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WO2025133252A1 - Domaines variables uniques d'immunoglobuline ciblant ceacam5 - Google Patents

Domaines variables uniques d'immunoglobuline ciblant ceacam5 Download PDF

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Publication number
WO2025133252A1
WO2025133252A1 PCT/EP2024/088111 EP2024088111W WO2025133252A1 WO 2025133252 A1 WO2025133252 A1 WO 2025133252A1 EP 2024088111 W EP2024088111 W EP 2024088111W WO 2025133252 A1 WO2025133252 A1 WO 2025133252A1
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WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
isvd
ceacam5
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PCT/EP2024/088111
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English (en)
Inventor
Björn NIEBEL
Evelyn De Tavernier
Emmanuelle Vigne
Anand Kumar
Alexey RAK
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Ablynx NV
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Ablynx NV
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Publication of WO2025133252A1 publication Critical patent/WO2025133252A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • C07K2317/90—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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5, also known as CEA) has an N-terminal IgV-like domain followed by six IgC-like domains and normally functions as an adhesion molecule but it also has roles in regulating differentiation, immune modulation, and inhibiting anoikis (Thomas J. et al., “CEACAMS 1, 5, and 6 in disease and cancer: interactions with pathogens”, Genes Cancer, 2023, 14:12-29).
  • antibodies targeting the A1B1 domains shared by CEACAM5 and CEACAM6 were shown to affect cell migration, cell invasion, and cell adhesion and affect metastasis and host survival (Blumenthal R.D., Hansen H. J, Goldenberg D.M., “Inhibition of adhesion, invasion, and metastasis by antibodies targeting CEACAM6 (NCA-90) and CEACAM5 (Carcinoembryonic Antigen)”, Cancer Res 1 October 2005; 65 (19): 8809–8817).
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 9; ⁇ CDR2 (AbM numbering) consists of one of the amino acid sequences of SEQ ID NO: 10; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 11; or ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 234; ⁇ CDR2 (AbM numbering) consists of one of the amino acid sequences of SEQ ID NO: 235; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 236.
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NOs: 115-117, 160-170.
  • the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NOs: 115-117, 160-170.
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of GLTFSTYTXG (SEQ ID NO: 333), wherein the amino acid residue X is selected from V or M; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of GLTFSTYTXG (SEQ ID NO: 333), wherein the amino acid residue X is selected from V or M; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences GLTFSTYTXG (SEQ ID NO: 333), wherein the amino acid residue X is selected from V or M; and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SX1IX2SGS
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 1; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 336; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 339; or ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 334; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 337; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 340.
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NOs: 118-119, 171-174.
  • the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NOs: 118-119, 171-174.
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTFSDXAMG (SEQ ID NO: 356), wherein the amino acid residue X is selected from F or Y; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTFSDXAMG (SEQ ID NO: 356), wherein the amino acid residue X is selected from F or Y; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTFSDXAMG (SEQ ID NO: 356), wherein the amino acid residue X is selected from F or Y; and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence AINWSGGWTY (SEQ ID NO:
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 359; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 357; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 360; or ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 234; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 357; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 361.
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NOs: 122 or 123.
  • the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NOs: 122 or 123.
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTFNINEYHLA (SEQ ID NO: 363); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTFNINEYHLA (SEQ ID NO: 363); c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTFNINEYHLA (SEQ ID NO: 363); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence AIWWSTGNKI (SEQ ID NO: 364); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence AIWWSTGNKI (SEQ ID NO: 364); c ) amino acid
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 363; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 364; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 365.
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NO: 124.
  • the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NO: 124.
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence AIWWSTGNRI (SEQ ID NO: 369); b) amino acid sequences that have at least 80%
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 368; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 369; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 370.
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NO: 125.
  • the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NO: 125.
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of GDXLNSYHMA (SEQ ID NO: 373), wherein the amino acid residue X is selected from T or N; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of GDXLNSYHMA (SEQ ID NO: 373), wherein the amino acid residue X is selected from T or N; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences of GDXLNSYHMA (SEQ ID NO: 373), wherein the amino acid residue X is selected from T or N; and -
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 376; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 377; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 365; or ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 378; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 379; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 380.
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NOs: 126 or 127.
  • the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NOs: 126 or 127.
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GX1TFSX2YAMG (SEQ ID NO: 386), wherein the amino acid residue X1 is selected from G or R, and wherein the amino acid residue X2 is selected from S or N; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GX 1 TFSX 2 YAMG (SEQ ID NO: 386), wherein the amino acid residue X 1 is selected from G or R, and wherein the amino acid residue X2 is selected from S or N; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences GX1TFSX2YAMG (SEQ ID NO: 386), wherein the amino acid residue X1 is selected
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 389; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 387; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 390; or ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 391; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 387; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 392.
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NOs: 128, 129, or 134.
  • the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NOs: 128, 129, or 134.
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GGTVSTYTMG (SEQ ID NO: 397); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GGTVSTYTMG (SEQ ID NO: 397); c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GGTVSTYTMG (SEQ ID NO: 397); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence SIIWSGSTTV (SEQ ID NO: 398); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence SIIWSGSTTV (SEQ ID NO: 398); c ) amino acid sequences that have
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 397; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 398; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 399.
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NOs: 130.
  • the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NOs: 130.
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence AIWWSTGNTI (SEQ ID NO: 379); b) amino acid sequences that have at least 80%
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 368; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 379; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 365.
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NO: 131.
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 1; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 336; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 339, or ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 334; ⁇ CDR2 (AbM numbering) consists of the amino acid sequences of SEQ ID NO: 337; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 340; or ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 1; ⁇ CDR2 (AbM numbering) consists of
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NOs: 8, 31, 32, 118-119, 130, 150-159, 171- 174.
  • the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NOs: 8, 31, 32, 118-119, 130, 150-159, 171-174.
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from a) the amino acid sequence GRTFDNX1AMG (SEQ ID NO: 593); wherein the amino acid residue X1 is selected from H, L or E; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTFDNX1AMG (SEQ ID NO: 593); wherein the amino acid residue X1 is selected from H, L or E; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTFDNX1AMG (SEQ ID NO: 593); wherein the amino acid residue X1 is selected from H, L or E; and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTFDN
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR3 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 610; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 610; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 610.
  • the immunoglobulin single variable domain of the present technology comprises four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from a) the amino acid sequence GFTFSSYDMS (SEQ ID NO: 611); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GFTFSSYDMS (SEQ ID NO: 611); c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GFTFSSYDMS (SEQ ID NO: 611); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence TINRGGSSTS (SEQ ID NO: 612); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GTINRGGSSTS (SEQ ID NO: 612); c ) amino
  • the immunoglobulin single variable domain of the present technology may comprise four framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: ⁇ CDR1 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 611; ⁇ CDR2 (AbM numbering) consists of one of the amino acid sequences of SEQ ID NO: 612; and ⁇ CDR3 (AbM numbering) consists of the amino acid sequence of SEQ ID NO: 610.
  • the immunoglobulin single variable domain of the present technology has an amino acid sequence with a sequence identity of more than 80%, more than 90% (such as more than 95% or even more then 99%) with one of SEQ ID NOs: 547, 549-563.
  • the polypeptide of the present technology comprises or consists of the amino acid sequence of SEQ ID NOs: 547, 549-563.
  • the immunoglobulin single variable domain of the present technology may essentially consist of a VHH, such as a humanized VHH, or a VH, such as a camelized VH, a human VH, a camelized human VH, a domain antibody, a single domain antibody, and/or a dAb.
  • the present technology also relates to polypeptides and constructs comprising such immunoglobulin single variable domain, and optionally further comprising one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers.
