into an amino acid with a reactivity that is orthogonal to the reactivity 7 of the other functional groups present.
  • a specific cysteine within a rare sequence context can be enzymatically converted in an aldehyde (see Frese MA and Dierks T. 2009. Chem Bio Chem. 10:425-427).
  • It is also possible to obtain a desired amino acid modification by utilizing the specific enzymatic reactivity of certain enzymes with a natural amino acid in a sequence see, e.g., Taki M et al. 2004. Prot Eng Des Sei. 17: 119-126: Gautier A. et al.
  • the moiety 7 may also be a synthetic peptide or peptide mimic.
  • amino acids with orthogonal chemical reactivity 7 can be incorporated during such synthesis (see. e.g., de Graaf AJ et al. 2009. Bioconjug Chem. 20: 1281-1295). Since a great variety of orthogonal functional groups is at stake and can be introduced into a synthetic peptide, conjugation of such peptide to a linker is standard chemistry.
  • the invention relates to pharmaceutical compositions that comprise a single domain variable antibody (sdAb) comprising a modified amino acid sequence corresponding to the C-terminal endogenous sdAb amino acid residues described herein and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances.
  • sdAb single domain variable antibody
  • suitable carrier, diluent or excipient i.e., suitable for pharmaceutical use
  • Such compositions, carriers, diluents, or excipients can for example be as described in WO 08/020079 for pharmaceutical compositions that comprise a sdAb protein or polypeptide that comprises at least one VH or VHH polypeptide.
  • the invention relates to pharmaceutical compositions that comprise a single domain variable antibody (sdAb) comprising a modified amino acid sequence corresponding to the C-terminal endogenous sdAb amino acid residues described herein and at least one suitable carrier, diluent or excipient (i.e. , suitable for pharmaceutical use), and optionally one or more further active substances for use for use in therapy of a disease.
  • sdAb single domain variable antibody
  • the invention relates to pharmaceutical compositions that comprise a method of treatment which comprises administering to a human subject (e g., to a patient in need of such treatment) a single domain variable antibody (sdAb) comprising or a pharmaceutical composition that comprises at least one such sdAb, protein or polypeptide, as described herein.
  • a human subject e g., to a patient in need of such treatment
  • sdAb single domain variable antibody
  • the therapeutic use of the sdAbs, proteins and polypeptides described herein are a very important aspect of the invention, as such therapeutic use (or the clinical development of such sdAbs, proteins and polypeptides for such therapeutic use) may involve the use of ADA assays to determine whether said sdAbs, protein or polypeptide is immunogenic or has reduced or eliminated immunogenicity to preexisting endogenous antibodies (i.e. can give rise to ADA's when administered to a human subject).
  • a sdAb, protein, polypeptide or pharmaceutical composition as described herein is intended for treatment of a chronic disease in a human being, and/or such sdAb.
  • protein, polypeptide as described herein is intended to be present in the circulation of the subject (i.e. at pharmacologically active levels) to which it is administered (i.e.
  • sdAb, protein, polypeptide as described herein is such that it has a half-life (preferably expressed as tty-beta) in a human subject of at least 3 days, such as at least one week, and up to 10 days or more; and/or such a sdAb, protein, polypeptide or pharmaceutical composition as described herein is intended to be administered to a human being as two or more doses that are administered over a period of at least 3 days, such as at least one week, for example at least two weeks or at least one month, or even longer (i.e. at least 3 months, at least 6 months or at least one year), or even chronically administered.
  • the sdAb molecules of the present disclosure are produced by recombinant DNA technology.
  • a nucleic acid sequence encoding the desired polypeptide is incorporated into an expression vector suitable for the host cell in which expression will be accomplish, the nucleic acid sequence being operably linked to one or more expression control sequences encoding by the vector and functional in the target host cell.
  • the recombinant protein may be recovered through disruption of the host cell or from the cell medium if a secretion leader sequence (signal peptide) is incorporated into the polypeptide.
  • the sdAb molecule is produced by recombinant methods using a nucleic acid sequence encoding the sdAb molecule (or fusion protein comprising the sdAb molecules).
  • the nucleic acid sequence encoding the desired sdAb molecule can be synthesized by chemical means using an oligonucleotide synthesizer.
  • the nucleic acid molecules are not limited to sequences that encode polypeptides; some or all of the non-coding sequences that he upstream or downstream from a coding sequence can also be included.
  • Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can. for example, be generated by the treatment of genomic DNA with restriction endonucleases, or by the performance of the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • nucleic acid molecule is a ribonucleic acid (RNA)
  • RNA ribonucleic acid
  • the nucleic acid molecules encoding the sdAb molecule may contain naturally occurring sequences or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide.
  • These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids.
  • the nucleic acid molecules can be double-stranded or single-stranded (i. e. , either a sense or an antisense strand).
  • Nucleic acid sequences encoding the sdAb molecule may be obtained from various commercial sources that provide custom-made nucleic acid sequences.
  • Amino acid sequence variants of the sdAb molecules of the present disclosure are prepared by introducing appropriate nucleotide changes into the coding sequence based on the genetic code which is well-known in the art. Such variants represent insertions, substitutions, and/or specified deletions of, residues as noted. Any combination of insertion, substitution, and/or specified deletion is made to arrive at the final construct, provided that the final construct possesses the desired biological activity 7 as defined herein.
  • Methods for constructing a DNA sequence encoding a sdAb molecule and expressing those sequences in a suitably transformed host include, but are not limited to, using a PCR- assisted mutagenesis technique. Mutations that consist of deletions or additions of amino acid residues to a sdAb molecule can also be made with standard recombinant techniques. In the event of a deletion or addition, the nucleic acid molecule encoding a sdAb molecule is optionally digested with an appropriate restriction endonuclease. The resulting fragment can either be expressed directly or manipulated further by, for example, ligating it to a second fragment.
  • the ligation may be facilitated if the two ends of the nucleic acid molecules contain complementary 7 nucleotides that overlap one another, but blunt-ended fragments can also be ligated.
  • PCR-generated nucleic acids can also be used to generate various mutant sequences.
  • a sdAb molecule of the present disclosure may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, for example, a signal sequence or other polypeptide having a specific cleavage site at the N-terminus or C-terminus of the mature sdAb molecules.
  • the signal sequence may be a component of the vector, or it may be a part of the coding sequence that is inserted into the vector.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the inclusion of a signal sequence depends on whether it is desired to secrete the sdAb molecule from the recombinant cells in which it is made. If the chosen cells are prokary otic, it generally is preferred that the DNA sequence not encode a signal sequence.
  • the recombinant host cell is ayeast cell such as Saccharomyces cerevisiae
  • the alpha mating factor secretion signal sequence may be employed to achieve extracellular secretion of the sdAb molecule into the culture medium as described in Singh, United States Patent No. 7,198,919 Bl issued April 3. 2007.
  • the sdAb molecule to be expressed is to be expressed as a chimera (e.g., a fusion protein comprising a sdAb molecule and a heterologous polypeptide sequence)
  • the chimeric protein can be encoded by a hybrid nucleic acid molecule comprising a first sequence that encodes all or part of the sdAb molecule and a second sequence that encodes all or part of the heterologous polypeptide.
  • subject sdAb molecules described herein may be fused to a hexa/octa-histidine tag to facilitate purification of bacterially expressed protein, or to a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells.
  • first and second it should not be understood as limiting to the orientation of the elements of the fusion protein and a heterologous polypeptide can be linked at either the N-terminus and/or C- terminus of the sdAb molecules.
  • the N-terminus may be linked to a targeting domain and the C-terminus linked to a hexa-histidine tag purification handle.
  • the complete amino acid sequence of the polypeptide (or fusion/chimera) to be expressed can be used to construct a back-translated gene.
  • a DNA oligomer containing a nucleotide sequence coding a sdAb molecule can be synthesized.
  • several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated.
  • the individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • the nucleic acid sequence encoding the sdAb molecule may be ’ codon-optimized" to facilitate expression in a particular host cell type.
  • Techniques for codon optimization in a wide variety of expression systems, including mammahan, yeast, and bacterial host cells, are well known in the art and online tools are publicly available to provide for codon-optimized sequences for expression in a variety of host cell types. See, e.g., Hawash, et al., (2017) 9:46-53 and Mauro and Chappell in Recombinant Protein Expression in Mammalian Cells: Methods and Protocols, edited by David hacker (Human Press New York). Additionally, there are a variety of web-based online software packages that are freely available to assist in the preparation of codon-optimized nucleic acid sequences.
  • an expression vector For uses in various host cells are available and are typically selected based on the host cell for expression.
