EP4448574A1 - Polypeptides comprising immunoglobulin single variable domains targeting tcralphabeta, cd33 and cd123 - Google Patents

Polypeptides comprising immunoglobulin single variable domains targeting tcralphabeta, cd33 and cd123

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Publication number
EP4448574A1
EP4448574A1 EP22844638.1A EP22844638A EP4448574A1 EP 4448574 A1 EP4448574 A1 EP 4448574A1 EP 22844638 A EP22844638 A EP 22844638A EP 4448574 A1 EP4448574 A1 EP 4448574A1
Authority
EP
European Patent Office
Prior art keywords
seq
amino acid
polypeptide
acid sequence
isvd
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22844638.1A
Other languages
German (de)
English (en)
French (fr)
Inventor
Helène BONNEVAUX
Melissa DULLAERS
Annelies Roobrouck
Stephanie Staelens
Diane Van Hoorick
Judith VERHELST
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ablynx NV
Sanofi SA
Original Assignee
Ablynx NV
Sanofi SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ablynx NV, Sanofi SA filed Critical Ablynx NV
Publication of EP4448574A1 publication Critical patent/EP4448574A1/en
Pending legal-status Critical Current

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Classifications

    • 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
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present technology relates to polypeptides targeting TCRa
  • AML acute myeloid leukemia
  • AML treatment approaches are aiming at directing cytotoxic T cells to AML cells.
  • the CD33 and the CD123 antigen were found to be overexpressed on blast and leukemic stem cells of most cases of AML and were thus used as suitable tumor-associated target antigens for antibody-based therapies.
  • Mylotarg (Gemtuzumab Ozogamicin; an antiCD33-antibody drug-conjugate) was the first targeted compound registered for the treatment of AML.
  • Recent therapies are based on dual specific antibody constructs targeting one of the tumor antigens CD33 or CD123 on the AML cells and CD3 on cytotoxic T cells.
  • dual-specific antibodies can anchor on CD33+ or CD123+ AML cells and at the same time can bind to CD3 on T cells.
  • the T cells are brought into close proximity to the tumor cell.
  • Multiple binding of the dual-specific antibodies to the tumor antigen (CD33 or CD123) on the tumor cell and simultaneous binding to the TCR-associated CD3 molecules on T cells leads to TCR clustering. This ultimately results in efficient T cell activation irrespective of TCR specificity. Cytotoxic T cell activation near AML cells may then result in tumor cell killing.
  • Dual specific antibody constructs presently tested in clinical trials are, e.g., Flotetuzumab (MGD006; CD3/CD123 DART), AMG330 or AMG673 (both CD3/CD33 BiTEs).
  • the present inventors found that a polypeptide (or ISVD construct) dual targeting CD33 and CD123 on acute myeloid leukemia (AML) cells combined with targeting the T cell receptor a
  • the killing activity on CD33/CD123 double expressing cells was comparable to single targeting benchmarks such as CD33/CD3 AMG 330 BiTE or CD123/CD3 MGD006 DART.
  • the polypeptide of the invention showed robust killing activity on CD33 and CD123 single expressing cells, whereas the single targeting benchmarks showed only activity against the cells expressing their specific target.
  • the polypeptide of the invention induces similar or even lower levels of inflammatory cytokines compared to benchmarks.
  • the polypeptides of the present technology are efficiently produced (e.g. in microbial hosts) and showed low viscosity at high concentrations which is advantageous and convenient for subcutaneous administration. Furthermore, such polypeptides have limited reactivity to pre-existing antibodies in the subject to be treated (i.e. antibodies present in the subject before the first treatment with the antibody construct). In preferred embodiments such polypeptides exhibit a half-life in the subject to be treated that is long enough such that consecutive treatments can be conveniently spaced apart.
  • the polypeptide of the present technology comprises or consists of at least three immunoglobulin single variable domains (ISVDs), wherein at least one ISVD specifically binds to TCRaP, at least one ISVD specifically binds to CD33 and at least one ISVD specifically bind to CD123 (an exemplary polypeptide is illustrated in Figure 1).
  • ISVDs immunoglobulin single variable domains
  • 3 specifically binds to human TCRa
  • the at least one ISVD binding to CD33 specifically binds to human CD33
  • the at least one ISVD binding to CD123 specifically binds to human CD123.
  • the polypeptide preferably further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
  • the binding unit can be an ISVD that binds to a serum protein, preferably to a human serum protein such as human serum albumin.
  • nucleic acid molecule capable of expressing the polypeptide of the present technology, a nucleic acid or vector comprising the nucleic acid, and a composition comprising the polypeptide, the nucleic acid or the vector.
  • the composition is preferably a pharmaceutical composition.
  • a host or host cell comprising the nucleic acid or vector that encodes the polypeptide according to the present technology.
  • a method for producing the polypeptide according to present technology comprising the steps of: a. expressing, optionally in a suitable host cell or host organism or in another suitable expression system, a nucleic acid sequence encoding the polypeptide according to the present technology optionally followed by: b. isolating and/or purifying the polypeptide according to the present technology.
  • the present technology provides the polypeptide, the composition comprising the polypeptide, or the composition comprising the nucleic acid or vector comprising the nucleotide sequence that encodes the polypeptide, for use as a medicament.
  • the polypeptide or composition is for use in the treatment of acute myeloid leukemia (AML), wherein preferably the AML is relapsed and/or refractory AML.
  • AML acute myeloid leukemia
  • a method of treating AML comprising administering, to a subject in need thereof, a pharmaceutically active amount of the polypeptide or a composition according to the present technology.
  • the AML is preferably relapsed and/or refractory AML.
  • the method further comprises administering one or more additional therapeutic agents.
  • polypeptide or composition of the present technology in the preparation of a pharmaceutical composition for treating AML, wherein AML is preferably relapsed and/or refractory AML.
  • the present technology provides the following embodiments:
  • Embodiment 1 A polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid comprising a nucleotide sequence that encodes the polypeptide, for use as a medicament, wherein the polypeptide comprises or consists of at least three immunoglobulin single variable domains (ISVDs), wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), optionally linked via one or more peptidic linkers; and wherein: a) a first ISVD specifically binds to T cell receptor a
  • ISVDs immunoglobulin single variable domains
  • a CDR1 which has the amino acid sequence of SEQ ID NO: 6 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 6
  • a CDR2 which has the amino acid sequence of SEQ ID NO: 10 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 10
  • a CDR3 which has the amino acid sequence of SEQ ID NO: 14 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 14
  • a second ISVD specifically binds to CD33 and comprises iv. a CDR1 which has the amino acid sequence of SEQ ID NO: 7 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 7; v.
  • a CDR2 which has the amino acid sequence of SEQ ID NO: 11 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 11; and vi. a CDR3 which has the amino acid sequence of SEQ ID NO: 15 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 15; and c) a third ISVD specifically binds to CD123 and comprises vii. a CDR1 which has the amino acid sequence of SEQ ID NO: 8 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 8; viii. a CDR2 which has the amino acid sequence of SEQ ID NO: 12 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 12; and ix. a CDR3 which has the amino acid sequence of SEQ ID NO: 16 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 16, wherein the ISVDs are in the order starting from the N-terminus.
  • Embodiment 2 The composition for use according to embodiment 1, which is a pharmaceutical composition which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprises one or more further pharmaceutically active polypeptides and/or compounds.
  • Embodiment 3 The polypeptide or composition for use according to embodiment 1 or 2, wherein: a) said first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:6, a CDR2 having the amino acid sequence of SEQ ID NO: 10 and a CDR3 having the amino acid sequence of SEQ ID NO: 14; b) said second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO: 11 and a CDR3 having the amino acid sequence of SEQ ID NO: 15; and c) said third ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO: 12 and a CDR3 having the amino acid sequence of SEQ ID NO: 16.
  • Embodiment 4 The polypeptide or composition for use according to any of embodiments 1 to 3, wherein: a) the amino acid sequence of said first ISVD has a sequence identity of more than 90% with SEQ ID NO: 2; b) the amino acid sequence of said second ISVD has a sequence identity of more than 90% with SEQ ID NO: 3; and c) the amino acid sequence of said third ISVD has a sequence identity of more than 90% identity with SEQ ID NO: 4.
  • Embodiment 6 The polypeptide or composition for use according to any of embodiments 1 to 5, wherein said polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
  • Embodiment 7 The polypeptide or composition for use according to embodiment 6, in which said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life is chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.
  • Embodiment s The polypeptide or composition for use according to any one of embodiments 6 to 7, in which said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life is chosen from the group consisting of binding units that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).
  • serum albumin such as human serum albumin
  • IgG serum immunoglobulin
  • Embodiment 9 The polypeptide or composition for use according to embodiment 8, in which said binding unit that provides the polypeptide with increased half-life is an ISVD that can bind to human serum albumin.
  • Embodiment 10 The polypeptide or composition for use according to embodiment 9, wherein the ISVD binding to human serum albumin comprises i. a CDR1 which has the amino acid sequence of SEQ ID NO: 9 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 9; ii. a CDR2 which has the amino acid sequence of SEQ ID NO: 13 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 13; and iii. a CDR3 which has the amino acid sequence of SEQ ID NO: 17 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 17.
  • Embodiment 11 The polypeptide or composition for use according to any of embodiments 9 to 10, wherein the ISVD binding to human serum albumin comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 13 and a CDR3 having the amino acid sequence of SEQ ID NO: 17.
  • Embodiment 12 The polypeptide or composition for use according to any of embodiments 9 to 11, wherein the amino acid sequence of said ISVD binding to human serum albumin has a sequence identity of more than 90% with SEQ ID NO: 5.
  • Embodiment 13 The polypeptide or composition for use according to any of embodiments 9 to 12, wherein said ISVD binding to human serum albumin has the amino acid sequence of SEQ ID NO: 5.
  • Embodiment 14 The polypeptide or composition for use according to any of embodiments 1 to 13, wherein the amino acid sequence of the polypeptide has a sequence identity of more than 90% with SEQ ID NO: 1.
  • Embodiment 15 The polypeptide or composition for use according to any of embodiments 1 to 14, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1.
  • Embodiment 16 The polypeptide or composition for use according to any of claims 1 to 15, for use in the treatment of AML.
  • Embodiment 17 The polypeptide or composition for use according to claim 16, wherein the AML is relapsed and/or refractory AML.
  • Embodiment 18 A polypeptide that comprises or consists of at least three immunoglobulin single variable domains (ISVDs), wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), optionally linked via one or more peptidic linkers; and wherein: a) a first ISVD specifically binds to T cell receptor a
  • ISVDs immunoglobulin single variable domains
  • a CDR3 which has the amino acid sequence of SEQ ID NO: 14 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 14;
  • a second ISVD specifically binds to CD33 and comprises iv. a CDR1 which has the amino acid sequence of SEQ ID NO: 7 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 7;
  • v. a CDR2 which has the amino acid sequence of SEQ ID NO: 11 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 11; and vi.