  • the immunoglobulin single variable domains of the present technology and polypeptides and constructs comprising said immunoglobulin single variable domains showed a high binding affinity in FACS binding assay on and a high internalization efficacy in FACS internalization assay of BxPC-3 cell line expressing CEACAM5, and could be efficiently produced (e.g., in microbial hosts such as Pichia, e.g., P. pastoris, or in E. coli).
  • the immunoglobulin single variable domain, polypeptide or construct of the present technology 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 ISVD, polypeptide or construct with increased half- life, compared to the corresponding ISVD, polypeptide or construct without said one or more other groups, residues, moieties or binding units.
  • Said one or more other groups, residues, moieties or binding units that provide the ISVD, polypeptide or construct with increased half-life may be chosen from the group consisting of a polyethylene glycol molecule (PEG), 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.
  • the binding units that provide the ISVD, polypeptide or construct with increased half-life may 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), e.g., human serum albumin.
  • the binding unit that can bind to serum albumin is an ISVD.
  • the ISVD binding to human serum albumin essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which CDR1 is SEQ ID NO: 80, CDR2 is SEQ ID NO: 81, CDR3 is SEQ ID NO: 82.
  • the ISVD binding to human serum albumin comprises a sequence identity of more than 90% (such as 95%) with SEQ ID NO: 62.
  • the ISVD binding to human serum albumin comprises or consists of the amino acid sequence of SEQ ID NO: 62.
  • the ISVD binding to human serum albumin is selected from the group consisting of ALB8 (SEQ ID NO: 47), ALB23 (SEQ ID NO: 48), ALBX00001 (SEQ ID NO: 61) and ALB23002 (SEQ ID NO: 62).
  • the ISVD binding to human serum albumin is ALB23002 (SEQ ID NO: 62).
  • a nucleic acid molecule capable of expressing the immunoglobulin single variable domain or polypeptide of the present technology a vector comprising the nucleic acid, and a composition comprising the polypeptide, nucleic acid or vector.
  • a (non-human) host or host cell comprising such a nucleic acid.
  • the composition can be 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.
  • the immunoglobulin single variable domain, polypeptide or construct of the present technology, a composition comprising the immunoglobulin single variable domain, polypeptide or construct, and a composition comprising a nucleic acid comprising a nucleotide sequence that encodes the immunoglobulin single variable domain or polypeptide can be used as a medicament.
  • the immunoglobulin single variable domain, polypeptide, construct or composition of the present technology can be used in the treatment.
  • the immunoglobulin single variable, polypeptide, construct or composition can be used in the treatment of cancer. Accordingly, the present technology also encompasses a method of treating cancer. In some embodiments, the present technology encompasses a method of treating cancer, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of an immunoglobulin single variable domain, polypeptide or construct of the present technology, a nucleic acid encoding an ISVD or polypeptide of the present technology or a composition comprising the same. Accordingly, the present technology also encompasses the use of an immunoglobulin single domain, polypeptide or construct of the present technology in the preparation of a pharmaceutical composition for treating cancer.
  • Figure 2 Quality check of unpurified VHH-HA-His produced in FreeStyle 293-F cells with SDS-PAGE. Following clones are shown: line 1: 14R04-02 ; line 2 : 14R04-03 ; line 3 : 14R04-04; line 4: 14R04- 05; line 5: 14R04-06; line 6: 14R04-07; line 7: 14R04-08; line 8: 14R04-09; line 9: 14R04-10; line 10: 14R04-11; line 11: 14R04-12; line 12: 14R04-13; line 13: 14R04-14; line 14: 14R04-15; line 15: 14R04-16; line 16: 14R04-17; line 17: 14R04-18; line 18: 14R04-19; line 19: 14R04-20; line 20: 14R04-21; line 21: 14R04-22.
  • Figure 3A and 3B EC50 determination of anti-CEACAM5 VHHs by ELISA on hCEACAM5 and cCEACAM5.
  • the ELISA assay was conducted with serial dilutions of non-purified VHHs produced in FreeStyle 293-F cells.
  • Figure 4A and 4B Evaluation of the affinity constant (KD) and maximum binding capacity (Bmax) to MKN45 cell line by flow cytometry.
  • the FACS assay was conducted with serial dilutions of non- purified VHHs produced in FreeStyle 293-F cells.
  • Figure 5 EC50 determination of anti-CEACAM5 VHHs by ELISA on (A) hCEACAM5 and (B) hCEACAM6.
  • Figure 6 Characterization by flow cytometry of the binding of anti-CEACAM5 VHHs on LS174T cells.
  • Figure 7a and 7b Evaluation of the internalization of VHHs on LS174T cell line. The ISVD applied to the cell line are indicated in the legend. pHrhodo: only the pHrhodo anti-Flag antibody was applied; IRR: an ISVD not binding to CEACAM5 was applied to the cell line.
  • Figure 8 Detailed views of interactions between the A2-B2 domains of hCEACAM5 and ISVD T232000134.
  • Figure 9 Detailed views of interactions between the A2-B2 domains of hCEACAM5 and ISVD 2G08.
  • A Epitope-paratope key interacting residues between the A2-B2 domains of hCEACAM5 and 2G08, dotted line represents H-bond interactions (dark gray). The residues from ISVD 2G08 are indicated with “VHH”.
  • B Key interacting residues of paratope located at ⁇ 3.8 ⁇ distance from the hCEACAM5, backbone is in brown, and residues are shown with side chains in Yellow.
  • C Key interacting residues of epitope located at ⁇ 3.8 ⁇ distance from IVSD 2G08, interacting residues are shown in colored in light see brown. 5 Detailed description of the present technology 5.1 Definitions Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person.
  • sequence as used herein (for example in terms like “immunoglobulin sequence”, “antibody sequence”, “variable domain sequence”, “VHH sequence” or “protein sequence”), should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation. Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code. Reference is made to Table A-2 on page 48 of WO 08/020079.
  • a nucleic acid or amino acid is considered to be “(in) (essentially) isolated (form)” - for example, compared to the reaction medium or cultivation medium from which it has been obtained - when it has been separated from at least one other component with which it is usually associated in said source or medium, such as another nucleic acid, another protein/polypeptide, another biological component or macromolecule or at least one contaminant, impurity or minor component.
  • a nucleic acid or amino acid is considered “(essentially) isolated” when it has been purified at least 2-fold, in particular at least 10-fold, more in particular at least 100-fold, and up to 1000-fold or more.
  • a nucleic acid or amino acid that is “in (essentially) isolated form” is preferably essentially homogeneous, as determined using a suitable technique, such as a suitable chromatographical technique, such as polyacrylamide-gel electrophoresis.
  • a suitable technique such as a suitable chromatographical technique, such as polyacrylamide-gel electrophoresis.
  • this may mean that the latter nucleotide sequence or amino acid sequence has been incorporated into the first mentioned nucleotide sequence or amino acid sequence, respectively, but more usually this generally means that the first mentioned nucleotide sequence or amino acid sequence comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, that has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, irrespective of how the first mentioned sequence has actually been generated or obtained (which may for example be by any suitable method described herein).
  • a polypeptide when said polypeptide is said to comprise an immunoglobulin single variable domain, this may mean that said immunoglobulin single variable domain sequence has been incorporated into the sequence of the polypeptide, but more usually this generally means that the polypeptide contains within its sequence the sequence of the immunoglobulin single variable domains irrespective of how said polypeptide has been generated or obtained.
  • the first mentioned nucleic acid or nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g., a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, that the latter nucleotide sequence is in the same reading frame as the first mentioned, larger nucleic acid or nucleotide sequence).
  • an expression product e.g., a polypeptide
  • the later nucleic acid sequence or amino acid sequence either is exactly the same as the polypeptide (e.g., the CDR region; the ISVD) or corresponds to the polypeptide (e.g., the CDR region; the ISVD) which has a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the immunoglobulin single variable domain.