  • An expression vector typically includes, but is not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • Vectors include viral vectors, plasmid vectors, integrating vectors, and the like. Plasmids are examples of non- viral vectors.
  • nucleic acid sequence encoding the polypeptide sequence to be expressed is operably linked to transcriptional and translational regulatory’ control sequences that are functional in the chosen expression host.
  • Expression vectors usually contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.
  • Expression vectors for sdAb molecules of the present disclosure contain a regulatory sequence that is recognized by the host organism and is operably linked to the nucleic acid sequence encoding the sdAb molecules.
  • the terms ‘'regulatory control sequence,” “regulatory sequence” or “expression control sequence” are used interchangeably herein to refer to promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press.
  • Regulatory sequences include those that direct constitute expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of the desired protein, and the like. In selecting an expression control sequence, a variety of factors understood by one of skill in the art are to be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the subject a sdAb molecule, particularly as regards potential secondary structures.
  • the regulatory sequence is a promoter, which is selected based on, for example, the cell type in which expression is sought. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of a particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. Many promoters recognized by a variety of potential host cells are well known.
  • a T7 promoter can be used in bacteria, a polyhedrin promoter can be used in insect cells, and a cytomegalovirus or metallothionein promoter can be used in mammalian cells. Also, in the case of higher eukaryotes, tissue-specific, and cell-type-specific promoters are widely available. These promoters are so named for their ability to direct the expression of a nucleic acid molecule in each tissue or cell type within the body. Skilled artisans are aware of numerous promoters and other regulatory elements which can be used to direct the expression of nucleic acids.
  • Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus (such as human adenovirus serotype 5), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (such as murine stem cell virus), hepatitis-B virus and most preferably Simian Virus 40 (SV40). from heterologous mammalian promoters, e.g...
  • viruses such as polyomavirus, fowlpox virus, adenovirus (such as human adenovirus serotype 5), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus (such as murine stem cell virus), hepatitis-B virus and most preferably Simian Virus 40 (SV40). from heterologous mamma
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus.
  • Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence but is preferably located at a site 5' from the promoter.
  • Expression vectors used in eukary otic host cells will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and. occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. Construction of suitable vectors containing one or more of the above-listed components employs standard techniques.
  • vectors can contain origins of replication, and other genes that encode a selectable marker.
  • neomycin-resistance (neoR) gene imparts G418 resistance to cells in which it is expressed, and thus permits phenotypic selection of the transfected cells.
  • marker or reporter genes include beta-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding betagalactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT).
  • CAT chloramphenicol acetyltransferase
  • ADA adenosine deaminase
  • DHFR dihydrofolate reductase
  • HPH hygromycin-B-phosphotransferase
  • TK thymidine kinase
  • lacZ encoding betagalactosidase
  • XGPRT xanthine guanine phosphoribosyltransferas
  • the present disclosure further provides prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule that encodes a sdAb molecule.
  • a cell of the present disclosure is a transfected cell, i.e., a cell into which a nucleic acid molecule, for example a nucleic acid molecule encoding a mutant IL-2 polypeptide, has been introduced by means of recombinant DNA techniques.
  • the progeny of such a cell are also considered within the scope of the present disclosure.
  • Host cells are typically selected in accordance with their compatibility with the chosen expression vector, the toxicity 7 of the product coded for by the DNA sequences of this invention, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the DNA sequences.
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells.
  • the recombinant sdAb molecule can also be made in eukaryotes, such as yeast or human cells.
  • Suitable eukaryotic host cells include insect cells (examples of Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology’ 170:31-39)); yeast cells (examples of vectors for expression in yeast S. cerenvisiae include pYepSecl (Baldari et al.
  • Examples of useful mammalian host cell lines are mouse L cells (L-M[TK-], ATCC#CRL-2648), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or HEK293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO); mouse sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCE 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells;
  • the sdAb molecule may be produced in a prokaryotic host, such as the bacterium E. coli, or in a eukaryotic host, such as an insect cell (e.g., an Sf21 cell), or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, it matters only that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).
  • a sdAb molecule obtained will be glycosylated or nonglycosylated depending on the host organism used to produce the mutein. If bacteria are chosen as the host cell then the a sdAb molecule produced will be nonglycosylated. Eukaryotic cells, on the other hand, will typically result in glycosylation of the sdAb molecules.