  • a CDR3 which has the amino acid sequence of SEQ ID NO: 15 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 15; and c) a third ISVD specifically binds to CD123 and comprises vii. a CDR1 which has the amino acid sequence of SEQ ID NO: 8 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 8; viii. a CDR2 which has the amino acid sequence of SEQ ID NO: 12 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 12; and ix. a CDR3 which has the amino acid sequence of SEQ ID NO: 16 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 16, wherein the ISVDs are in the order starting from the N-terminus.
  • Embodiment 19 The polypeptide according to embodiment 18, wherein: a) said first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:6, a CDR2 having the amino acid sequence of SEQ ID NO: 10 and a CDR3 having the amino acid sequence of SEQ ID NO: 14; b) said second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO: 11 and a CDR3 having the amino acid sequence of SEQ ID NO: 15; and c) said third ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO: 12 and a CDR3 having the amino acid sequence of SEQ ID NO: 16.
  • Embodiment 20 The polypeptide according to any of embodiments 18 or 19, wherein: a) the amino acid sequence of said first ISVD has a sequence identity of more than 90% with SEQ ID NO: 2; b) the amino acid sequence of said second ISVD has a sequence identity of more than 90% with SEQ ID NO: 3; and c) the amino acid sequence of said third ISVD has a sequence identity of more than 90% identity with SEQ ID NO: 4.
  • Embodiment 21 The polypeptide according to any of embodiments 18 to 20, wherein: a) said first ISVD has the amino acid sequence of SEQ ID NO: 2; b) said second ISVD has the amino acid sequence of SEQ ID NO: 3; and c) said third ISVD has the amino acid sequence of SEQ ID NO: 4.
  • Embodiment 22 The polypeptide according to any of embodiments 18 to 21, wherein said polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
  • Embodiment 23 The polypeptide according to embodiment 22, in which said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life is chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.
  • Embodiment 24 The polypeptide according to any one of embodiments 22 to 23, in which said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life is chosen from the group consisting of binding units that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).
  • serum albumin such as human serum albumin
  • serum immunoglobulin such as IgG
  • Embodiment 25 The polypeptide according to embodiment 24, in which said binding unit that provides the polypeptide with increased half-life is an ISVD that can bind to human serum albumin.
  • Embodiment 26 The polypeptide according to embodiment 25, wherein the ISVD binding to human serum albumin comprises i. a CDR1 which has the amino acid sequence of SEQ ID NO: 9 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 9; ii. a CDR2 which has the amino acid sequence of SEQ ID NO: 13 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 13; and iii. a CDR3 which has the amino acid sequence of SEQ ID NO: 17 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 17.
  • Embodiment 27 The polypeptide according to any of embodiments 25 to 26, wherein the ISVD binding to human serum albumin comprises a CDR1 having the amino acid sequence of SEQ ID NO:9, a CDR2 having the amino acid sequence of SEQ ID NO: 13 and a CDR3 having the amino acid sequence of SEQ ID NO: 17.
  • Embodiment 28 The polypeptide according to any of embodiments 25 to 27, wherein the amino acid sequence of said ISVD binding to human serum albumin has a sequence identity of more than 90% with SEQ ID NO: 5.
  • Embodiment 29 The polypeptide according to any of embodiments 25 to 28, wherein said ISVD binding to human serum albumin has the amino acid sequence of SEQ ID NO: 5.
  • Embodiment 30 The polypeptide according to any of embodiments 18 to 29, wherein the amino acid sequence of the polypeptide has a sequence identity of more than 90% with SEQ ID NO: 1.
  • Embodiment 31 The polypeptide according to any of embodiments 18 to 29, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1.
  • Embodiment 32 A nucleic acid comprising a nucleotide sequence that encodes a polypeptide according to any of embodiments 18 to 31.
  • Embodiment 33 A host or host cell comprising a nucleic acid according to embodiment 32.
  • Embodiment 34 A method for producing a polypeptide according to any of embodiments 18-31, said method at least comprising the steps of: a) expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid according to embodiment 32; optionally followed by: b) isolating and/or purifying the polypeptide according to any of embodiments 18 to 31.
  • Embodiment 35 A composition comprising at least one polypeptide according to any of embodiments 18 to 31, or a nucleic acid according to embodiment 32.
  • Embodiment 36 The composition according to embodiment 35, which is a pharmaceutical composition which further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprises one or more further pharmaceutically active polypeptides and/or compounds.
  • Embodiment 37 A method of treating AML, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of a polypeptide according to any of claims 18 to 31 or a composition according to any of claims 35 to 36.
  • Embodiment 38 The method according to claim 37, wherein the AML is relapsed and/or refractory AML.
  • Embodiment 39 Use of a polypeptide according to any of claims 18 to 31 or a composition according to any of claims 35 to 36, in the preparation of a pharmaceutical composition for treating AML.
  • Embodiment 40 Use of the polypeptide or a composition according to claims 39, wherein the AML is relapsed and/or refractory AML.
  • Figure 1 Schematic presentation of the multispecific ISVD construct according to the invention showing from the N-terminus to the C-terminus the monovalent building blocks/ISVDs TCRaP, CD33, CD123, and Alb connected via linkers.
  • Figure 2 Binding of the monovalent CD33 (left) and CD123 binding BB (right) to human (top) or cynomolgus (bottom) transfected CD33, respectively CD123 cells.
  • Figure 3 Binding of A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO.: 1) to human CD33 and/or human CD123 expressing cells.
  • Figure 4 Dose dependent inhibition of A025001562, TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO.: 1 (black squares) and reference TCR (grey dots) in the competition assays on primary T cells in the absence (dotted curves) or presence (full curves) of clinical grade HSA.
  • Figure 5 Dose dependent human (top) or cynomolgus (bottom) T cell mediated killing of corresponding species CD33 (left) or CD123 (right) transfected cells, using an effector to target ratio of 15 to 1 in an impedance-based assay (xCELLigence) in the presence of 50 pM HSA.
  • FIG. 6 Dose dependent human (left) or cynomolgus (right) T cell mediated MOLM-13 cell killing in a flow cytometry-based assay using an effector to target ratio of 10:1.
  • the % TO- PRO®-3 positive target cells is plotted against the concentration of the ISVD.
  • Figure 7 Dose dependent human T cell mediated cell killing using an effector to target ratio of 15 to 1 in an impedance-based assay (xCELLigence). The cell index (Cl) after 32-35 h of incubation is plotted against the concentration of the ISVDs.
  • Figure 8 A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO.: 1) inhibition of Molml3-luc AML tumor growth by in vivo bioluminescence imaging.
  • Figure 9 A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO.: 1) inhibition of Molml3-luc AML tumor growth by ex vivo bioluminescence imaging.
  • Figure 10 Dose dependent human T cell mediated killing of the ISVDs according to the invention compared to CD123 and CD33 positive controls in MOLM-13 cells.
  • Figure 11 Dose dependent human T cell mediated killing of the ISVDs according to the invention compared to CD123 and CD33 positive controls in KG-la cells.
  • Figure 12 Dose dependent human T cell mediated killing of the ISVDs according to the invention compared to CD123 and CD33 positive controls in U-937 cells.
  • Figure 13 Dose dependent monocyte depletion of the ISVD according to the invention compared to CD123 and CD33 positive controls as well as a negative control (untargeted TCE) within human peripheral blood mononuclear cells (PBMCs).
  • Figure 14 Dose dependent cytokine release of the ISVD according to the invention compared to CD123 and CD33 positive controls as well as a negative control (untargeted TCE) in human PBMCs from a healthy donor using a panel of different cytokines.
  • D. IL-2 Dose dependent monocyte depletion of the ISVD according to the invention compared to CD123 and CD33 positive controls as well as a negative control (untargeted TCE) within human peripheral blood mononuclear cells (PBMCs).
  • Figure 15 AML blast killing of the ISVD according to the invention versus CD33 and CD123 positive controls in all tested patients.
  • Figure 16 Scatter plot depicting the percentage of CD33 or CD123 positive cells per AML sample.
  • Figure 17 Cell viability of primary blasts from AML patients with a broad range of disease subtypes to the ISVD according to the invention compared to CD123 and CD33 positive controls as well as a negative control.
  • Figure 18 Individual absolute cell counts of total CD123+ T cells (A), monocytes CD33+ cells (B), CD4+ T cells (C) and CD8+ T cells (D) as measured in peripheral blood of cynomolgus monkey treated with the ISVD according to the invention over time.
  • Animals Ml and M2 received 0.04 pg/kg, while M3 and M4 received 0.4 pg/kg of a single 1-hour continuous intravenous infusion of a solution of the ISVD according to the invention.
  • the present technology aims at providing a novel type of drug for treating acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the present inventors found that a polypeptide (or ISVD construct) dual targeting of CD33 and CD123 on acute myeloid leukemia (AML) cells combined with targeting the T cell receptor a
  • the killing activity was comparable to or even higher compared to single targeting benchmarks such as CD33/CD3 AMG 330 BiTE or CD123/CD3 MGD006 DART. Due to the high heterogeneity of CD33 and CD123 expression on AML cells in intra- and inter-patient samples, the polypeptide of the present invention provides a broader patient coverage compared to the single-targeting benchmarks.
  • the polypeptides of the present technology are efficiently produced (e.g. in microbial hosts) and showed low viscosity at high concentrations which is advantageous and convenient for subcutaneous administration. Furthermore, such polypeptides have limited reactivity to pre-existing antibodies in the subject to be treated (i.e. antibodies present in the subject before the first treatment with the antibody construct). In preferred embodiments such polypeptides exhibit a half-life in the subject to be treated that is long enough such that consecutive treatments can be conveniently spaced apart.
  • the polypeptide is at least bispecific, but can also be e.g., trispecific, tetraspecific or pentaspecific. Moreover, the polypeptide is at least tetravalent, but can also be e.g. pentavalent or hexavalent, etc.
  • bispecific all fall under the term “multispecific” and refer to binding to two, three, four or five different target molecules, respectively.
  • the terms “bivalent”, “trivalent”, “tetravalent” , “pentavalent”, or “hexavalent” all fall under the term “multivalent” and indicate the presence of two, three, four or five binding units (such as ISVDs), respectively.
  • the polypeptide may be tetrapecific- tetravalent, such as a polypeptide comprising or consisting of four ISVDs, wherein one ISVD binds to human TCRa
  • a polypeptide may at the same time be biparatopic, for example if two ISVDs bind two different epitopes on the same target, e.g., if two ISVDs bind to TCRa
  • the term "biparatopic" refers to binding to two different parts (e.g., epitopes) of the same target molecule.