  • 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 identity a sequence identity between two amino acid sequences in accordance with the calculation method outlined hereinabove
  • amino acid sequence with the greatest number of amino acid residues will be taken as the “first” amino acid sequence
  • amino acid sequence with the greatest number of amino acid residues 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-à-vis a reference sequence.
  • an “amino acid difference” is a substitution.
  • amino acid substitutions are conservative substitutions.
  • Such conservative substitutions 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 Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.
  • conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.
  • aliphatic index refers to the relative volume occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine)-
  • the “GRAVY value” (grand average of hydropathicity value), as used herein, is calculated as the sum of hydropathy value of all the amino acids, divided by the number of residues in the sequence (Kyte, J. "A simple method for displaying the hydropathic character of a protein.” J. Mol. Biol.268 (1993): 10558-10563).
  • VHH family refers to a group of VHH sequences that have identical lengths (i.e., they have the same number of amino acids within their sequence) and of which the amino acid sequence between position 8 and position 106 (according to Kabat numbering) has an amino acid sequence identity of 89% or more.
  • epitopope and antigenic determinant refer to the part of a macromolecule, such as a polypeptide or protein that is recognized by antigen-binding molecules, such as immunoglobulins, conventional antibodies, or immunoglobulin single variable domains, and more particularly by the antigen-binding site of said molecules.
  • Epitopes define the minimum binding site for an immunoglobulin, and thus represent the target of specificity of an immunoglobulin.
  • the part of an antigen-binding molecule (such as an immunoglobulin, a conventional antibody, an immunoglobulin single variable domain) that recognizes the epitope is called a “paratope”.
  • a polypeptide such as an immunoglobulin, an antibody, an immunoglobulin single variable domain, or generally an antigen binding molecule or a fragment thereof
  • a polypeptide that can “bind to” or “specifically bind to”, that “has affinity for” and/or that “has specificity for” a certain epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be "against” or “directed against” said epitope, antigen or protein or is a “binding” molecule with respect to such epitope, antigen or protein, or is said to be “anti”-epitope, “anti”-antigen or “anti”-protein (e.g., “anti”- CEACAM5).
  • 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, 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 by the KD, or dissociation constant, which has 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 lower 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, 10 -7 to 10 -12 moles/liter or less, or 10 -8 to 10 -12 moles/liter (i.e., with an association constant (KA) of 10 5 to 10 12 liter/moles or more, 10 7 to 10 12 liter/moles or more, or 10 8 to 10 12 liter/moles).
  • KD dissociation constant
  • KA association constant
  • Any KD value greater than 10 -4 mol/liter (or any KA value lower than 10 4 liters/mol) 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 10 ⁇ M) to 10 -12 moles/liter (0.001 nM or 1 pM) or less. Accordingly, specific/selective binding may mean that -using the same measurement method, e.g., SPR- a binding unit (or polypeptide comprising the same) binds to CEACAM5 with a KD value of 10- 5 to 10 -12 moles/liter or less and binds to related CEACAMs members with a KD value greater than 10 -4 moles/liter.
  • CEACAM1 An example of a related CEACAMs member is CEACAM1, CEACAM6, CEACAM7 and CEACAM8.
  • the ISVD binds to (human) CEACAM5 with a KD value of 10 -5 to 10 -12 moles/liter or less and binds to CEACAM1, CEACAM6, CEACAM7 or CEACAM8 of the same species with a KD value greater than 10 -4 moles/liter.
  • 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.
  • binding unit or a polypeptide comprising the same can also specifically bind to CEACAM5 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 further herein.
  • the dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person.
  • An amino acid sequence is said to be “cross-reactive” for two different antigens or antigenic determinants (such as e.g., serum albumin from two different species of mammal, such as e.g., human serum albumin and cyno serum albumin, such as e.g., CEACAM5 from different species of mammal, such as e.g., human CEACAM5, cyno CEACAM5 and mouse CEACAM5) if it is specific for (as defined herein) these different antigens or antigenic determinants.
  • serum albumin from two different species of mammal
  • cyno serum albumin such as e.g., CEACAM5 from different species of mammal, such as e.g., human CEACAM5, cyno CEACAM5 and mouse CEACAM5
  • block is used interchangeably herein to mean the ability of an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent to interfere with the binding of another protein, polypeptides, ligand or binding agent to a given target.
  • the extent to which an immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent is able to interfere with the binding of another ligand to the target, and therefore whether it can be said to “block”, can be determined using competition binding assays.
  • FACS fluorescence-activated cell sorting
  • a FACS competition experiment can be performed using cells (such as, e.g., Flp-InTM-293 cells) overexpressing human CEACAM5 and the parental cells as background cell line.
  • Different detection reagents can be used including, e.g., monoclonal ANTI-FLAG® M2 antibody (Sigma- Aldrich, cat# F1804), monoclonal anti-C-myc antibody (Sigma-Aldrich, cat# WH0004609M2), monoclonal ANTI-HIS TAG antibody (Sigma-Aldrich, cat# SAB1305538), each labeled differently.
  • fluorophores can be used as labels in flow cytometry (such as, e.g., PE (R- Phycoerythrin), 7-aminoactinomycin D (7-AAD), Acridine Orange, various forms of Alexa Fluor, Allophycocyanin (APC), AmCyan, Aminocoumarin, APC Cy5, APC Cy7, APC-H7, APC/Alexa Fluor 750, AsRed2, Azami-Green, Azurite, B ODIPY FL C5-ceramide, BCECF-AM, Bis-oxonol DiBAC2(3), BODIPY- FL, Calcein, Calcein AM, Caroxy-H2DCFDA, Cascade Blue, Cascade Yellow, Cell Tracker Green, Cerulean, CFSE, Chromomycin A3, CM-H2DCFDA, Cy2, Cy3, Cy3.5, Cy3B, Cy5, Cy5.5, Cy7, CyPet, DAF-FM DAF-FM dia
  • Fluorophores are typically attached to the antibody (e.g., the immunoglobulin single variable domain) that recognizes CEACAM5 or to the antibody that is used as detection reagent.
  • Various conjugated antibodies are available, such as (without being limiting) for example antibodies conjugated to Alexa Fluor®, DyLight®, Rhodamine, PE, FITC, and Cy3.
  • Alexa Fluor® conjugated to Alexa Fluor®
  • DyLight® Rhodamine
  • PE FITC
  • Cy3 Cy3
  • Each fluorophore has a characteristic peak excitation and emission wavelength.
  • the combination of labels which can be used will depend on the wavelength of the lamp(s) or laser(s) used to excite the fluorophore and on the detectors available.
  • binding agent B* a dilution series of cold (without any label) binding agent A is added to (e.g., 200000) cells together with the labeled binding agent B*.
  • concentration of binding agent B* in the test mix should be high enough to readily saturate the binding sites on CEACAM5 expressed on the cells.
  • concentration of binding agent B* that saturates the binding sites for that binding agent on CEACAM5 expressed on the cells can be determined with a titration series of binding agent B* on the CEACAM5 cells and determination of the EC50 value for binding.
  • binding agent B* can be used at 100x the EC50 concentration.
  • a reduction of fluorescence for the cells incubated with the mixture of binding agent A and B* compared to the fluorescence for the cells incubated with the separate solution of binding agent B* indicates that binding agent A blocks binding by binding agent B* to CEACAM5 expressed on the cells.
  • a cross-blocking immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent is one which will bind to the CEACAM5 in the above competition FACS such that during the assay and in the presence of the second binding agent the recorded fluorescence is between 80% and 0.1% (e.g., 80% to 4%) of the maximum fluorescence (measured for the separate labelled immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent), specifically between 75% and 0.1% (e.g., 75% to 4%) of the maximum fluorescence, and more specifically between 70% and 0.1% (e.g., 70% to 4%) of maximum fluorescence (as just defined above).