  • an amino acid sequence (particularly a CDR sequence) of a sdAb to be incorporated into a sdAb molecule may contain a glycosylation motif, particularly an N-linked glycosylation motif of the sequence Asn-X-Ser (N-X-S) or Asn- X-Thr (N-X-T), wherein X is any amino acid except for proline.
  • N-X-Ser N-X-Ser
  • N-X-Thr N-X-Thr
  • X is any amino acid except for proline.
  • the N-linked glycosylation motif is disrupted by the incorporation of conservative amino acid substitution of the Asn (N) residue of the N-linked glycosylation motif.
  • the expression constructs of the disclosure can be introduced into host cells to thereby produce a sdAb molecule disclosed herein.
  • the expression vector comprising a nucleic acid sequence encoding sdAb molecule is introduced into the prokaryotic or eukaryotic host cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory' Manual (2d ed., Cold Spring Harbor Laboratory' Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.
  • the target cell may be exposed directly with the non-viral vector may’ under conditions that facilitate uptake of the non-viral vector.
  • Examples of conditions which facilitate uptake of foreign nucleic acid by mammalian cells are well known in the art and include but are not limited to chemical means (such as Lipofectamine®, Thermo-Fisher Scientific), high salt, and magnetic fields (electroporation).
  • Cells may be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • Mammalian host cells may be cultured in a variety of media.
  • DMEM Dulbecco's Modified Eagle's Medium
  • Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics, trace elements, and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.
  • Recombinantly produced sdAb molecule polypeptides can be recovered from the culture medium as a secreted polypeptide if a secretion leader sequence is employed.
  • the sdAb molecule polypeptides can also be recovered from host cell lysates.
  • a protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) may be employed during the recovery phase from cell lysates to inhibit proteolytic degradation during purification, and antibiotics may be included to prevent the grow th of adventitious contaminants.
  • PMSF phenyl methyl sulfonyl fluoride
  • affinity chromatography makes use of the highly specific binding sites usually present in biological macromolecules, separating molecules on their ability to bind a particular ligand. Covalent bonds attach the ligand to an insoluble, porous support medium in a manner that overtly presents the ligand to the protein sample, thereby using natural specific binding of one molecular species to separate and purify’ a second species from a mixture.
  • Antibodies are commonly used in affinity chromatography.
  • Size selection steps may also be used, for example, gel filtration chromatography (also known as size-exclusion chromatography or molecular sieve chromatography) is used to separate proteins according to their size.
  • a recombinantly sdAb molecule by the transformed host can be purified according to any suitable method.
  • Recombinant sdAb molecules can be isolated from inclusion bodies generated in E. coli, or from conditioned medium from either mammalian or yeast cultures producing a given mutein using cation exchange, gel filtration, and or reverse phase liquid chromatography.
  • the substantially purified forms of the recombinant a sdAb molecule can be purified from the expression system using routine biochemical procedures, and can be used, for example, as therapeutic agents, as described herein.
  • this purification handle may be used for isolation of the sdAb molecule from the cell lysate or cell medium.
  • the purification tag is a chelating peptide
  • methods for the isolation of such molecules using immobilized metal affinity chromatography are well known in the art. See. e.g., Smith, et al. United States Patent 4.569,794.
  • the biological activity of the sdAb molecules recovered can be assayed for activating by any suitable method known in the art and may be evaluated as substantially purified forms or as part of the cell lysate or cell medium when secretion leader sequences are employed for expression.
  • compositions including pharmaceutical compositions.
  • Such compositions typically include the polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration and is compatible with the therapeutic use for which the sdAb molecule is to be administered to the subject in need of treatment or prophylaxis.
  • Carriers include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by using a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by using surfactants, such as sodium dodecyl sulfate.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.). or phosphate-buffered saline (PBS).
  • buffer includes buffers such as acetates, citrates, or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as mono- and/or di-basic sodium phosphate, hydrochloric acid, or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5).
  • acids or bases such as mono- and/or di-basic sodium phosphate, hydrochloric acid, or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5).
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the pharmaceutical formulations for parenteral administration to a subject should be sterile and should be fluid to facilitate easy syringability. It should be stable under the conditions of manufacture and storage and be preserved against contamination. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Sterile solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Some embodiments of the therapeutic methods of the present disclosure involve the administration of a pharmaceutical formulation comprising a sdAb molecule (and/or nucleic acids encoding the sdAb molecule or recombinantly modified host cells expressing the sdAb molecules) to a subject in need of treatment.