  • first ISVD first ISVD
  • second ISVD second ISVD
  • third ISVD etc.
  • the polypeptide can further comprise another ISVD binding to human serum albumin that can be a fourth ISVD located C- terminally to the at least three ISVDs.
  • the polypeptide can further comprise another ISVD that can even be located between e.g., the "second ISVD” and "third ISVD”.
  • the invention provides a polypeptide comprising or consisting of at least three ISVDs, wherein at least one ISVD specifically binds to TCRa
  • the components, preferably ISVDs, of the polypeptide may be linked to each other by one or more suitable linkers, such as peptidic linkers.
  • linkers to connect two or more (poly)peptides is well known in the art. Exemplary peptidic linkers are shown in Table A-5. One often used class of peptidic linker are known as the "Gly-Ser” or “GS” linkers. These are linkers that essentially consist of glycine (G) and serine (S) residues, and usually comprise one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 77) motif (for example, having the formula (Gly-Gly-Gly-Gly-Gly-Ser)n in which n may be 1, 2, 3, 4, 5, 6, 7 or more).
  • GGGGS SEQ ID NO: 77
  • 3 is positioned at the N- terminus of the polypeptide.
  • the ISVD specifically binding to CD33 is positioned C- terminally to the ISVD specifically binding to TCRa
  • ISVD specifically binding to CD123 is positioned C- terminally to the ISVD specifically binding to CD33, which is itself C-terminally positioned to the ISVD specifically binding to TCRa
  • the polypeptide comprises or consists of the following, in the order starting from the N-terminus of the polypeptide: a first ISVD specifically binding to TCRaP, a second ISVD specifically binding to CD33 and a third ISVD specifically binding to CD123, and an optional binding unit providing the polypeptide with increased half-life as defined herein.
  • the binding unit providing the polypeptide with increased half-life is preferably an ISVD.
  • the polypeptide comprises or consists of the following, in the order starting from the N-terminus of the polypeptide: an ISVD specifically binding to TCRa
  • Such configurations of the polypeptide can provide for increased production yield, good CMC characteristics as well as optimized functionality and stronger potency with regard to modulation of an immune response.
  • the polypeptide of the present technology exhibits reduced binding by preexisting antibodies in human serum.
  • the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD, but preferably in each ISVD.
  • the polypeptide comprises an extension of 1 to 5 (preferably naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C- terminal ISVD.
  • the C-terminus of an ISVD is normally VTVSS (SEQ ID NO: 93).
  • polypeptide comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD.
  • ISVD comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) in at least one ISVD.
  • the C-terminus of the ISVD is VKVSS (SEQ ID NO: 94), VQVSS (SEQ ID NO: 95), VTVKS (SEQ ID NO: 96), VTVQS (SEQ ID NO: 97), VKVKS (SEQ ID NO: 98), VKVQS (SEQ ID NO: 99), VQVKS (SEQ ID NO: 100), or VQVQS (SEQ ID NO: 101) such that after addition of a single alanine the C-terminus of the polypeptide for example comprises the sequence VTVSSA (SEQ ID NO: 102), VKVSSA (SEQ ID NO: 103), VQVSSA (SEQ ID NO: 104), VTVKSA (SEQ ID NO: 105), VTVQSA (SEQ ID NO: 106), VKVKSA (SEQ ID NO: 107), VKVQSA (SEQ ID NO: 108), VQVKSA (SEQ ID NO: 109), or VQV
  • the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD, optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and comprises an extension of 1 to 5 (preferably naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C-terminal ISVD (such that the C-terminus of the polypeptide for example comprises the sequence VTVSSA (SEQ ID NO: 102), VKVSSA (SEQ ID NO: 103) or VQVSSA (SEQ ID NO: 104), preferably VTVSSA (SEQ ID NO: 102)).
  • VTVSSA SEQ ID NO: 102
  • VKVSSA SEQ ID NO: 103
  • VQVSSA SEQ ID NO: 104
  • the polypeptide of the invention comprises or consists of an amino acid sequence having a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 1, wherein the CDRs of the four ISVDs are as defined in items A to D (or A' to D' if using the Kabat definition) set forth in sections "Immunoglobulin single variable domains" and "(In vivo) half-life extension” below, respectively, wherein in particular:
  • 3 has a CDR1 having the amino acid sequence of SEQ ID NO: 6, a CDR2 having the amino acid sequence of SEQ ID NO: 10 and a CDR3 having the amino acid sequence of SEQ ID NO: 14;
  • the ISVD specifically binding to CD33 has a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 11 and a CDR3 having the amino acid sequence of SEQ ID NO: 15;
  • the ISVD specifically binding to CD123 has a CDR1 having the amino acid sequence of SEQ ID NO: 8, a CDR2 having the amino acid sequence of SEQ ID NO: 12 and a CDR3 having the amino acid sequence of SEQ ID NO: 16; and • the ISVD binding to human serum albumin has a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 13 and a CDR3 having the amino acid sequence of SEQ ID NO: 17, or alternatively if using the Kabat definition:
  • 3 has a CDR1 having the amino acid sequence of SEQ ID NO: 34, a CDR2 having the amino acid sequence of SEQ ID NO: 38 and a CDR3 having the amino acid sequence of SEQ ID NO: 42;
  • the ISVD specifically binding to CD33 has a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 39 and a CDR3 having the amino acid sequence of SEQ ID NO: 43;
  • the ISVD specifically binding to CD123 has a CDR1 having the amino acid sequence of SEQ ID NO: 36, a CDR2 having the amino acid sequence of SEQ ID NO: 40 and a CDR3 having the amino acid sequence of SEQ ID NO: 44; and
  • the ISVD binding to human serum albumin has a CDR1 having the amino acid sequence of SEQ ID NO: 37, a CDR2 having the amino acid sequence of SEQ ID NO: 41 and a CDR3 having the amino acid sequence of SEQ ID NO: 45.
  • polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 1.
  • polypeptide consists of the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide of the invention preferably has at least half the binding affinity, more preferably at least the same binding affinity, to human TCRa
  • immunoglobulin single variable domain (ISVD), interchangeably used with “single variable domain”, defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from “conventional” immunoglobulins (e.g. monoclonal antibodies) or their fragments (such as Fab, Fab', F(ab')z, scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site.
  • conventional immunoglobulins e.g. monoclonal antibodies
  • fragments such as Fab, Fab', F(ab')z, scFv, di-scFv
  • VH heavy chain variable domain
  • VL light chain variable domain
  • CDRs complementarity determining regions
  • the antigen-binding domain of a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
  • a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
  • a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of
  • immunoglobulin single variable domains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain.
  • the binding site of an immunoglobulin single variable domain is formed by a single VH, a single VHH or single VL domain.
  • the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a Vn-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).
  • a light chain variable domain sequence e.g., a VL-sequence
  • a heavy chain variable domain sequence e.g., a Vn-sequence or VHH sequence
  • An immunoglobulin single variable domain can for example be a heavy chain ISVD, such as a VH, VHH, including a camelized VH or humanized VHH. Preferably, it is a VHH, including a camelized VH or humanized VHH.
  • Heavy chain ISVDs can be derived from a conventional four-chain antibody or from a heavy chain antibody.
  • the immunoglobulin single variable domain may be a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb” or dAb (or an amino acid sequence that is suitable for use as a dAb) (as defined herein, and including but not limited to a NANOBODY® ISVD); other single variable domains, or any suitable fragment of any one thereof.
  • the immunoglobulin single variable domain may be a NANOBODY® ISVD, including a humanized VHH or camelized VH, or a suitable fragment thereof.
  • NANOBODY®, NANOBODIES® and NANOCLONE® are registered trademarks of Ablynx N.V.
  • VHH domains also known as VHHS, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin variable domain of "heavy chain antibodies” (i.e., of "antibodies devoid of light chains”; Hamers-Casterman et al. Nature 363: 446-448, 1993).
  • VHH domain has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains").
  • VHH'S For a further description of VHH'S, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V.
  • immunoglobulins typically involve the immunization of experimental animals, fusion of immunoglobulin producing cells to create hybridomas and screening for the desired specificities.
  • immunoglobulins can be generated by screening of naive or synthetic libraries e.g. by phage display.
  • immunoglobulin sequences such as NANOBODY® ISVDs
  • WO 94/04678 Hamers- Casterman et al. 1993
  • Muyldermans et al. 2001 Reviews in Molecular Biotechnology 74: 277-302, 2001
  • camelids are immunized with the target antigen in order to induce an immune response against said target antigen.
  • the repertoire of NANOBODY® ISVDs obtained from said immunization is further screened for ISVDs that bind the target antigen.
  • Antigens can be purified from natural sources, or in the course of recombinant production.
  • Immunization and/or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.
  • the invention may use immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences.
  • the invention also includes fully human, humanized or chimeric sequences.
  • the invention comprises camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g. camelized dAb as described by Ward et al (see for example WO 94/04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. Eng., 9:531- 537, 1996).
  • the invention also uses fused immunoglobulin sequences, e.g.
  • a multivalent and/or multispecific construct for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001, as well as to for example WO 96/34103 and WO 99/23221), and immunoglobulin sequences comprising tags or other functional moieties, e.g. toxins, labels, radiochemicals, etc., which are derivable from the immunoglobulin sequences of the present invention.
  • a “humanized VHH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized” , i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g. indicated above).
  • This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art (e.g. WO 2008/020079).
  • humanized VHHS can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.
  • a “camelized VH” comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been "camelized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody.
  • This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art (e.g. WO 2008/020079).
  • the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is preferably a VH sequence from a mammal, more preferably the VH sequence of a human being, such as a VH3 sequence.
  • camelized VH can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.
  • one or more immunoglobulin sequences may be linked to each other and/or to other amino acid sequences (e.g. via disulphide bridges) to provide peptide constructs that may also be useful in the invention (for example Fab' fragments, F(ab')2 fragments, scFv constructs, "diabodies” and other multispecific constructs).
  • peptide constructs that may also be useful in the invention (for example Fab' fragments, F(ab')2 fragments, scFv constructs, "diabodies” and other multispecific constructs).
  • a preferred structure of an immunoglobulin single variable domain sequence can be considered to be comprised of four framework regions ("FRs”), which are referred to in the art and herein as “Framework region 1" (“FR1”); as “Framework region 2” (“FR2”); as “Framework region 3” ("FR3”); and as “Framework region 4" ("FR4"), respectively; which framework regions are interrupted by three complementary determining regions (“CDRs”), which are referred to in the art and herein as “Complementarity Determining Region 1" (“CDR1”); as “Complementarity Determining Region 2" (“CDR2”); and as “Complementarity Determining Region 3" (“CDR3”), respectively.