  • the competition between two test binding agents (termed A* and B*) for binding to CEACAM5 can also be evaluated by adding both binding agents, each labeled with a different fluorophore, to the CEACAM5 expressing cells. After incubation and cells wash, read out can be performed on a FACS. A gate is set for each fluorophore and the total amount of channel fluorescence is recorded. Reduction and/or absence of fluorescence of one of the fluorophores indicates blocking by the binding agents for binding to CEACAM5 expressed on the cells.
  • Cell culture-based potency assays are often the preferred format for determining biological activity since they measure the physiological response elicited by the agent and can generate results within a relatively short period of time.
  • Various types of cell-based assays based on the mechanism of action of the product, can be used, such as e.g., in a FACS assay analysing the binding of the immunoglobulin single variable domains of the present technology to human CEACAM5 expressed on BxPC-3 cells, which is a human pancreatic adenocarcinoma cell line (as further described in the Example section).
  • the “efficacy” of an agent such as an ISVD or polypeptide, measures the maximum strength of the effect itself, at saturating agent concentrations.
  • Efficacy indicates the maximum response achievable from the agent. It refers to the ability of the agent to produce the desired (therapeutic) effect.
  • the efficacy of an agent can be evaluated using in vitro functional assays or in vivo models.
  • 5.2 Immunoglobulin single variable domains The present technology aims at providing a novel type of drug for treating diseases associated with CEACAM5.
  • an immunoglobulin single variable domain comprising a CDR1 (AbM numbering) consisting of the amino acid sequence of SEQ ID NO:1 ; a CDR2 (AbM numbering) consisting of the amino acid sequence of SEQ ID NO: 2 (AbM); and a CDR3 (AbM numbering) consisting of the amino acid sequence of SEQ ID NO: 230, or an immunoglobulin single variable domain comprising a CDR1 (AbM numbering) consisting of the amino acid sequence of SEQ ID NO: 231; a CDR2 (AbM numbering) consisting of the amino acid sequence of SEQ ID NO: 232; and a CDR3 (AbM numbering) consisting of the amino acid sequence of SEQ ID NO: 233; or an immunoglobulin single variable domain comprising a CDR1 (AbM numbering) consisting of the amino acid sequence of SEQ ID NO: 242; a CDR2 (AbM numbering) consisting of the amino acid sequence of SEQ
  • immunoglobulin single variable domains are highly specific for CEACAM5 and do not bind related CEACAM family members. They are thus a highly specific tool that can be used in the detection, targeting and/or treatment of diseases associated with CEACAM5.
  • 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.
  • immunoglobulin single variable domains apart from “conventional” immunoglobulins (e.g., monoclonal antibodies) or their fragments (such as Fab, Fab’, F(ab’)2, scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site.
  • a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e., a total of 6 CDRs will be involved in antigen binding site formation.
  • 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 disulfide 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
  • 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 VH- 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 VH- sequence or VHH sequence
  • An immunoglobulin single variable domain can for example be a heavy-chain ISVD, such as a VHH, including a humanized VHH, a VH, including a camelized VH and a human VH. In one embodiment, it is a VHH, 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) or a NANOBODY® ISVD (as defined herein and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof.
  • the immunoglobulin single variable domain may be a NANOBODY® ISVD (such as a VHH, including a humanized VHH or camelized VH) or a suitable fragment thereof.
  • VHH domains also known as VHHs, VHH antibody fragments, 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 For a further description of VHH’s, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001).
  • the generation of immunoglobulin sequences, such as VHHs has been described extensively in various publications, among which WO 94/04678, Hamers-Casterman et al.1993 and 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 VHHs obtained from said immunization is further screened for VHHs 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. Immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences can be used in the present technology. Also, fully human, humanized or chimeric sequences can be used. For example, camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g., camelized dAb as described by Ward et al.
  • the ISVDs can be fused forming 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. 2001 (J. Biol. Chem., Vol. 276, 10.
  • the ISVD comprised in the present technology 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 camelid, mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), “camelized” (as defined herein) immunoglobulin sequences (and in particular camelized VH sequences), as well as ISVDs 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.
  • “humanized” as defined herein
  • immunoglobulin sequences such as partially or fully humanized camelid, mouse or rabbit immunoglobulin sequences,
  • 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
  • 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 prior art (e.g., WO 2008/020079).
  • 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 (camelid) heavy chain antibody.
  • the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is a VH sequence from a mammal, such as the VH sequence of a human being, such as a VH3 sequence.
  • a mammal such as 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.
  • the 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
  • the amino acid residues of an ISVD 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. This means that, generally, 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.
  • FR1 of an ISVD comprises the amino acid residues at positions 1-25
  • CDR1 of an ISVD comprises the amino acid residues at positions 26-35
  • FR2 of an ISVD comprises the amino acids at positions 36- 49
  • CDR2 of an ISVD comprises the amino acid residues at positions 50-58
  • FR3 of an ISVD comprises the amino acid residues at positions 59-94
  • CDR3 of an ISVD comprises the amino acid residues at positions 95-102
  • FR1 of an ISVD comprises the amino acid residues at positions 1-30
  • CDR1 of an ISVD comprises the amino acid residues at positions 31-35
  • FR2 of an ISVD comprises the amino acids at positions 36-49
  • CDR2 of an ISVD comprises the amino acid residues at positions 50-65
  • FR3 of an ISVD comprises the amino acid residues at positions 66-94
  • CDR3 of an ISVD comprises the amino acid residues at positions 95-102
  • FR4 of an ISVD comprises the amino acid residues at positions 103-113.
  • the framework sequences are (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 VL-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 methods described herein may contain one or more of hallmark residues (as defined herein), such that the ISVD sequence is a NANOBODY® ISVD, such as, e.g., a VHH, including a humanized VHH, or camelized VH.
  • a NANOBODY® ISVD such as, e.g., a VHH, including a humanized VHH, or camelized VH.
  • 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
  • Hallmark residues as described herein
  • 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.
  • Table 1 Hallmark Residues in NANOBODY® ISVDs P osition Human VH3 Hallmark Residues 11 L, V; predominantly L L, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P, I; preferably L 3 7 V, I, F; usually V F(1), Y, V, L, A, H, S, I, W, C, N, G, D, T, P, preferably F(1) or Y 4 4(8) G E(3), Q(3), G(2), D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G (2) , E (3) or Q (3) ;most preferably G (2) or Q (3) .
  • sequences such as TERE (for example TEREL), TQRE (for example TQREL), KECE (for example KECEL or KECER), KQCE (for example KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREF or RQREW), QERE (for example QEREG), QQRE, (for example QQREW, QQREL or QQREF), KGRE (for example KGREG), KDRE (for example KDREV) are possible.
  • Some other possible, but less preferred sequences include for example DECKL and NVCEL. ( 4) With both GLEW at positions 44-47 and KERE or KQRE at positions 43-46.
  • the GLEW group also contains GLEW-like sequences at positions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and ELEW.
  • the present technology relates to ISVDs that specifically bind to CEACAM5.
  • the target molecules for the ISVDs is CEACAM5.
  • Examples are mammalian CEACAM5.
  • human CEACAM5 SEQ ID NO: 26
  • the versions from other species are also amenable to the present technology, for example CEACAM5 from mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates, such as cynomolgus monkeys (also referred to herein as “cyno”), or camelids, such as llama or alpaca.
  • the ISVDs of the present technology bind to human CEACAM5 (hCEACAM5; SEQ ID NO: 26).
  • the ISVDs of the present technology bind to human CEACAM5 and cyno CEACAM5 (cCEACAM5; SEQ ID NO: 27). Sequences of human, and cyno CEACAM5 are depicted in Table A-1 (SEQ ID NOs: 26 and 27).
  • the immunoglobulin single variable domains of the present technology show a highly affinity on CEACAM5 (e.g., as measured by SPR) as a (monovalent) single variable domain.