  • the pharmaceutical formulation comprising a sdAb molecule of the present disclosure may be administered to a subject in need of treatment or prophylaxis by a variety of routes of administration, including parenteral administration, oral, topical, or inhalation routes.
  • the methods of the present disclosure involve the parenteral administration of a pharmaceutical formulation comprising a sdAb molecule (and/or nucleic acids encoding the sdAb molecule or recombinantly modified host cells expressing the sdAb molecules) to a subject in need of treatment.
  • parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration.
  • Parenteral formulations comprising solutions or suspensions used for parenteral application can include vehicles, carriers, and buffers.
  • compositions for parenteral administration include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the formulation is provided in a prefilled syringe.
  • the methods of the present disclosure involve the oral administration of a pharmaceutical formulation comprising a sdAb molecule (and/or nucleic acids encoding the sdAb molecule or recombinantly modified host cells expressing the sdAb molecules) to a subject in need of treatment.
  • Oral compositions if used, generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, such as gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. 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, PrimogelTM, or com starch; a lubricant such as magnesium stearate or SterotesTM; 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.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PrimogelTM, or com starch
  • a lubricant such as magnesium stearate or SterotesTM
  • a glidant such as colloidal silicon dioxide
  • the methods of the present disclosure involve the inhaled administration of a pharmaceutical formulation comprising a sdAb molecule (and/or nucleic acids encoding the sdAb molecule or recombinantly modified host cells expressing the sdAb molecules) to a subject in need of treatment.
  • a pharmaceutical formulation comprising a sdAb molecule (and/or nucleic acids encoding the sdAb molecule or recombinantly modified host cells expressing the sdAb molecules) to a subject in need of treatment.
  • subject sdAb molecules, or the nucleic acids encoding them 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.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • the methods of the present disclosure involve the mucosal or transdermal administration of a pharmaceutical formulation comprising a sdAb molecule (and/or nucleic acids encoding the sdAb molecule or recombinantly modified host cells expressing the sdAb molecules) to a subject in need of treatment.
  • a pharmaceutical formulation comprising a sdAb molecule (and/or nucleic acids encoding the sdAb molecule or recombinantly modified host cells expressing the sdAb molecules) to a subject in need of treatment.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • 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 using nasal sprays or suppositories suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art and may incorporate permeation enhancers such as ethanol or lanolin.
  • the sdAb molecule is administered to a subject in need of treatment in a formulation to provide extended release of the sdAb molecule agent.
  • extended-release formulations of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the subject sdAb molecules or nucleic acids are prepared with carriers that will protect the sdAb molecules 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. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals. Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) 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 U.S. Pat. No. 4,522,811.
  • delivery’ of the sdAb molecule to a subject in need of treatment is achieved by the administration of a nucleic acid encoding the sdAb molecules.
  • Methods for the administration of nucleic acid encoding the sdAb molecule to a subject is achieved by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (Nature (2002) 418:6893), Xia et al. (Nature Biotechnol. (2002) 20: 1006-1010), or Putnam (Am. J. Health Syst. Pharm. (1996) 53: 151-160 erratum at Am. J. Health Syst.
  • the sdAb molecule is administered to a subject by the administration of a pharmaceutically acceptable formulation of recombinant expression vector comprising a nucleic acid sequence encoding the sdAb molecule operably linked to one or more expression control sequences operable in a mammalian subject.
  • the expression control sequence may be selected that is operable in a limited range of cell types (or single cell type) to facilitate the selective expression of the sdAb molecule in a particular target cell type.
  • the recombinant expression vector is a viral vector.
  • the recombinant vector is a recombinant viral vector.
  • the recombinant viral vector is a recombinant adeno-associated virus (rAAV) or recombinant adenovirus (rAd), in particular a replication-deficient adenovirus derived from human adenovirus serotypes 3 and/or 5.
  • the replication-deficient adenovirus has one or more modifications to the El region which interfere with the ability of the virus to initiate the cell cycle and/or apoptotic pathways in a human cell.
  • the replication-deficient adenoviral vector may optionally comprise deletions in the E3 domain.
  • the adenovirus is a replication-competent adenovirus.
  • the adenovirus is a replication- competent recombinant virus engineered to selectively replicate in the target cell type.