  • CDRs complementary determining regions
  • amino acid residues of an immunoglobulin single variable domain can be numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see for example Figure 2 of this publication).
  • the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (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 Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
  • the total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.
  • FR1 comprises the amino acid residues at positions 1-25
  • CDR1 comprises the amino acid residues at positions 26-35
  • FR2 comprises the amino acids at positions 36-49
  • CDR2 comprises the amino acid residues at positions 50-58
  • FR3 comprises the amino acid residues at positions 59-94
  • CDR3 comprises the amino acid residues at positions 95-102
  • FR4 comprises the amino acid residues at positions 103-113.
  • CDR regions may also be done according to different methods.
  • FR1 of an immunoglobulin single variable domain comprises the amino acid residues at positions 1-30
  • CDR1 of an immunoglobulin single variable domain comprises the amino acid residues at positions 31-35
  • FR2 of an immunoglobulin single variable domain comprises the amino acids at positions 36-49
  • CDR2 of an immunoglobulin single variable domain comprises the amino acid residues at positions 50-65
  • FR3 of an immunoglobulin single variable domain comprises the amino acid residues at positions 66-94
  • CDR3 of an immunoglobulin single variable domain comprises the amino acid residues at positions 95-102
  • FR4 of an immunoglobulin single variable domain comprises the amino acid residues at positions 103-113.
  • the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.
  • the framework sequences are preferably (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization).
  • the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a V sequence) and/or from a heavy chain variable domain (e.g. a VH- sequence or VHH sequence).
  • the framework sequences are either framework sequences that have been derived from a VHH-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences that have been camelized (as defined herein).
  • the framework sequences present in the ISVD sequence used in the invention may contain one or more of hallmark residues (as defined herein), such that the ISVD sequence is a NANOBODY® immunoglobulin variable domain, including a humanized VHH or camelized VH.
  • hallmark residues as defined herein
  • the ISVD sequence is a NANOBODY® immunoglobulin variable domain, including a humanized VHH or camelized VH.
  • suitable fragments or combinations of fragments of any of the foregoing, such as fragments that contain one or more CDR sequences, suitably flanked by and/or linked via one or more framework sequences (for example, in the same order as these CDR's and framework sequences may occur in the full-sized immunoglobulin sequence from which the fragment has been derived).
  • the invention is not limited as to the origin of the ISVD sequence (or of the nucleotide sequence used to express it), nor as to the way that the ISVD sequence or nucleotide sequence is (or has been) generated or obtained.
  • the ISVD sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences.
  • the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized” (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), "camelized” (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.
  • immunoglobulin sequences such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences
  • camelized as defined herein immunoglobulin
  • nucleotide sequences may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.
  • a suitable naturally occurring template e.g. DNA or RNA isolated from a cell
  • nucleotide sequences that have been isolated from a library and in particular, an expression library
  • nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence using any suitable technique known per
  • an ISVD may be a NANOBODY® VHH or a suitable fragment thereof.
  • NANOBODY® ISVDs For a general description of NANOBODY® ISVDs, reference is made to the further description below, as well as to the prior art cited herein. In this respect, it should however be noted that this description and the prior art mainly described NANOBODY® ISVDs of the so-called "VH3 class" (i.e. NANOBODY® ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51 or DP-29).
  • VH3 class i.e. NANOBODY® ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51 or DP-29.
  • NANOBODY® ISVD can generally use any type of NANOBODY® ISVD, and for example also uses the NANOBODY® ISVDs belonging to the so-called "VH4 class" (i.e. NANOBODY® ISVDs with a high degree of sequence homology to human germline sequences of the VH4 class such as DP-78), as for example described in WO 2007/118670.
  • VH4 class i.e. NANOBODY® ISVDs with a high degree of sequence homology to human germline sequences of the VH4 class such as DP-78
  • NANOBODY® ISVDs in particular VHH sequences, including (partially) humanized VHH sequences and camelized VH sequences
  • VHH sequences including (partially) humanized VHH sequences and camelized VH sequences
  • a NANOBODY® ISVD can be defined as an immunoglobulin sequence with the (general) structure
  • FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined herein.
  • NANOBODY® ISVD can be an immunoglobulin sequence with the (general) structure
  • FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein.
  • NANOBODY® ISVD can be an immunoglobulin sequence with the (general) structure
  • FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which: one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table 1 below.
  • the present technology inter alia uses ISVDs that can specifically bind to TCRa
  • binding to a certain target molecule has the usual meaning in the art as understood in the context of antibodies and their respective antigens.
  • the polypeptide of the present technology may comprise one or more ISVDs specifically binding to TCRa
  • the ISVDs used in the present technology form part of a polypeptide of the present technology, which comprises or consists of at least three ISVDs, such that the polypeptide can specifically bind to TCRa
  • the polypeptide can anchor on CD33+CD123+ leukemia stem cells (LSCs) and acute myeloid leukemia (AML) blasts and at the same time can bind to TCRa
  • LSCs leukemia stem cells
  • AML acute myeloid leukemia
  • T cell activation in proximity to the LSCs and AML blasts may lead to efficient tumor cell killing and consequently to therapeutic efficacy in AML patients.
  • the target molecules of the at least three ISVDs as used in the polypeptide of the present technology are TCRa
  • Examples are mammalian CD33, CD123 and TCRa
  • 3 Uniprot accession
  • human CD33 Uniprot accession
  • human CD123 Uniprot accession
  • the versions from other species are also amenable to the present technology, for example TCRa
  • 3, CD123 and CD123 that can be used in the present technology are as described in the following items A to C:
  • An ISVD that specifically binds to human TCRa
  • 3 have one or more, and preferably all, framework regions as indicated for the TCRa
  • 3 may have a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 2, wherein optionally the CDRs are as defined in the preceding item A.
  • 3 preferably has the amino acid sequence of SEQ ID NO: 2.
  • the ISVD when such an ISVD specifically binding to TCRa
  • a CDR2 which has the amino acid sequence SEQ ID NO: 11 or has 2 or 1 amino acid difference with SEQ ID NO: 11; and iii. a CDR3 which has the amino acid sequence SEQ ID NO: 15 or has 2 or 1 amino acid difference with SEQ ID NO: 15, preferably a CDR1 having the amino acid sequence of SEQ ID NO: 7, a CDR2 having the amino acid sequence of SEQ ID NO: 11 and a CDR3 having the amino acid sequence of SEQ ID NO: 15.
  • Preferred examples of such an ISVD that specifically binds to human CD33 have one or more, and preferably all, framework regions as indicated for CD33-VHH in Table A-2 (in addition to the CDRs as defined in the preceding item B), and most preferred is an ISVD having the full amino acid sequence of CD33-VHH (SEQ ID NO: 3, see Table A-l and A-2).
  • the amino acid sequence of an ISVD specifically binding to human CD33 may have a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 3, wherein optionally the CDRs are as defined in the preceding item B.
  • the ISVD binding to CD33 preferably has the amino acid sequence of SEQ ID NO: 3.
  • the ISVD when such an ISVD specifically binding to CD33 has 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (item B above), the ISVD preferably has at least half the binding affinity, more preferably at least the same binding affinity, to human CD33 as construct CD33-VHH (SEQ ID NO: 3), wherein the binding affinity is measured using the same method, such as SPR.
  • An ISVD that specifically binds to human CD123 and comprises i. a CDR1 which has the amino acid sequence of SEQ ID NO: 8 or has 2 or 1 amino acid difference with SEQ ID NO: 8; ii. a CDR2 which has the amino acid sequence of SEQ ID NO: 12 or has 2 or 1 amino acid difference with SEQ ID NO: 12; and iii. a CDR3 which has the amino acid sequence of SEQ ID NO: 16 or has 2 or 1 amino acid difference with SEQ ID NO: 16, preferably a CDR1 having the amino acid sequence of SEQ ID NO: 8, a CDR2 having the amino acid sequence of SEQ ID NO: 12 and a CDR3 having the amino acid sequence of SEQ ID NO: 16.
  • Preferred examples of such an ISVD that specifically binds to human CD123 have one or more, and preferably all, framework regions as indicated for CD123-VHH in Table A-2 (in addition to the CDRs as defined in the preceding item C), and most preferred is an ISVD having the full amino acid sequence of CD123-VHH (SEQ ID NO: 4, see Table A-l and A-2).
  • the amino acid sequence of an ISVD specifically binding to human CD123 may have a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 4, wherein optionally the CDRs are as defined in the preceding item C.
  • the ISVD binding to CD123 preferably has the amino acid sequence of SEQ ID NO: 4.
  • the ISVD when such an ISVD specifically binding to CD123 has 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (item C above), the ISVD preferably has at least half the binding affinity, more preferably at least the same binding affinity, to human CD123 as CD123-VHH (SEQ ID NO: 4), wherein the binding affinity is measured using the same method, such as SPR.
  • each of the ISVDs as defined under items A to C above is comprised in the polypeptide of the invention.
  • a polypeptide of the invention comprising each of the ISVDs as defined under items A to C above preferably has at least half the binding affinity, more preferably at least the same binding affinity, to human TCRa
  • the SEQ ID NOs referred to in the above items A to C are based on the CDR definition according to the AbM definition (see Table A-2). It is noted that the SEQ ID NOs defining the same CDRs according to the Kabat definition (see Table A-2.1) can likewise be used in the above items A to C.
  • An ISVD that specifically binds to human TCRa
  • 3 have one or more, and preferably all, framework regions as indicated for TCROP-VHH in Table A-2.1 (in addition to the CDRs as defined in the preceding item A'), and most preferred is an ISVD having the full amino acid sequence of TCROI
  • An ISVD that specifically binds to human CD33 and comprises i. a CDR1 which has the amino acid sequence SEQ ID NO: 35 or has 2 or 1 amino acid difference with SEQ ID NO: 35; ii. a CDR2 which has the amino acid sequence SEQ ID NO: 39 or has 2 or 1 amino acid difference with SEQ ID NO: 39; and iii. a CDR3 which has the amino acid sequence SEQ ID NO: 43 or has 2 or 1 amino acid difference with SEQ ID NO: 43, preferably a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 39 and a CDR3 having the amino acid sequence of SEQ ID NO: 43.
  • Preferred examples of such an ISVD that specifically binds to human CD33 have one or more, and preferably all, framework regions as indicated for CD33-VHH in Table A-2.1 (in addition to the CDRs as defined in the preceding item B'), and most preferred is an ISVD having the full amino acid sequence of CD33-VHH (SEQ ID NO: 3, see Table A-l and A-2.1).