  • the immunoglobulin single variable domains of the present technology show a high potency for binding CEACAM5 on cells (e.g., as measured in (FACS) binding assay) as a (monovalent) single variable domain.
  • the immunoglobulin single variable domains of the present technology show a high potency for internalization of CEACAM5 expressing cells (e.g., as measured in internalization assay, for example as described herein) as a (monovalent) single variable domain.
  • the immunoglobulin single variable domains of the present technology are efficiently produced (e.g., in microbial hosts, such as Pichia, e.g., P. pastoris), even when encompassed with binders to other targets, and have good stability. Because of their small size, (monovalent) immunoglobulin single variable domains are more efficient for the penetration of epithelial tissues.
  • the immunoglobulin single variable domains of the present technology show a highly affinity on CEACAM5 as a (monovalent) single variable domain.
  • the affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see for example Ober et al. 2001, Intern. Immunology 13: 1551-1559).
  • SPR surface plasmon resonance
  • 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
  • a change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since the interactions can be measured in real-time, association and dissociation rates and affinities can be determined.
  • BLI can for example be performed using the 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 refers to a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules.
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of GLTFSTYTMG (SEQ ID NO:1); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of GLTFSTYTMG (SEQ ID NO: 1); c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences GLTFSTYTMG (SEQ ID NO: 1); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of AIIWSGSNTY (SEQ ID NO: 2); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of AIIWSGSNTY (
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 1, 2, and/or 230.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 8, 31, 32, 150-159.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 3.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 33.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 249.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 250.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 251.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 252.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 253.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 254.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315007E07 (SEQ ID NO: 8) and sequence optimized variants thereof in Table A-4 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of A0315007E07 (SEQ ID NO: 8); T028501789 (SEQ ID NO: 31), or T028501789 (E1D) (SEQ ID NO: 32), A031500251 (SEQ ID NO: 150), A031500252 (SEQ ID NO: 151), A031500253 (SEQ ID NO: 152), A031500254 (SEQ ID NO: 153), A031500379 (SEQ ID NO: 154), A031500382 (SEQ ID NO: 155), A031500383 (SEQ ID NO: 156), A031500385 (SEQ ID NO: 157), A031500387 (SEQ ID NO: 158), and A031500394 (SEQ ID NO: 159) (see Table A-2, Table A-4).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of TYTMG (SEQ ID NO: 17); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of TYTMG (SEQ ID NO: 17); c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences of TYTMG (SEQ ID NO: 17); and - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of AIIWSGSNTYYADSVKG (SEQ ID NO: 18); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of AIIWSGSNTYYADSVKG (SEQ ID NO:
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 17, 18, and/or 230.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 8, 31, 32, 150-159.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 3.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 33.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 249.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 250.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 251.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 252.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 253.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 254.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315007E07 (SEQ ID NO: 8) and sequence optimized variants thereof in Table A-4 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of A0315007E07 (SEQ ID NO: 8); T028501789 (SEQ ID NO: 31), or T028501789 (E1D) (SEQ ID NO: 32), A031500251 (SEQ ID NO: 150), A031500252 (SEQ ID NO: 151), A031500253 (SEQ ID NO: 152), A031500254 (SEQ ID NO: 153), A031500379 (SEQ ID NO: 154), A031500382 (SEQ ID NO: 155), A031500383 (SEQ ID NO: 156), A031500385 (SEQ ID NO: 157), A031500387 ((SEQ ID NO: 158), and A031500394 (SEQ ID NO: 159) (see Table A-2, Table A-4).
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 8, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 8.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 31, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 31.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 32, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 32.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 150, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 150.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 151, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 151.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 152, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 152.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 153, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 153.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 154, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 154.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 157, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 157.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 158, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 158.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 159, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 159.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 8, 31, 32, 150-159, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 8, 31, 32, 150-159, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 8, 31, 32, 150-159
  • the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 8, 31, 32, 150-159, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 8, 31, 32, 150-159
  • the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 8, 31, 32, 150-159, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 8, 31, 32, 150-159, the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 8, 31, 32, 150-159, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 8, 31, 32, 150-159, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8 or other members of the CEACAM family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a dissociation constant (K D ) of 10 -7 to 10 -11 moles/litre or less, and preferably 10 -8 to 10 -10 moles/litre or less and more preferably 1x10 -8 to 1x10 -9 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a KD of about 1.34x10 -9 moles/litre or 1.24x10 -9 moles/litre, as determined by SPR.
  • K D dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a k on -rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , such as between 1x10 5 M -1 s -1 and 5x10 5 M -1 s -1 , even more preferably of about 3.17x10 5 M -1 s -1 or 7.16x10 5 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a dissociation constant (K D ) of 10 -7 to 10 -10 moles/litre or less, and preferably 5x10 -8 to 10 -9 moles/litre or less and more preferably 5x10 -8 to 1x10 -8 moles/litre, even more preferably wherein the ISVD specifically binds to cyno CEACAM5 with a K D of about 1.76x10 -8 moles/litre, as determined by SPR.
  • K D dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD). In preferred embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM1, CEACAM6, CEACAM7 or CEACAM8.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 10 -8 M or lower, or even of 10 -9 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -8 M, such as between 10- 10 M and 10 -8 M, between 5x10 -9 M and 10 -8 M or between 10 -9 M and 10 -8 M.
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 10 -7 M or lower, more preferably of 5x10 -8 M or lower, or even of 10 -8 M or lower, such as between 10 -7 M and 10 -10 M, between 10 -7 M and 10 -9 M, between 5x10 -8 M and 10 -9 M or between 5x10 -8 M and 10 -8 M, for example, as measured in a FACS internalization assay on BxPC-3 cells expressing human CEACAM5.
  • EC50 value CEACAM5-mediated internalization potency
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of GX1TFSX2YAX3G (SEQ ID NO: 231), wherein the amino acid residue X1 is selected from R or H, the amino acid residue X2 is selected from E or D and the amino acid residue X3 is selected from L or M; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of GX1TFSX2YAX3G (SEQ ID NO: 231), wherein the amino acid residue X1 is selected from R or H, the amino acid residue X2 is selected from E or D and the amino acid residue X3 is selected from L or M; c ) amino acid sequences that
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 231, 232, and/or 233.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 16, 39, 40, 120, 121, 135-149.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 9, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 10 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 11.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 234, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 235 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 236.
  • the ISVD comprises or consists of the full amino acid sequence of A0315001C02 (SEQ ID NO.: 120) or A0315026D05 (SEQ ID NO.: 121), see Table A-2.
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 240, 241, and/or 233.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 145, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 145.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 146, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 146.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 135-149, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 135-149, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 135-149, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 135-149
  • the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 135-149, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 135-149
  • the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 135- 149, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 135-149, the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 135-149, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 135-149, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with dissociation constant (KD) of 10 -7 to 10 -10 moles/litre or less, and preferably 5x10 -8 to 10 -9 moles/litre or less and more preferably 5x10 -8 to 5x10 -9 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a K D of about 1.06x10 -8 moles/litre, 4,86x10 -9 moles/litre or 8.9x10 -9 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with kon-rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , such as between 1x10 5 M -1 s -1 and 5x10 5 M -1 s -1 , even more preferably of about 1.2x10 5 M -1 s -1 , 1.6x10 5 M -1 s -1 , 1.98x10 5 M -1 s, or 2.38x10 5 M -1 s , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a dissociation constant (KD) of 10 -7 to 10 -11 moles/litre or less, and preferably 10 -8 to 10 -10 moles/litre or less and more preferably 5x10 -9 to 10 -9 moles/litre, even more preferably wherein the ISVD specifically binds to cyno CEACAM5 with a KD of about 6.94x10 -9 moles/litre, 2.28x10 -9 moles/litre or 4.86x10 -9 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a kon-rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , such as between 2x10 5 M -1 s -1 and 1x10 6 M -1 s -1 , even more preferably of about 5.9x10 5 M -1 s -1 , 9.94x10 5 M -1 s -1 , or 8.62x10 5 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD). In preferred embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM1, CEACAM6, CEACAM7 or CEACAM8.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 5x10 -8 M or lower, or even of 10 -8 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -7 M, such as between 10 -10 M and 10 -7 M, between 10 -9 M and 5x10 -8 M or between 5x10 -9 M and 5x10 -8 M.