  • the nucleic acid encoding the sdAb molecule may be delivered to the subject by the administration of a recombinantly modified bacteriophage vector encoding the sdAb molecules.
  • bacteriophage vector encoding the sdAb molecules.
  • Bacteriophage selectively infects procaryotic cells, restricting the expression of the sdAb molecule to procaryotic cells in the subject while avoiding expression in mammalian cells.
  • the phage is modified to remove adj acent motifs (PAM). Elimination of the of Cas9 sequences from the phage genome reduces the ability of the Cas9 endonuclease of the target procaryotic cell to neutralize the invading phage encoding the SdAb molecules.
  • delivery' of the sdAb molecule to a subject in need of treatment is achieved by the administration of recombinant host cells modified to express the sdAb molecule may be administered in the therapeutic and prophylactic applications described herein.
  • the recombinant host cells are mammalian cells, such as human cells.
  • the nucleic acid sequence encoding the sdAb molecule may be maintained extrachromosomally in the recombinantly modified host cell for administration.
  • the nucleic acid sequence encoding the sdAb molecule may be incorporated into the genome of the host cell to be administered using at least one endonuclease to facilitate incorporating an insertion of a nucleic acid sequence into the genomic sequence of the cell.
  • the term “endonuclease” is used to refer to a wild-type or variant enzyme capable of catalyzing the cleavage of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule.
  • Endonucleases are referred to as “rare-cutting” endonucleases when such endonucleases have a polynucleotide recognition site greater than about 12 base pairs (bp) in length, more preferably of 14-55 bp.
  • Rare-cutting endonucleases can be used for inactivating genes at a locus or to integrate transgenes by homologous recombination (HR) i.e. by inducing DNA doublestrand breaks (DSBs) at a locus and insertion of exogenous DNA at this locus by gene repair mechanism.
  • HR homologous recombination
  • DSBs DNA doublestrand breaks
  • rare-cutting endonucleases include homing endonucleases (Grizot, et al (2009) Nucleic Acids Research 37( 16):5405-5419), chimeric Zinc-Finger nucleases (ZFN) resulting from the fusion of engineered zine-finger domains (Porteus M and Carroll D., Gene targeting using zinc finger nucleases (2005) Nature Biotechnology 23(3):967-973, a TALEN- nuclease, a Cas9 endonuclease from CRISPR system as or a modified restriction endonuclease to extended sequence specificity (Eisenschmidt, et al. 2005; 33(22): 7039-7047).
  • the sdAb may be delivered to the subject by a recombinantly modified procary otic cell (e.g., Lactobacillus lacti).
  • a recombinantly modified procary otic cell e.g., Lactobacillus lacti
  • the use of engineered procaryotic cells for the delivery' of recombinant proteins to the intestinal tract are known in the art. See, for example, Lin et al. 2017. Microb Cell Fact 16: 148.
  • the engineered bacterial cell expressing the sdAb may be administered orally, typically in aqueous suspension, or rectally (e g., enema).
  • the present disclosure further provides methods of treating a subject suffering from a disease disorder or condition by the administration of a therapeutically effective amount of an SdAb molecule (or nucleic acid encoding sdAb including recombinant viruses encoding the sdAbs) of the present disclosure.
  • VTVSS (SEQ ID NO: 1) variants of the present disclosure, exemplified using an ILlORa-ILlORb VHH construct as a vehicle for modifying the endogenous VTVSS (SEQ ID NO: 1) motif.
  • ILlORa/ILlORb VHH dimers of SEQ ID NOs: 49-59 were used as test articles in the following experiments to evaluated the effect of the immunogenicity reducing the amino acid substitutions of the present disclosure.
  • the polypeptides molecules described in the Table 11 below were used in the following experiment.
  • the reference sequence DR2485aa (SEQ ID NO:49) retains the characteristic w ild type VTVSS (SEQ ID NO:1) C-terminal motif whereas the derivative molecules incorporate various amino acid substitutions to reduce immunogenicity as may be evidenced in reference to the amino acid sequences provided hereinabove.
  • IL1 ORa/ILl ORb VHH dimers dimers were produced recombinantly using the corresponding DNA sequences provided in a row of Table 11 above. Briefly, nucleic acid encoding the ILlORa/ILlORb VHH test article described in Table XXx further incorporated 5' sequence encoding the mouse IgH signal peptide (Uniprot Reference: Q99LA6) upstream of the ILlORa/ILlORb VHH dimers polypeptide coding sequence.