  • An ISVD that specifically binds to human CD123 and comprises i. a CDR1 which has the amino acid sequence of SEQ ID NO: 36 or has 2 or 1 amino acid difference with SEQ ID NO: 36; ii. a CDR2 which has the amino acid sequence of SEQ ID NO: 40 or has 2 or 1 amino acid difference with SEQ ID NO: 40; and iii. a CDR3 which has the amino acid sequence of SEQ ID NO: 44 or has 2 or 1 amino acid difference with SEQ ID NO: 44, preferably a CDR1 having the amino acid sequence of SEQ ID NO: 36, a CDR2 having the amino acid sequence of SEQ ID NO: 40 and a CDR3 having the amino acid sequence of SEQ ID NO: 44.
  • Preferred examples of such an ISVD that specifically binds to human CD123 have one or more, and preferably all, framework regions as indicated for CD123-VHH in Table A-2.1 (in addition to the CDRs as defined in the preceding item C'), and most preferred is an ISVD having the full amino acid sequence of CD123-VHH (SEQ ID NO: 4, see Table A-l and A-2.1).
  • the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (i.e. at a single position).
  • amino acid sequence with the greatest number of amino acid residues will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence.
  • amino acid difference refers to a deletion, insertion or substitution of a single amino acid residue vis-a-vis a reference sequence, and preferably is a substitution.
  • Amino acid substitutions are preferably conservative substitutions.
  • Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, lie, Vai and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; lie into Leu or into Vai; Leu into lie or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into lie; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Vai, into lie or into Leu.
  • binding specifically refers to the number of different target molecules, such as antigens, from the same organism to which a particular binding unit, such as an ISVD, can bind with sufficiently high affinity (see below). “Specificity”, “binding specifically” or “specific binding” are used interchangeably herein with “selectivity”, “binding selectively” or “selective binding”. Binding units, such as ISVDs, preferably specifically bind to their designated targets.
  • the specificity /selectivity of a binding unit can be determined based on affinity.
  • the affinity denotes the strength or stability of a molecular interaction.
  • the affinity is commonly given as by the KD, or dissociation constant, comprising units of mol/liter (or M).
  • the affinity can also be expressed as an association constant, KA, which equals 1/KD and has units of (mol/liter) 1 (or M 1 ).
  • the affinity is a measure for the binding strength between a moiety and a binding site on the target molecule: the lesser the value of the KD, the stronger the binding strength between a target molecule and a targeting moiety.
  • binding units used in the present technology will bind to their targets with a dissociation constant (KD) of 10 -5 to 10 12 moles/liter or less, and preferably 10 -7 to 10 12 moles/liter or less and more preferably IO -8 to 10 12 moles/liter (i.e. with an association constant (KA) of 10 5 to 10 12 liter/ moles or more, and preferably 10 7 to 10 12 liter/moles or more and more preferably 10 8 to 10 12 liter/moles).
  • KD dissociation constant
  • KA association constant
  • KD value greater than IO -4 mol/liter is generally considered to indicate non-specific binding.
  • the KD for biological interactions, such as the binding of immunoglobulin sequences to an antigen, which are considered specific are typically in the range of 10 -5 moles/liter (10000 nM or lOpM) to 10 12 moles/liter (0.001 nM or 1 pM) or less.
  • specific/selective binding may mean that - using the same measurement method, e.g. SPR - a binding unit (or polypeptide comprising the same) binds to TCRa
  • SPR - a binding unit or polypeptide comprising the same
  • the polypeptide of the present technology preferably has at least half the binding affinity, more preferably at least the same binding affinity, to human TCRa
  • Specific binding to a certain target from a certain species does not exclude that the binding unit can also specifically bind to the analogous target from a different species.
  • 3 does not exclude that the binding unit (or a polypeptide comprising the same) can also specifically bind to TCRa
  • specific binding to human CD33 or CD123 does not exclude that the binding unit (or a polypeptide comprising the same) can also specifically bind to CD33 or CD123 from cynomolgus monkeys ("cyno").
  • Specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
  • Scatchard analysis and/or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.
  • the dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned below. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more than IO -4 moles/liter or 10 -3 moles/liter (e.g. of 10 -2 moles/liter).
  • KA association constant
  • SPR surface plasmon resonance
  • surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding k on , k O ff measurements and hence KD (or KA) values.
  • This can for example be performed using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ).
  • BIAcore® system BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ.
  • bio-layer interferometry refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam).
  • reference beam an internal reference layer
  • signal beam a layer of immobilized protein on the biosensor tip
  • BLI can for example be performed using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).
  • affinities can be measured in Kinetic Exclusion Assay (KinExA) (see for example Drake et al. 2004, Anal. Biochem., 328: 35-43), using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA).
  • KinExA Kinetic Exclusion Assay
  • Equilibrated solutions of an antibody/antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).
  • the GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).
  • the polypeptide may further comprise one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased (in vivo) half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
  • In vivo half-life extension means, for example, that the polypeptide has an increased half-life in a mammal, such as a human subject, after administration.
  • Half-life can be expressed for example as tl/2beta.
  • the type of groups, residues, moieties or binding units is not generally restricted and may for example be chosen from the group consisting of a polyethylene glycol molecule, serum proteins or fragments thereof, binding units that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins.
  • said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life can be chosen from the group consisting of binding units that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG, and preferably is a binding unit that can bind to human serum albumin.
  • the binding unit is preferably an ISVD.
  • WO 04/041865 describes NANOBODY® ISVDs binding to serum albumin (and in particular against human serum albumin) that can be linked to other proteins (such as one or more other NANOBODY® ISVDs binding to a desired target) in order to increase the half-life of said protein.
  • NANOBODY® ISVDs against (human) serum albumin.
  • These NANOBODY® ISVDs include the NANOBODY® ISVD called Alb-1 (SEQ ID NO: 52 in WO 06/122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 06/122787). Again, these can be used to extend the half-life of therapeutic proteins and polypeptide and other therapeutic entities or moieties.
  • W02012/175400 describes a further improved version of Alb-1, called Alb-23.
  • the polypeptide comprises a serum albumin binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 and Alb-23, preferably Alb-8 or Alb-23 or its variants, as shown on pages 7-9 of W02012/175400 and the albumin binders described in WO2012/175741, WO2015/173325, W02017/080850, WO2017/085172, WO2018/104444, WO2018/134235, WO2018/134234.
  • Some preferred serum albumin binders are also shown in Table A-4.
  • a particularly preferred further component of the polypeptide of the present technology is as described in item C:
  • An ISVD that binds to human serum albumin and comprises i. a CDR1 comprising the amino acid sequence of SEQ ID NO: 9 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 9; ii. a CDR2 comprising the amino acid sequence of SEQ ID NO: 13 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 13; and iii.
  • a CDR3 comprising the amino acid sequence of SEQ ID NO: 17 or has 2 or 1 amino acid difference(s) with SEQ ID NO: 17; preferably a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a CDR2 comprising the amino acid sequence of SEQ ID NO: 13 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 17.
  • ISVD that binds to human serum albumin
  • Item C can be also described using the Kabat definition as:
  • An ISVD that binds to human serum albumin and comprises i. a CDR1 comprising the amino acid sequence of SEQ ID NO: 37 or has 2 or 1 amino acid difference with SEQ ID NO: 37; ii. a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 or has 2 or 1 amino acid difference with SEQ ID NO: 41; and iii. a CDR3 comprising the amino acid sequence of SEQ ID NO: 45 or has 2 or 1 amino acid difference with SEQ ID NO: 45; preferably a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 comprising the amino acid sequence of SEQ ID NO: 41 and a CDR3 comprising the amino acid sequence of SEQ ID NO: 45.
  • Preferred examples of such an ISVD that binds to human serum albumin have one or more, and preferably all, framework regions as indicated for construct ALB23002 in Table A-2.1 (in addition to the CDRs as defined in the preceding item C'), and most preferred is an ISVD comprising the full amino acid sequence of construct ALB23002 (SEQ ID NO: 5, see Table A-l and A-2.1).
  • the amino acid sequence of an ISVD binding to human serum albumin may have a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 5, wherein optionally the CDRs are as defined in the preceding item C.
  • the ISVD binding to human serum albumin preferably comprises the amino acid sequence of SEQ ID NO: 5.
  • the ISVD has at least half the binding affinity, preferably at least the same binding affinity to human serum albumin as construct ALB23002 set forth in SEQ ID NO: 5, wherein the binding affinity is measured using the same method, such as SPR.
  • an ISVD binding to human serum albumin when such an ISVD binding to human serum albumin has a C- terminal position it exhibits a C-terminal alanine (A) or glycine (G) extension and is preferably selected from SEQ ID NOs: 64, 65, 67, 69, 70, 71, 72, 73, 74, and 75 (see table A-4 below). If the ISVD binding to human serum albumin comprises another position than the C- terminal position (i.e. is not the C-terminal ISVD of the polypeptide of the present technology) and is selected from SEQ ID NOs: 5, 62, 63, 66, and 68 (see table A-4 below).
  • nucleic acid molecule encoding the polypeptide of the present technology.
  • a “nucleic acid molecule” (used interchangeably with “nucleic acid”) is a chain of nucleotide monomers linked to each other via a phosphate backbone to form a nucleotide sequence.
  • a nucleic acid may be used to transform/transfect a host cell or host organism, e.g. for expression and/or production of a polypeptide.
  • Suitable hosts or host cells for production purposes will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism.
  • a host or host cell comprising a nucleic acid encoding the polypeptide of the present technology is also encompassed by the present technology.
  • a nucleic acid may be for example DNA, RNA, or a hybrid thereof, and may also comprise (e.g. chemically) modified nucleotides, like PNA. It can be single- or double-stranded, and is preferably in the form of double-stranded DNA.
  • the nucleotide sequences of the present technology may be genomic DNA, cDNA.
  • nucleic acids of the present technology can be prepared or obtained in a manner known per se, and/or can be isolated from a suitable natural source.
  • Nucleotide sequences encoding naturally occurring (poly)peptides can for example be subjected to site-directed mutagenesis, so as to provide a nucleic acid molecule encoding polypeptide with sequence variation.
  • site-directed mutagenesis so as to provide a nucleic acid molecule encoding polypeptide with sequence variation.
  • nucleic acid also several nucleotide sequences, such as at least one nucleotide sequence encoding a targeting moiety and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner.
  • nucleic acids may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers.
  • restriction sites e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes
  • a vector comprising the nucleic acid molecule encoding the polypeptide of the present technology.
  • a vector as used herein is a vehicle suitable for carrying genetic material into a cell.
  • a vector includes naked nucleic acids, such as plasmids or mRNAs, or nucleic acids embedded into a bigger structure, such as liposomes or viral vectors.