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 5x10 -7 M or lower, more preferably of 10 -7 M or lower, or even of 5x10 -8 M or lower, such as between 10 -7 M and 10 -10 M, between 10- 7 M and 10 -9 M, between 8x10 -8 M and 10 -9 M or between 8x10 -8 M and 10 -8 M, for example, as measured in a FACS internalization assay on BxPC-3 cells expressing human CEACAM5.
  • EC50 value CEACAM5-mediated internalization potency
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTFXSYDMG (SEQ ID NO: 242), wherein the amino acid residue X is selected from N, E, G, P, S or T; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTFXSYDMG (SEQ ID NO: 242), wherein the amino acid residue X is selected from N, E, G, P, S or T; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTFXSYDMG (SEQ ID NO: 242), wherein the amino acid residue X is selected from N, E, G, P,
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 242, 256, and/or 257.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 114, 175-208.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 255, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 256 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 284, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 256 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 287, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 256 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 288, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 256 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises or consists of the full amino acid sequence of A0315001D07 (SEQ ID NO: 114) (see Table A-2 and Table A-7).
  • SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 289, 290, and/or 257.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315001D07 (SEQ ID NO: 114) and sequence optimized variants thereof in Table A-7 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of A031500014 (SEQ ID NO: 175), A031500015 (SEQ ID NO: 176), A031500016 (SEQ ID NO: 177), A031500017 (SEQ ID NO: 178), A031500018 (SEQ ID NO: 179), A031500019 (SEQ ID NO: 180), A031500020 (SEQ ID NO: 181), A031500021 (SEQ ID NO: 182), A031500022 (SEQ ID NO: 183), A031500023 (SEQ ID NO: 184), A031500024 (SEQ ID NO: 185), A031500025 (SEQ ID NO: 186), A031500026 (SEQ ID NO: 187), A031500027 (SEQ ID NO: 188), A031500028 (SEQ ID NO: 189), A031500029 (SEQ ID NO: 190), A031500030 (SEQ ID NO: 191)
  • the ISVD comprises or consists of the full amino acid sequence of A0315001D07 (SEQ ID NO: 114) (see Table A-2 and Table A-7).
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 114, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 114.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 175, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 175.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 176, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 176.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 177, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 177.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 178, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 178.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 179, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 179.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 180, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 180.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 181, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 181.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 182, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 182.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 187, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 187.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 188, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 188.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 189, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 189.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 190, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 190.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 191, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 191.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 192, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 192.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 193, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 193.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 194, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 194.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 195, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 195.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 196, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 196.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 197, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 197.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 198, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 198.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 199, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 199.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 200, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 200.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 201, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 201.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 202, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 202.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 203, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 203.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 204, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 204.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 205, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 205.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 206, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 206.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 207, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 207.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 208, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 208.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 114, 175-208, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 114, 175-208, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 114, 175-208, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 114, 175-208, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with at least 100-fold lower affinity (KD), such as a at least 400-fold lower affinity (KD). In preferred embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 10 -8 M or lower, or even of 10 -9 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -7 M, such as between 10- 10 M and 10 -8 M, between 10 -9 M and 5x10 -8 M or between 10 -9 M and 10 -8 M.
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 10 -7 M or lower, more preferably of 5x10 -8 M or lower, or even of 10 -8 M or lower, such as between 10 -7 M and 10 -10 M, between 10 -7 M and 10 -9 M, between 5x10 -8 M and 10 -9 M or between 10 -8 M and 10 -9 M, for example, as measured in a FACS internalization assay on BxPC-3 cells expressing human CEACAM5.
  • EC50 value CEACAM5-mediated internalization potency
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTFSDXAMG (SEQ ID NO: 356), wherein the amino acid residue X is selected from F or Y; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTFSDXAMG (SEQ ID NO: 356), wherein the amino acid residue X is selected from F or Y; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTFSDXAMG (SEQ ID NO: 356), wherein the amino acid residue X is selected from F or Y; and - CDR2 (AbM numbering) consists of an amino acid sequence selected from:
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 356, 357, and/or 358.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 122 or 123.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 359, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 357 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 360.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 234, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 357 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 361.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315001C11 (SEQ ID NO: 122), A0315004G01 (SEQ ID NO: 123), and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of DXAMG (SEQ ID NO:237), wherein the amino acid residue X is selected from Y or F; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of DXAMG (SEQ ID NO: 237), wherein the amino acid residue X is selected from Y or F; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences of DXAMG (SEQ ID NO: 237), wherein the amino acid residue X is selected from Y or F; and - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of AIN
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 122 or 123, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD).
  • the ISVD specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above)
  • the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, and CEACAM8.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 122 or 123, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 122 or 123, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD).
  • KD 100-fold lower affinity
  • the ISVD specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, and CEACAM8.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 122 or 123
  • the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 122 or 123, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 122 or 123
  • the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 122 or 123, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 122 or 123, the ISVD still specifically binds to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD).
  • the ISVD specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 122 or 123, the ISVD still specifically binds to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD).
  • the ISVD specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, and CEACAM8.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a dissociation constant (KD) of 10 -7 to 10 -11 moles/litre or less, and preferably 10 -8 to 10 -10 moles/litre or less and more preferably 1x10 -8 to 1x10 -9 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a KD of about 5.85x10 -9 moles/litre, or 6.01x10 -9 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a k on -rate of 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 4 M -1 s -1 and 10 6 M -1 s -1 , more preferably between 10 4 M -1 s -1 and 10 5 M -1 s -1 , such as between 5x10 4 M -1 s -1 and 1x10 5 M -1 s -1 , even more preferably of about 8.68x10 4 M -1 s -1 , or 8.07x10 4 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a k on -rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , such as between 1x10 5 M -1 s -1 and 5x10 5 M -1 s -1 , even more preferably of about 3.04x10 5 M -1 s -1 , or 3.18x10 5 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (K D ). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD). In a preferred embodiment, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM1, CEACAM6, or CEACAM8.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 10 -8 M or lower, or even of 10 -9 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -7 M, such as between 10- 10 M and 10 -7 M, between 10 -9 M and 10 -7 M or between 5x10 -9 M and 10 -7 M.
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 10 -7 M or lower, more preferably of 5x10 -8 M or lower, or even of 10 -8 M or lower, such as between 10 -7 M and 10 -10 M, between 10 -7 M and 10 -9 M, between 5x10 -8 M and 10 -9 M or between 5x10 -8 M and 10 -8 M, for example, as measured in a FACS internalization assay on BxPC-3 cells expressing human CEACAM5.
  • EC50 value CEACAM5-mediated internalization potency
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTFNINEYHLA (SEQ ID NO: 363); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTFNINEYHLA (SEQ ID NO: 363); c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTFNINEYHLA (SEQ ID NO: 363); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence AIWWSTGNKI (SEQ ID NO: 364); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence the amino acid AI
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 363, 364, and/or 365.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NO: 124.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 363, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 364 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 365.
  • CDR1 AbM numbering
  • CDR2 AbM numbering
  • CDR3 AbM numbering
  • Specific examples of such ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315025D01 (SEQ ID NO: 124) and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8).