  • the nucleic acid sequences encoding the signal peptide and ILlORa/ILlORb VHH dimers were inserted using the EcoRl and Notl restriction endonucleases downstream of the CMV promoter of the pExSYN2.0 plasmid, a derivative of the pcDNA3.4 (ThermoFisher) expression vector.
  • the resulting expression vector was used to used to transfect expi293 cells and the cells were cultured for 96 hours in Expi293 expression medium (ThermoFisher Scientific).
  • the proteins were purified by affinity chromatography using MabSelectTM VH3 resin (Cytiva) resin.
  • VHH dimers produced in accordance with the foregoing were diluted to 5 ug/mL in Phosphate Buffered Saline (PBS, Gibco, Cat. # 10010-031) and transferred to wells of a Standard 96-well Multi-Array MSD plate (Meso Scale Discovery, Cat. # L15XA-3). Plates were briefly shaken at room temperature on a plate shaker and then transferred to 4C and incubated overnight to coat the plates. Some w ells w ere left empty, as a negative control. After incubation, wells of all plates were washed three times with lx TRIS-buffered saline (TBS [20x], Thermo Scientific Pierce, Cat.
  • TRIS-buffered saline TRIS-buffered saline
  • Table 12 provides the assay results used to measure pre-existing human anti-VHH AD As. Mutations show varied efficacy in ameliorating the binding of pre-existing human ADAs against camehd VHHs, compared to the parental VHH. These results are also shown graphically in Figs. 1, 2, and 3.
  • Volbarilizumab (also known in the literature as ALX-0061) is bispecific fusion protein comprising an affinity matured, humanized, llama-derived anti-hIL6R (ocIL6R) VHH and an anti-HSA (ocHSA) VHH joined by a GGGGSGGGS (SEQ ID NO: 48) linker and produced in Pichia pastoris. Van Roy, et al.
  • vobarilizumab An analog of vobarilizumab was prepared comprising the amino acid sequences derived from the anti-IL6R (aIL6R) VHH and anti-HSA (ocHSA) VHH of vobarilizumab linked via a GGGS (SEQ ID NO: SEQ ID NO: 47) linker to form DR2514aa (SEQ ID NO: 84).
  • aIL6R anti-IL6R
  • ocHSA anti-HSA
  • the carboxy terminus of the aIL16R VHH domain of DR2514 aa is linked to the amino terminus of the aHS A VHH domain of DR2514aa via a GGGS (SEQ ID NO: 47) linker.
  • a series of aIL-6R-aHSA VHH dimers molecules containing modifications to reduce immunogenicity as described in the present disclosure were designed based on DR2514aa, the amino acid sequences of which are provided in Table 6.
  • the aIL-6R-aHSA VHH dimers polypeptides of Table 6 above were prepaared by generating a synthetic nucleic acid sequence encoding the polypeptide further comprising 5' sequence encoding the mouse IgH signal peptide (Uniprot Reference: Q99LA6) upstream of the aIL-6R-aHSA VHH dimers polypeptide coding sequence.
  • the nucleic acid sequences encoding the signal peptide and mature aIL-6R-aHSA VHH dimers were inserted using the EcoRl and Notl restriction endonucleases downstream of the CMV promoter of the pExSYN2.0 plasmid, a derivative of the pcDNA3.4 (ThermoFisher) expression vector.
  • the resulting expression vector was used to transfect expi293 cells and the cells were cultured for 96 hours in Expi293 expression medium (ThermoFisher Scientific).
  • the proteins were purified by affinity chromatography using MabSelectTM VH3 resin (Cytiva) resin.
  • the polypeptides corresponding to SEQ ID NOs: 84 and 94-109 were used in the following experiments as the polypeptides of Sequence ID NOs: 85-93 did not express efficiently in the foregoing system and were therefore not included in the following experiments.
  • Normalized binding activity 7 was measured in terms of measured bioluminescence (relative light units, or RLU). This data is presented in graphical form in Figure 4 of the attached drawings with relative luminescence units (RLU) plotted on the vertical axis.
  • Table 14 provides the results of a statistical analysis of the normalized binding activity relative to the parent molecule DR2514.