  • Vectors generally comprise at least one nucleic acid that is optionally linked to one or more regulatory elements, such as for example one or more suitable promoter(s), enhancer(s), terminator(s), etc.).
  • the vector preferably is an expression vector, i.e. a vector suitable for expressing an encoded polypeptide or construct under suitable conditions, e.g. when the vector is introduced into a (e.g. human) cell.
  • this usually includes the presence of elements for transcription (e.g. a promoter and a polyA signal) and translation (e.g. Kozak sequence).
  • said at least one nucleic acid and said regulatory elements are "operably linked" to each other, by which is generally meant that they are in a functional relationship with each other.
  • a promoter is considered “operably linked” to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being “under the control of” said promotor).
  • two nucleotide sequences when operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.
  • any regulatory elements of the vector are such that they are capable of providing their intended biological function in the intended host cell or host organism.
  • a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, by which is meant that for example said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence - e.g. a coding sequence - to which it is operably linked.
  • the present technology also provides a composition comprising at least one polypeptide of the present technology, at least one nucleic acid molecule encoding a polypeptide of the present technology or at least one vector comprising such a nucleic acid molecule.
  • the composition may be a pharmaceutical composition.
  • the composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. 5.7 Host organisms
  • the present technology also pertains to host cells or host organisms comprising the polypeptide of the present technology, the nucleic acid encoding the polypeptide of the present technology, and/or the vector comprising the nucleic acid molecule encoding the polypeptide of the present technology.
  • Suitable host cells or host organisms are clear to the skilled person, and are for example any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. The most preferred host is Pichia pastoris.
  • the present technology also provides a method for producing the polypeptide of the present technology.
  • the method may comprise transforming/transfecting a host cell or host organism with a nucleic acid encoding the polypeptide, expressing the polypeptide in the host, optionally followed by one or more isolation and/or purification steps.
  • the method may comprise: a) expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid sequence encoding the polypeptide; optionally followed by: b) isolating and/or purifying the polypeptide.
  • Suitable host cells or host organisms for production purposes will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. The most preferred host is Pichia pastoris.
  • the polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide of the present technology, nucleic acid molecule or vector -preferably the polypeptide or a composition comprising the same- are useful as a medicament.
  • the present technology provides the polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide of the present technology, nucleic acid molecule or vector for use as a medicament. Also provided is the polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide of the present technology, nucleic acid molecule or vector for use in the (prophylactic or therapeutic) treatment of acute myeloid leukemia (AML), preferably relapsed and/or refractory AML.
  • AML acute myeloid leukemia
  • a (prophylactic and/or therapeutic) method of treating AML comprises administering, to a subject in need thereof, a pharmaceutically active amount of the polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide of the present technology, nucleic acid molecule or vector.
  • polypeptide of the present technology a nucleic acid molecule or vector as described, or a composition comprising the polypeptide of the present technology, nucleic acid molecule or vector in the preparation of a pharmaceutical composition, preferably for treating AML.
  • the AML may be relapsed and/or refractory AML.
  • a "subject" as referred to in the context of the present technology can be any animal, preferably a mammal. Among mammals, a distinction can be made between humans and non-human mammals.
  • Non-human animals may be for example companion animals (e.g. dogs, cats), livestock (e.g. bovine, equine, ovine, caprine, or porcine animals), or animals used generally for research purposes and/or for producing antibodies (e.g. mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates, such as cynomolgus monkeys, or camelids, such as llama or alpaca).
  • companion animals e.g. dogs, cats
  • livestock e.g. bovine, equine, ovine, caprine, or porcine animals
  • animals used generally for research purposes and/or for producing antibodies e.g. mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates
  • the subject can be any animal, and more specifically any mammal, but preferably is a human subject.
  • Substances may be administered to a subject by any suitable route of administration, for example by enteral (such as oral or rectal) or parenteral (such as epicutaneous, sublingual, buccal, nasal, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, or transmucosal) administration.
  • enteral such as oral or rectal
  • parenteral such as epicutaneous, sublingual, buccal, nasal, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, or transmucosal
  • Parenteral administration such as intramuscular, subcutaneous or intradermal, administration is preferred. Most preferred is subcutaneous administration.
  • An effective amount of a polypeptide, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector can be administered to a subject in order to provide the intended treatment results.
  • One or more doses can be administered. If more than one dose is administered, the doses can be administered in suitable intervals in order to maximize the effect of the polypeptide, composition, nucleic acid molecule or vector.
  • Table A-l Amino acid sequences of the different monovalent VHH building blocks identified within the tetravalent polypeptide A025001562 (TCR-CD33-CD123 multispecific ISVD construct) ("ID" refers to the SEQ ID NO as used herein)
  • Table A-2 Sequences for CDRs according to AbM numbering and frameworks ("ID" refers to the given SEQ ID NO)
  • Table A-4 Serum albumin binding ISVD sequences ("ID” refers to the SEQ ID NO as used herein)
  • Multi-specific NANOBODY® ISVD proteins were expressed in P. pastoris.
  • the yeast expression vectors contain the AOX1 promoter and terminator, a resistance gene for Zeocin and the coding information for the Saccharomyces cerevisiae a-mating factor signal peptide.
  • the NANOBODY® ISVD monovalent building blocks (BBs) were combined with GS linkers and cloned in the expression vector via Golden Gate cloning (Engler C, Marillonnet S. Golden Gate cloning. Methods Mol Biol. 2014;1116:119-31).
  • the expression vectors contain two Bpil restriction sites for the cloning the PCR-amplified monovalent NANOBODY® ISVD building blocks together with the GS linkers included in one or multiple vectors. All these elements are flanked by Bpil sites.
  • the use of unique nucleotide overhangs for each position of the cloning cassette allows seamless ligation in a pre-defined order.
  • plasmid DNA derived from E. coli TOPIC was linearized and transformed by electroporation into in-house prepared hypercompetent P. pastoris, strain NRRL Y-11430 (ATCC 76273).
  • E. co// TGI cells (Lucigen, Cat. No.
  • FLAG3His6-tagged NANOBODY® ISVD proteins were purified by immobilized metal affinity chromatography (IMAC) on or NilDA/NTA (Genscript) resins with Imidazole (for the former) or acidic elution (for the latter) followed by a desalting step (PD columns with Sephadex G25 resin, GE Healthcare) and if necessary, preparative size exclusion chromatography (SEC) (Superdex 75 column, GE Healthcare) in D-PBS. To this end, robotic stations or AKTA purification systems were used.
  • IMAC immobilized metal affinity chromatography
  • NilDA/NTA Genescript
  • SEC preparative size exclusion chromatography
  • Tagless NANOBODY® ISVD proteins or constructs containing the ALB building block were purified on Amsphere A3 (JSR) or MabCaptureA (Poros) resins followed by a desalting step (PD columns with Sephadex G25 resin, GE Healthcare) and if necessary, preparative SEC (Superdex 75 column, GE Healthcare) in D-PBS.
  • 3-d-glucopyranoside (OGP; Alpha Aesar, Cat. No. J67390) treatment was implemented during purification/gel filtration chromatography whenever low LPS levels were required. Concentration was determined via OD280/OD340 measurement. Quality control was performed by SDS- PAGE and mass spectrometry.
  • 3-CD33-CD123 multispecific ISVD construct as set forth in SEQ ID NO: 1 was generated. 6.2.1 Affinity determination towards human and cynomolgus CD33 and CD123 proteins
  • 3-CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) towards recombinant huCD33L-Fc, cyCD33L-Fc (via capturing setup) or huCD123 (via capturing setup) proteins were measured at 37°C by means of a surface plasmon resonance (SPR)-based assay on a ProteOn XPR36 instrument (BioRad Laboratories, Inc.).
  • SPR surface plasmon resonance
  • Anti-huIgG (Fc) was immobilized on a GLH (long matrix, high capacity) sensor chip via amine coupling, using EDC (l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N- hydroxysuccinimide esters) chemistry.
  • EDC l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • NHS N- hydroxysuccinimide esters
  • Anti-huIgG (Fc) was immobilized on a GLH (long matrix, high capacity) sensor chip via amine coupling, using EDC (l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N- hydroxysuccinimide esters) chemistry.
  • EDC l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • NHS N- hydroxysuccinimide esters
  • Table 3 SPR based kinetic determination of TCRap-CD33-CD123 multispecific ISVD construct for human and cynomolgus CD123 protein
  • 3-CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) towards recombinant huTCR(2XN9)-zipper, cyTCR(AEA41865)-zipper (via coated setup) were evaluated at 37°C by means of an SPR based assay on a ProteOn XPR36 instrument (BioRad Laboratories, Inc.).
  • 3 huTCR(2XN9)-zipper, cyTCR(AEA41865)-zipper proteins were coated a GLC (short matrix, normal capacity) sensor chip.
  • Purified TCRa -CD33-CD123 multispecific ISVD construct as set forth in SEQ ID NO: 1 was injected at different concentrations (0.4 to 625 nM) for 120 s and dissociation was followed for 900 s.
  • Table 4 SPR based kinetic determination of TCRap-CD33-CD123 multispecific ISVD construct and reference TCR-ISVD construct for human and cynomolgus TCRap
  • Human, cynomolgus and mouse serum albumin were immobilized on a ProteOn GLC sensor chip using amine coupling using amine coupling, using EDC and NHS chemistry (running buffer used: HBS-EP+, pH 7.4).
  • the albumins were immobilized at two concentrations of 2.5 pg/mL (HSA & MSA) and 5 pg/mL (CSA) in pH4.5 Acetate buffer, giving rise to immobilization levels of up to 220 RUs for CSA, 150 RUs for MSA and 110 RUs for HSA.
  • VHHS Purified multivalent VHHS were injected for 2 minutes (flow rate 45 pL/min) at different concentrations (between 4.3 nM and 416 nM) and dissociation was followed for 900s. Regeneration between cycles consisted of an injection of 10 mM glycine-HCL, pH1.5 for 47 s at lOOpL/min.
  • 3-CD33-CD123 multispecific ISVD construct was evaluated using flow cytometry.
  • the target cell lines expressing CD123 are described in detail in W02018/091606A1.
  • Transfected CD33 cells were generated as follows. Stable CHO Fl p-l n (Invitrogen, R758-07) cell lines with recombinant overexpression of CD33, were generated using the Flp-lnTM site-directed recombination technology (Flp-lnTM System For Generating Stable Mammalian Expression Cell Lines by Flp Recombinase-Mediated Integration (Invitrogen, K601001, K601002)).
  • FRT Flp Recombination Target
  • pOG44 Flp recombinase
  • the Flp-lnTM host cell line and expression plasmid both contain this FRT site, thereby allowing a single homologous DNA recombination.