  • the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of INEYHLA (SEQ ID NO: 366); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of INEYHLA (SEQ ID NO: 366); c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences of INEYHLA (SEQ ID NO: 366); and - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of AIWWSTGNKIVADSVKG (SEQ ID NO: 367); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of AIWWSTGNKIVADSV
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 366, 367, and/or 365.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NO: 124.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 366, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 367 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 365.
  • CDR1 Kabat numbering
  • CDR2 Kabat numbering
  • CDR3 Kabat numbering
  • Specific examples of such ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315025D01 (SEQ ID NO: 124) and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 124, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 124.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 124, wherein the binding affinity is measured using the same method, such as e.g. SPR.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NO: 124, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 124, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NO: 124, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 124, the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 124, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a dissociation constant (KD) of 10 -7 to 10 -10 moles/litre or less, and preferably 10 -7 to 10 -9 moles/litre or less and more preferably 5x10 -7 to 1x10 -8 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a K D of about about 3.65x10 -8 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM1, CEACAM6, CEACAM7 or CEACAM8.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 10 -8 M or lower, or even of 10 -9 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -7 M, such as between 10- 10 M and 10 -7 M, between 5x10 -9 M and 8x10 -8 M or between 10 -9 M and 8x10 -8 M.
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 10 -7 M or lower, more preferably of 5x10 -8 M or lower, or even of 10 -8 M or lower, such as between 10 -7 M and 10 -10 M, between 10 -7 M and 10 -9 M, between 5x10 -8 M and 10 -9 M or between 5x10 -8 M and 10 -8 M, for example, as measured in a FACS internalization assay on BxPC-3 cells expressing human CEACAM5.
  • EC50 value CEACAM5-mediated internalization potency
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence AIWWSTGNRI (SEQ ID NO: 369); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence AIWWSTGN
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 368, 369, and/or 370.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NO: 125.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 368, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 369 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 370.
  • CDR1 AbM numbering
  • CDR2 AbM numbering
  • CDR3 AbM numbering
  • Specific examples of such ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315020C01 (SEQ ID NO: 125) and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8).
  • the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of DYHMA (SEQ ID NO: 371); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of DYHMA (SEQ ID NO: 371); c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences of DYHMA (SEQ ID NO: 371); and - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of AIWWSTGNRIVADSVKG (SEQ ID NO: 372); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of AIWWSTGNRIVADSVKG (SEQ ID NO: 372
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 371, 372, and/or 370.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NO: 125.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 371, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 372 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 370.
  • CDR1 Kabat numbering
  • CDR2 Kabat numbering
  • CDR3 Kabat numbering
  • Specific examples of such ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315020C01 (SEQ ID NO: 125) and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 125, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 125.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 125, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NO: 125, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 125, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NO: 125, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a k on -rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 4 M -1 s -1 and 10 6 M -1 s -1 , more preferably between 10 4 M -1 s -1 and 5x10 5 M -1 s -1 , such as between 5x10 4 M -1 s -1 and 1x10 5 M -1 s -1 , even more preferably of about 8.70x10 4 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -7 M, such as between 10 -10 M and 10 -7 M, between 5x10 -9 M and 8x10 -8 M or between 10 -9 M and 8x10 -8 M.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315026B01 (SEQ ID NO: 126) and A0315008B03 (SEQ ID NO: 127), and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence SYHMA (SEQ ID NO: 381); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence SYHMA (SEQ ID NO: 381); c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences SYHMA (SEQ ID NO: 381); and - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence AIWWSTGNXIVADSVKG (SEQ ID NO: 382), wherein the amino acid residue X is selected from A or T; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 381, 382, and/or 375.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 126 or 127.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 127, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 127.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 126 or 127, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 126 or 127, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 126 or 127, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 126 or 127, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 126 or 127
  • the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 126 or 127, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 126 or 127
  • the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 126 or 127, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 126 or 127, the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 126 or 127, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 126 or 127, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8 or other members of the CEACAM family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a dissociation constant (KD) of 10 -7 to 10 -11 moles/litre or less, and preferably 10 -7 to 10 -9 moles/litre or less and more preferably 1x10 -7 to 1x10 -8 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a KD of about 4.11x10 -8 moles/litre, or 2.67x10 -8 moles/litre, as determined by Surface Plasmon Resonance.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a k on rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , such as between 1x10 5 M -1 s -1 and 5x10 5 M -1 s -1 , even more preferably of about 1.93x10 5 M -1 s -1 , or 2.18x10 5 M -1 s -1 , as determined by Surface Plasmon Resonance .
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a dissociation constant (K D ) of 10 -6 to 10 -10 moles/litre or less, and preferably 5x10 -7 to 10 -9 moles/litre or less and more preferably 5x10 -7 to 1x10 -8 moles/litre, even more preferably wherein the ISVD specifically binds to cyno CEACAM5 with a KD of about 4.71x10 -8 moles/litre, or 2.72x10 -7 moles/litre, as determined by Surface Plasmon Resonance.
  • K D dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a kon rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 4 M -1 s -1 and 10 6 M -1 s -1 , more preferably between 5x10 4 M -1 s -1 and 10 6 M -1 s -1 , such as between 5x10 4 M -1 s -1 and 5x10 5 M -1 s -1 , even more preferably of about 6.61x10 4 M -1 s -1 , or 1.22x10 5 M -1 s -1 , as determined by Surface Plasmon Resonance.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD). In preferred embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM1, CEACAM6, CEACAM7 or CEACAM8.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GX1TFSX2YAMG (SEQ ID NO: 386), wherein the amino acid residue X 1 is selected from G or R, and wherein the amino acid residue X 2 is selected from S or N; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GX1TFSX2YAMG (SEQ ID NO: 386), wherein the amino acid residue
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 386, 387, and/or 388.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 128, 129, or 134.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 389, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 387 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 390.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 391, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 387 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 392.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315003D09 (SEQ ID NO: 128), A0315009B07 (SEQ ID NO: 129), and A031500085 (SEQ ID NO: 134), and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9).
  • the ISVDs provided by the present technology can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence XYAMG (SEQ ID NO: 393), wherein the amino acid residue X is selected from S or N; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence XYAMG (SEQ ID NO: 393), wherein the amino acid residue X is selected from S or N; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences XYAMG (SEQ ID NO: 393), wherein the amino acid residue X is selected from S or N;
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 393, 394, and/or 388.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 128, 129 or 134.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 395, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 394 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 390.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 396, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 394 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 392.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315003D09 (SEQ ID NO: 128), A0315009B07 (SEQ ID NO: 129) and A031500085 (SEQ ID NO: 134), and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 128, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 128.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 128, 129, or 134
  • the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 128, 129, or 134, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (K D ). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 10-fold lower affinity (KD). In preferred embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno, CEACAM6 or CEACAM7.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 10 -8 M or lower, or even of 10 -9 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -8 M, such as between 10- 10 M and 10 -8 M, between 10 -9 M and 10 -8 M or between 5x10 -10 M and 5x10 -9 M.
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 10 -7 M or lower, more preferably of 10 -8 M or lower, or even of 5x10 -9 M or lower, such as between 10 -7 M and 10 -10 M, between 10 -8 M and 10 -10 M, between 5x10 -9 M and 10 -10 M or between 5x10 -9 M and 10 -9 M, for example, as measured in a FACS internalization assay on BxPC-3 cells expressing human CEACAM5.
  • EC50 value CEACAM5-mediated internalization potency
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GGTVSTYTMG (SEQ ID NO: 397); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GGTVSTYTMG (SEQ ID NO: 397); c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GGTVSTYTMG (SEQ ID NO: 397); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence SIIWSGSTTV (SEQ ID NO: 398); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence SIIWSGST
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 397, 398, and/or 399.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NO: 130.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 397, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 398 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 399.
  • CDR1 AbM numbering
  • CDR2 AbM numbering
  • CDR3 AbM numbering
  • Specific examples of such ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315003F04 (SEQ ID NO: 130) and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8).