  • a series of molecules based on a parent IL 18 surrogate cytokine agonist were prepared that contained modifications to reduce immunogenicity of the present disclosure. These molecules comprised a VHH molecule that binds to the IL18Ra receptor subunit and a second VHH that binds to the IL18Rb receptor subunit linked via a 10 amino acid linker of the sequence GGGGSGGGGS (SEQ ID NO: 27).
  • the amino acid sequences of the IL18 VHH dimer parent molecule (DR3905) (SEQ ID NO: 110) and the derivative molecules (SEQ ID NOS: 111-134) containing the amino acid substitutions to reduce immunogenicity are provided in Table 7 above.
  • the test article polypeptides of Table 8 were prepared by generating synthetic nucleic acid sequences encoding the above polypeptides as provided in Table 9 below.
  • the name of the nucleic acid sequence in Table 8 (e.g., DR3905- dna) reflects the sequence encoding the corresponding polypeptide of Table 7 (e.g. DR3905aa).
  • nucleic sequences of Table 9 encoding the mature IL18 VHH dimers were further modified by the addition of a 5' sequence encoding the mouse IgH signal peptide (Uniprot Reference: Q99LA6).
  • the nucleic acid sequences encoding the signal peptide and mature IL18 VHH dimers coding sequences of Table 8 were inserted using the EcoRl and Notl restriction endonucleases downstream of the CMV promoter of the pExSYN2.0 plasmid, a derivative of the pcDNA3.4 (ThermoFisher) expression vector.
  • the resulting expression vector was used to used to transfect expi293 cells and the cells were cultured for 96 hours in Expi293 expression medium (ThermoFisher Scientific).
  • the proteins were purified by affinity chromatography using MabSelectTM VH3 resin (Cytiva) resin.
  • the polypeptides corresponding to SEQ ID NOs: 110-134 were used in the following experiments.
  • Table 16 provides the results of a statistical analysis of the normalized binding activity relative to the parent molecule DR3905aa.
  • VHH anti-HSA VHH monomers were prepared based one the anti-HSA VHH of DR2514aa.
  • the parent VHH anti- HSA VHH monomer molecule, DR2830aa (SEQ ID NO: 159) and the derivatives thereof SEQ ID NO: 160-194) which incorporate the amino acid sequence modifications of the present disclosure are provided in Table 9 above.
  • the aHSA VHH monomer polypeptides of SEQ ID NOs: 169-194 of Table 9 above were prepared by generating a synthetic nucleic acid sequence encoding the polypeptide further comprising 5' sequence encoding the mouse IgH signal peptide (Uniprot Reference: Q99LA6) upstream of the aHSA VHH monomer polypeptide coding sequence.
  • the nucleic acid sequences encoding the signal peptide and aHSA VHH monomer were inserted using the EcoRl and Notl restriction endonucleases downstream of the CMV promoter of the pExSYN2.0 plasmid, a derivative of the pcDNA3.4 (ThermoFisher) expression vector.
  • the resulting expression vector was used to transfect expi293 cells and the cells were cultured for 96 hours in Expi293 expression medium (ThermoFisher Scientific).
  • the proteins were purified by affinity chromatography using MabSelectTM VH3 resin (Cytiva) resin.
  • the polypeptides corresponding to SEQ ID NOs: 159, 169-177 and 190-194 were used in the following experiments as the polypeptides of Sequence ID NOs: 178-189 did not express efficiently in the foregoing system and were therefore not included in the following experiments. Immunogenicity was evaluated in substantial accordance with the teaching of Example 2.
  • the data is presented in Figure 6 of the attached drawings. As illustrated by the data presented in Figure 6, the inclusion of the modifications of the present disclosure significantly reduced the immunogenicity of the anti-HSA monomer VHH.

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Abstract

La présente divulgation concerne de manière générale des anticorps à domaine unique (sdAb), tels que des fragments polypeptidiques VH, VHH et scFv, comprenant des modifications C-terminales qui ont une liaison réduite ou éliminée à des anticorps préexistants endogènes, des polypeptides isolés comprenant de telles modifications, des nucléotides codant pour de tels polypeptides, et des procédés d'utilisation de tels polypeptides d'anticorps.
PCT/US2024/033155 2023-06-07 2024-06-07 Domaines lourds variables d'immunoglobuline modifiés ayant une immunogénicité réduite Pending WO2024254562A2 (fr)

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IL324785A IL324785A (en) 2023-06-07 2024-06-07 Modified immunoglobulin variable heavy domains having reduced immunogenicity
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