  • the sequence for human CD33 was derived from NCBI RefSeq NP_001763, the sequence of cynomolgus CD123 was derived from NCBI genbank no. XP_005590138.
  • cells were harvested and transferred to a V-bottom 96-well plate (5xl0 4 cells per well) and incubated with a serial dilution of TCRa
  • FACS buffer D-PBS (Gibco, 14190) with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)
  • 3-CD33-CD123 multispecific ISVD construct towards primary T cells was evaluated using flow cytometry in a competition setup using the FLAG3Hise tagged monovalent TCR ISVD as ligand.
  • human or cynomolgus primary T cells were thawed and transferred to a V- bottom 96-well plate (7.5xl0 4 cells per well in 100 pL) and incubated with a serial dilution of TCRa
  • FACS buffer D-PBS (Gibco, 14190) with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)
  • the concentration of ligand used in the assay was below its binding EC50. After an incubation period of 90 min at 4°C, the level of ligand binding was determined via flow cytometry. Thereto, cells were washed 3 times and incubated with 1 pg/ml mouse monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich, F1804) for 30 min at 4°C, washed again, and incubated for 30 min at 4°C with 5 pg/ml Allophycocyanin (APC) AffiniPure Goat Anti-Mouse IgG (subclasses l+2a+2b+3), Fey fragment specific (Jackson Immunoresearch, 115-136-071), in a final volume of 100 pL.
  • APC Allophycocyanin
  • the human-cynomolgus cross-reactivity was evaluated by testing the binding of the monovalent CD33 building block (SEQ ID NO: 3) and the monovalent CD123 building block (SEQ ID NO: 4) to human or cynomolgus CD33 transfected cell lines or to human or cynomolgus CD123 transfected cell lines using flow cytometry as described above. The results are graphically represented in Figure 2. The EC50 values of the different experiments are summarized in Table 6 (CD33 transfected target cells) and Table 7 (CD123 transfected target cells).
  • Table 6 EC50 values for binding of the CD33 building block (SEQ ID NO: 3) to cells expressing huCD33 or cyCD33.
  • Table 7 EC S o values for binding of the CD123 building block (SEQ ID NO: 4) to cells expressing huCD123 or cyCD123.
  • FIG. 4 An exemplary example of the DRC is depicted in Figure 4.
  • the ISVD constructs were tested on multiple healthy donorT cells.
  • Global IC50 is shown in Table 8.
  • Table 8 Global IC 50 (M) of TCRap-CD33-CD123 multispecific ISVD construct and reference TCR-ISVD construct in the human and cynomolgus T cell competition assay as determined in flow cytometry
  • ISVD constructs were characterized for redirected T cell mediated killing in an impedancebased cytotoxicity assay (e.g. as described in W02018091606A1) using human or cynomolgus primary effector T cells and adherent target cells. Changes in impedance induced by the adherence of target cells to the surface of an electrode were measured using the xCELLigence instrument (Roche). T cells are non-adherent and therefore do not impact the impedance measurements.
  • the xCELLigence® RTCA MP instrument quantifies the changes in electrical impedance, displaying them as a dimensionless parameter termed cell index, which is directly proportional to the total area of tissue-culture well that is covered by cells.
  • ISVD constructs were characterized for redirected T cell mediated killing in a flow cytometry-based cytotoxicity assay using human or cynomolgus primary T cells as effector cells and non-adherent target cells.
  • Target cells were labelled with 4 pM PKH26 membrane dye using the PKH26 red fluorescent cell linker kit (Sigma, PKH26GL-1KT) according to manufacturer's instruction.
  • Effector cells 2.5 x 10 5 cells/well
  • PKH- labelled target cells 2.5 x 10 4 cells/well
  • assay medium of the target cell line target growth medium with 1% Penicillin/streptomycin (Life Technologies, 15140) and 50 pM Alburex HSA (CSL Behring, 2160-679)
  • serial dilutions of ISVD constructs in target assay medium were added to the cells and incubated for 18 h in a 5% CO2 atmosphere at 37°C.
  • FACS buffer D-PBS (Gibco, 14190) with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)
  • FACS buffer D-PBS (Gibco, 14190) with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)
  • FACS buffer D-PBS (Gibco, 14190) with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)
  • the percent specific lysis ((% TO-PRO-3+no construct - % TO-PRO-3+with construct)/ % TO-PRO-3+no construct)) xlOO.
  • 3-CD33-CD123 multispecific ISVD construct towards CD33, CD123 and TCR was determined in the impedance-based cytotoxicity assay (xCELLigence) using human or cynomolgus primary T cells and adherent human or cynomolgus transfected CD33 or CD123 cells.
  • the assay results comparing human versus cynomolgus primary T cells are graphically illustrated in Figure 5 and 7.
  • the evaluation of human T cell mediated cell killing of human CD33 and human CD123 transfected cells was performed using T cells from different human donors, which allowed to calculate the global IC50 values (Table 9).
  • the evaluation of cynomolgus T cell mediated killing of human CD33 and human CD123 transfected cells was performed using T cells from 1 cynomolgus monkey.
  • the IC50 values are summarized in Table 10.
  • Table 9 Global IC 50 (M) of TCRap-CD33-CD123 in the impedance-based (xCELLigence) human T cell mediated CD33+ or CD123+ cell killing assay using an effector to target ratio of 15 to 1 in the presence of 50 pM HSA.
  • Global ICso's towards human target expressing cells were 5,4.10 -11 M and 2,5.10 -11 M respectively for CD33 and CD123.
  • Table 10 IC 50 (M) of TCRap-CD33-CD123 in the impedance-based (xCELLigence) cynomolgus T cell mediated CD33+ or CD123+ cell killing assay using an effector to target ratio of 15 to 1 in the presence of 50 pM HSA.
  • 3-CD33-CD123 ISVD construct was evaluated in a flow cytometry-based T cell mediated MOLM-13 cell killing using either human or primary T cells in combination with the CD33, CD123 double expressing human MOLM-13 target cell line in the presence of 50 pM HSA as described above.
  • Graphical illustration of these results is shown in Figure 6. Additional data of the TCRa
  • Table 11 Global EC S0 (M) of A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO.: 1) in the flow cytometry human T cell mediated MOLM-13 cell killing assay using an effector to target ratio of 10 to 1 in the presence of 50 pM HSA.
  • Table 12 EC50 (M) for TCRap-CD33-CD123 ISVD construct in the cynomolgus T cell mediated MOLM-13 cell killing assay using an effector to target ratio of 10 to 1.
  • 3-CD33-CD123 ISVD construct was functional both in the human and cynomolgus human target cell mediated killing assay.
  • the global killing potency of the described ISVD towards the CD33/CD123 double positive AML cell line was 1,8.10 11 M.
  • the human AML-derived cell lines expressing CD123 Molm-13 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). Molm-13 cells were grown in culture (37°C, 5% CO2, 95% humidity) in RPMI1640 Glutamax medium (completed with foetal cow serum 20%). Cells were infected with a luciferase vector (SV40-PGL4-Puro) carried by a non-replicative lentivirus; polyclonal Molm-13-luc were selected using 2 pg/ml puromycin.
  • SV40-PGL4-Puro luciferase vector carried by a non-replicative lentivirus
  • polyclonal Molm-13-luc were selected using 2 pg/ml puromycin.
  • PBMCs peripheral blood mononuclear cells
  • PBS phosphate-buffered saline
  • the total viable PBMCs number was defined by Vi-CELL counter (Beckman Coulter Life Sciences, Brea, CA, USA).
  • the pellet was recovered in autoMACS Running Buffer (Miltenyi Biotec). T cells were isolated from PBMCs using the Pan T Cell Isolation Kit (Miltenyi Biotec) and autoMACS according to manufacturer instructions.
  • T cells were activated and expanded in vitro over 14 days using the T Cell TRANSACT matrix Activation/Expansion Kit (Miltenyi Biotec) based on CD3 and CD28 co stimulation.
  • the activation protocol involved culturing T cells for 2 weeks in presence of TRANSACT matrix in TexMACS medium (Miltenyi Biotec), supplemented with 20 000 IU of soluble IL-2 and 1% penicillin-streptomycin (Gibco).
  • T cells were harvested and resuspended in PBS at a final concentration of 5 x 10 7 cells/ml, with 10 7 cells administered through intraperitoneal (IP) injection to each animal. T cell viability prior to animal injection being tested to be higher than 85%.
  • IP intraperitoneal
  • 3-CD33-CD123 multispecific ISVD construct (SEQ ID NO:1) anti-tumor activity was evaluated after Molml3-luc AML engraftment in non-irradiated NOD.Cg-Prkdc scid Il2rgtmiwji/s z j (NSG) mice (Charles River Laboratories, Saint-Germain-Nuelles, France).
  • NSG non-irradiated NOD.Cg-Prkdc scid Il2rgtmiwji/s z j mice (Charles River Laboratories, Saint-Germain-Nuelles, France).
  • Female animals aged 6-8 weeks were implanted intravenously (IV) with 10 6 /0.2 ml Molm- 13-luc cells per mouse on day 0. Same animals were intraperitoneally implanted with 10 7 /0.2 ml human T cells per mouse on day 1.
  • mice were distributed among groups based on all-body bioluminescence imaging (BLI) signal homogeneity and tumor myeloid engraftment evaluated by long bones signal segmentation on day 3.
  • Mice were treated IV with TCRa
  • Animal body weight was monitored from day 3 to the end of assay to follow impact of therapy. A dosage producing a 20% weight loss or 15% weight loss for 3 consecutive day or 10% or more drug deaths, was considered an excessively toxic dosage. Animal body weights included the tumor weights.
  • Tumor growth was assessed by in vivo BLI using the MS Lumina XRMS imager (PerkinElmer, Waltham, MA, USA) with the Living Image 4.5.2 acquisition software (PerkinElmer) through luciferase activity measurement in vivo on days 3, 7, 10, and 14 post-tumor injections, using beetle luciferin potassium salt 160 mg/kg Ip injection 15 minutes before image processing of animals anesthetized with ketamine® /Xylazine® (120 mg/kg; 6 mg/kg IM, 5 ml/kg) 5 minutes before image. Tumor growth was based on bioluminescence signal curves (expressed in photons/second).
  • Tumor growth was followed in all body and long bones in posteriors legs by BLI signal measurements at days 7, 10 and 14 after tumor implantation.
  • the primary efficacy end points are ratio of change in tumor signal changes from baseline between treated and control groups (T/C), partial regression (PR) and complete regression (CR).
  • Tumor growth based on bioluminescence signal curves (expressed in Phot/sec) in time was drawn for each animal of each treatment group and represented as median curve ⁇ MAD, both for all body (linear scale) and bone segmented signals (Log scale). Changes in tumor bioluminescence signal for each treated (T) and control (C) are calculated for each animal and each day by subtracting the tumor signal on the day of first treatment (staging day) from the tumor signal on the specified observation day. The median T is calculated for the treated group and the median C is calculated for the control group. Then the ratio T/C is calculated and expressed as a percentage: dT/dC [ (median T end - median T day3)/ (median C end - median C day 3) ]x 100
  • the dose is considered as therapeutically active when dT/dC is lower than 42% and very active when dT/dC is lower than 10%. If dT/dC is lower than 0, the dose is considered as highly active and the percentage of regression is dated:
  • Percent tumor regression is defined as the % of tumor signal decrease in the treated group at a specified observation day compared to its signal on the first day of treatment.
  • % regression is calculated at specific time-points. Considering the risk of signal variability due to luciferin kinetics variability with ip miss injection, true regressions are considered when they are observed at least at two consecutive time points for each animal.
  • Partial regression Regressions are defined as partial if the tumor Signal decreases bellow the tumor Signal at the start of treatment at two consecutive time points, one being below 50% of start signal.
  • Complete regression Regressions are defined as Complete if the tumor Signal decreases below 80% of start signal.
  • ANOVA-Type two non-parametric two-way analysis of variance-Type
  • two contrast analyses with Bonferroni-Holm adjustment for multiplicity were performed with repeated measures by day: p>0.05: NS, 0.05 ⁇ p > 0.01: * , p ⁇ 0.01: **.
  • a one-way ANOVA with factor group on rank-transformed bioluminescence signal was performed. Descriptive statistics with medians ⁇ median absolute deviation was provided by group and day of measurement: p>0.05: NS, 0.05 ⁇ p > 0.01: * , p ⁇ 0.01: **.
  • 3-CD33-CD123 multispecific ISVD construct induces anti-leukemic effects in Molm- 13-luc AML xenograft model in vivo ( Figure 8A).
  • a sum of the longest diameter (LD) for all target lesions was the baseline sum LD.
  • the baseline sum LD was used as reference by which to characterize the objective tumor response.
  • 3-CD33-CD123 multispecific ISVD construct significantly inhibited tumor growth in liver (p ⁇ 0.0001), spleen (p ⁇ 0.0001) and ovaries (p ⁇ 0.0001) but not in abdominal fat at all tested doses, when reference ISVD TCR-HLE was inactive in all tissues, and CD123/CD3 positive control inhibited significantly the tumor load in liver (p ⁇ 0.0001) and spleen (p ⁇ 0.0001) but not in ovaries (NS) or abdominal fat tissues.
  • the ISVD constructs were characterized for redirected T cell mediated killing in a flow cytometry-based cytotoxicity assay using human primary T cells as effector cells and nonadherent target cells.
  • CD123 and/or CD33 positive target cells (MOLM-13, DSMZ ACC 554, U-937, ATCC® CRL1593.2, and KG-la, ATCC® CCL246.1TM) were labelled with 4 pM PKH-26 membrane dye using the PKH26 red fluorescent cell linker kit (Sigma, PKH26GL-1KT) according to manufacturer's instructions.
  • Effector cells 2.5 x 10 5 cells/well
  • PKH- labelled target cells 2.5 x 10 4 cells/well
  • assay medium of the target cell line target growth medium without antibiotics
  • serial dilutions of compounds in target assay medium were added to the cells and incubated for 18 h in a 5% CO2 atmosphere at 37°C. After incubation, cells were pelleted by centrifugation and washed with FACS buffer (D-PBS from Gibco, with 10% FBS from Sigma and 0.05% sodium azide from Merck).
  • FACS buffer D-PBS from Gibco, with 10% FBS from Sigma and 0.05% sodium azide from Merck
  • 3-CD33-CD123 ISVD construct according to the invention was compared to corresponding constructs, where either the CD33 binding ISVD or the CD123 binding ISVD was replaced by an irrelevant ISVD IRR (not binding to CD33 and not binding to CD123; Table 23) as well as a CD123/CD3 positive control and a CD33/CD3 positive control. Results are shown in Figures 10, 11 and 12.
  • Table 24 EC50 (M) and % lysis for the trivalent NANOBODY® ISVDs in the human T cell mediated U-937 or KG-la cells cell killing assay using an effector to target ratio of 10 to 1.
  • the TCR-CD123 single targeting ISVD hardly induced killing of CD123- U-937 cells, as was also observed for the CD123/CD3 positive control.
  • the CD33 single targeting ISVD induced only low level killing of CD33-/+ KG-la cells, as was also observed for the CD33/CD3 positive control.
  • 3-CD33-CD123 ISVD (construct A) demonstrated potent tumor cell killing on both CD33- and CD123- cell lines, thus exemplifying the advantage of the dual-targeting approach.
  • 3-CD33-CD123 multispecific ISVD construct according to the invention was determined in an autologous healthy donor PBMC assay. In parallel, autologous depletion of monocytes within human PBMCs was evaluated. The functionality of the ISVD according to the invention was compared with 2 tool molecules: a CD123/CD3 positive control and a CD33/CD3 positive control. An untargeted T cell engager (TCE) ISVD was taken along as negative control. 6.7.1 Materials and methods
  • PBMCs 100 pL @ 2x106 cells/mL in culture medium
  • LeucosepTM tube containing LymphoprepTM solution were transferred to 96-well V-bottom clear well plates (Greiner CELLSTAR® 96 well plates; 651 180).
  • PBMCs were incubated at 37°C at 5% CO2 for 20h. After overnight (20h) incubation, PBMCs were centrifuged at 300g for 2 min. The supernatant was collected and transferred to a new 96 well storage plate, frozen at -20°C for cytokine measurements. The cell pellets were suspended in 100 pL cold FACS buffer and washed 1 time with 100 pL FACS buffer.
  • PBMCs were centrifuged at 300g for 2 min at 4°C. Supernatant was discarded and cells were resuspended in 30 pL diluted Fc block (1/200 in FACS buffer) (BD, 564220) and incubated for 10 minutes at room temperature.
  • the frozen supernatant from the overnight incubation of the human PBMCs was used to measure a panel of cytokines including IL-2, IL-6, IFNy, TNFa using a multiplex bead assay from Bio-rad.
  • the assay was performed according to the manufacturer's guidelines. Data from the reaction were acquired using the Luminex FlexMAP 3D system ( Figure 14).
  • 3-CD33-CD123 multispecific ISVD construct induced depletion of monocytes associated with cytokine production in all donors.
  • the untargeted TCE did not show any killing nor cytokine release (see Figure 13).
  • the data shown in Figure 13 is data from 1 donor, which is representative of the data of all 6 donors that provided PBMCs.
  • both the potencies and the top levels of the cytokines induced by the ISVD construct according to the invention were substantially lower compared to the CD123/CD3 control compound.
  • IL-6 and TNFa production induced by the ISVD construct according to the invention were comparable.
  • the level of IL-2 and IFNy were higher than with the CD33/CD3 control, the levels were still acceptable.
  • the ISVD construct according to the invention does not have a higher risk to induce CRS than other CD123 or CD33 targeting compounds and should be safe for use in humans.
  • Lysis of AML blasts mediated by CD33/CD3 positive control was tested as follows: AML samples from patients with a primary diagnosis or relapse were used in an ex vivo coculture system without the addition of any other cells. Hence, the E:T ratio was determined by the number of residual T cells within the primary AML sample.
  • Whole blood samples from AML patients were provided by public hospitals in Marseille (La Conception, AP-HM) or adjoin or a central lab.
  • 300 pL of AML blast cells were added to 700 pL of culture medium per well in a 6 well plate in the presence of 1 mL (2x) saturating concentration of TCRa
  • Zombie Violet viability marker (diluted 1/100 in PBS) was added to the cell pellets and incubated for 5 minutes at room temperature in the dark.
  • cells were washed and stained with an antibody cocktail (CD45, CD33, CD34, CD38, CD14, CD123, CD11 antibodies) and incubated 10 minutes on ice.
  • an antibody cocktail CD45, CD33, CD34, CD38, CD14, CD123, CD11 antibodies
  • the percentage of CD33 or CD123 positive cells per AML sample are shown in Figure 16. As can be seen, all patients had a high number of both CD33 and CD123 positive cells. Additionally, the AML patients had a wide range of disease subtypes. Therefore, the AML samples were suitable for use in this assay.
  • the cell viability of the blast cells treated with the ISVD according to the invention was, on average, the lowest compared to the positive controls. Cells from all patients showed a clear response, although the response varied from patient to patient. This shows that the ISVD according to the invention is capable of targeted cell killing of CD123 and CD33 positive tumor cells.
  • Example 9 Nonhuman primate (NHP) study The pharmacokinetic (PK), pharmacodynamic (PD) and nonclinical safety profile of the TCRa
  • PK pharmacokinetic
  • PD pharmacodynamic
  • 3-CD33-CD123 multispecific ISVD construct according to the invention was evaluated in studies in cynomolgus monkeys.
  • the ISVD construct according to the invention was administered to a total of 4 males Cynomolgus monkeys as a single dose 1-hour continuous intravenous (IV) infusion followed by a 21-day observation period.
  • IV intravenous
  • the systemic exposure and potential toxicity of the ISVD was determined, and PD endpoints (immune cells evaluation) were evaluated.
  • the study design is shown in Table 25.
  • AUC Area Under the Curve from 0 to infinity
  • AUCIast area under plasma concentration versus time curve from 0 to the time of the last measurable concentration
  • CL clearance
  • Ceoi plasma concentration at the end of infusion
  • tl/2 terminal half-life
  • Hast last measurable concentration
  • tmax first time to reach Cmax
  • Vss volume of distribution at steady state
  • the single dose IV infusion of the ISVD construct to cynomolgus monkeys at 0.04 and 0.4 pg/kg induced changes in CD123+ cell populations and CD33+ monocytes at both dose levels. These changes consisted of: a total decrease in total CD123+ cells number observed for all animals starting from 6 hours after the start of the infusion. This decrease was maintained for all animals throughout the study. a total decrease in monocytes CD33+ cells number observed for all animals starting from 6 hours after the start of the infusion (and up to day 1 for animal Ml receiving the ISVD construct at 0.04pg/kg). A full recovery was observed at Day 1 for animal M3.
  • CD4+ and CD8+ cells numbers were also observed for all animals starting from 6 hours after the start of the infusion.
  • CD8+ cells a full recovery was observed at Day 3 with a rebound effect for animal M4.
  • CD4+ cells a full recovery was observed on Day 3 for animal M4.
  • polypeptides, nucleic acid molecules encoding the same, vectors comprising the nucleic acids and compositions described herein may be used for example in the treatment of subjects suffering from acute myeloid leukemia.

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