  • the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of TYTMG (SEQ ID NO: 17); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence TYTMG (SEQ ID NO: 17); c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences TYTMG (SEQ ID NO: 17); and - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence SIIWSGSTTVYADSVKG (SEQ ID NO: 400); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence SIIWSGSTTVYADSVKG (SEQ ID NO: 400);
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 17, 400, and/or 399.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NO: 130.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 400 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 399.
  • CDR1 Kabat numbering
  • CDR2 Kabat numbering
  • CDR3 Kabat numbering
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 130, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 130.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 130, wherein the binding affinity is measured using the same method, such as e.g.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NO: 130, wherein the binding affinity is measured using the same method, such as e.g. SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 50-fold lower affinity (KD).
  • the ISVD specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above)
  • the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM6 and CEACAM7.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 130, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NO: 130, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 50-fold lower affinity (KD).
  • KD 50-fold lower affinity
  • the ISVD specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM6 and CEACAM7.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 130, the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 130, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 130, the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NO: 130, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 130, the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 130, the ISVD still specifically binds to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 50-fold lower affinity (KD).
  • KD 50-fold lower affinity
  • the ISVD specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 130, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM6 and CEACAM7.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a dissociation constant (K D ) of 10 -7 to 10 -11 moles/litre or less, and preferably 10 -9 to 10 -11 moles/litre or less and more preferably 1x10 -9 to 1x10 -10 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a K D of about 7.90x10 -10 moles/litre, as determined by SPR.
  • K D dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a kon rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 6 M -1 s -1 and 10 7 M -1 s -1 , such as between 1x10 6 M -1 s -1 and 5x10 6 M -1 s -1 , even more preferably of about 1.19x10 6 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a dissociation constant (K D ) of 10 -7 to 10 -10 moles/litre or less, and preferably 1x10 -8 to 10 -10 moles/litre or less and more preferably 1x10 -8 to 1x10 -9 moles/litre, even more preferably wherein the ISVD specifically binds to cyno CEACAM5 with a KD of about 6.61x10 -9 moles/litre, as determined by SPR.
  • K D dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a k on rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , such as between 5x10 5 M -1 s -1 and 1x10 6 M -1 s -1 , even more preferably of about 9.2x10 5 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (K D ). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 50-fold lower affinity (KD). In preferred embodiments, the immunoglobulin single variable domains specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM6 or CEACAM7.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 10 -8 M or lower, or even of 10 -9 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -8 M, such as between 10- 10 M and 10 -8 M, between 5x10 -10 M and 10 -8 M or between 10 -9 M and 10 -8 M.
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 10 -7 M or lower, more preferably of 10 -8 M or lower, or even of 5x10 -9 M or lower, such as between 10 -7 M and 10 -10 M, between 10 -8 M and 10 -10 M, between 5x10 -9 M and 10 -10 M or between 5x10 -9 M and 10 -9 M, for example, as measured in a FACS internalization assay on BxPC-3 cells expressing human CEACAM5.
  • EC50 value CEACAM5-mediated internalization potency
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTLNDYHMA (SEQ ID NO: 368); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence AIWWSTGNTI (SEQ ID NO: 379); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence AIWW
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 368, 379, and/or 365.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NO: 131.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 368, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 379 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 365.
  • CDR1 AbM numbering
  • CDR2 AbM numbering
  • CDR3 AbM numbering
  • Specific examples of such ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315021A01 (SEQ ID NO: 131) and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8).
  • the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of DYHMA (SEQ ID NO: 371); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence DYHMA (SEQ ID NO: 371); c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences DYHMA (SEQ ID NO: 371); and - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence AIWWSTGNTIVADSVKG (SEQ ID NO: 385); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence AIWWSTGNTIVADSVKG (SEQ ID NO:
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 371, 385, and/or 365.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NO: 131.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 371, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 385 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 365.
  • CDR1 Kabat numbering
  • CDR2 Kabat numbering
  • CDR3 Kabat numbering
  • Specific examples of such ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315021A01 (SEQ ID NO: 131) and sequence optimized variants thereof (in addition to the CDRs as defined above).
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 131, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 131.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 131, wherein the binding affinity is measured using the same method, such as e.g. SPR.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NO: 131, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 131, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NO: 131, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 131
  • the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NO: 131, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 131
  • the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NO: 131, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 131, the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 131, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NO: 131, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8 or other members of the CEACAM family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a dissociation constant (K D ) of 10 -7 to 10 -11 moles/litre or less, and preferably 10 -7 to 10 -9 moles/litre or less and more preferably 1x10 -7 to 1x10 -8 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a K D of about 3.12x10 -8 moles/litre, as determined by SPR.
  • K D dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a kon rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , such as between 1x10 5 M -1 s -1 and 5x10 5 M -1 s -1 , even more preferably of about 2.16x10 5 M -1 s -1 ,as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a dissociation constant (K D ) of 10 -7 to 10 -10 moles/litre or less, and preferably 1x10 -7 to 10 -9 moles/litre or less and more preferably 1x10 -7 to 10 -8 moles/litre, even more preferably wherein the ISVD specifically binds to cyno CEACAM5 with a KD of about 3.45x10 -8 moles/litre, as determined by SPR.
  • K D dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a kon rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 4 M -1 s -1 and 10 6 M -1 s -1 , more preferably between 10 4 M -1 s -1 and 10 5 M -1 s -1 , such as between 5x10 4 M -1 s -1 and 1x10 5 M -1 s -1 , even more preferably of about 7.16x10 4 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD). In preferred embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM1, CEACAM6, CEACAM7 or CEACAM8.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 10 -8 M or lower, or even of 10 -9 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 5x10 -8 M, such as between 10 -10 M and 5x10 -8 M, between 5x10 -9 M and 5x10 -8 M or between 10 -9 M and 5x10 -8 M.
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 10 -6 M or lower, more preferably of 5x10 -6 M or lower, or even of 10 -7 M or lower, such as between 10 -6 M and 10 -10 M, between 10 -6 M and 10 -9 M, between 5x10 -6 M and 10 -9 M or between 5x10 -6 M and 10 -8 M, for example, as measured in a FACS internalization assay on BxPC-3 cells expressing human CEACAM5.
  • EC50 value CEACAM5-mediated internalization potency
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GX1TFSX2X3AX4G (SEQ ID NO: 530); wherein the amino acid residue X 1 is selected from R or H; wherein the amino acid residue X 2 is selected from E or D; wherein the amino acid residue X 3 is selected from F or Y; and wherein the amino acid residue X 4 is selected from L or M; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GX1TFSX2X3AX4G (SEQ ID NO: 530); wherein the amino acid residue X1 is selected from R or H; wherein the amino acid residue X
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID Nos: 530, 531, and/or 532.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 9, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 10 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 11.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 234, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 235 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 236.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 234, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 238 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 239.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 359, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 357 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 360.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 234, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 357 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 361.
  • CDR1 AbM numbering
  • CDR2 AbM numbering
  • CDR3 AbM numbering
  • Specific examples of such ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315024B02 (SEQ ID NO: 16) and sequence optimized variants thereof in Table A-3 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of A0315024B02 (SEQ ID NO: 16); T028501817 (SEQ ID NO: 39), or T028501817(E1D) (SEQ ID NO: 40), A031500086 (SEQ ID NO: 135), A031500087 (SEQ ID NO: 136), A031500088 (SEQ ID NO: 137), A031500089 (SEQ ID NO: 138), A031500090 (SEQ ID NO: 139), A031500091 (SEQ ID NO: 140), A031500092 (SEQ ID NO: 141), A031500093 (SEQ ID NO: 142), A031500094 (SEQ ID NO: 143), A031500095 (SEQ ID NO: 144), A031500096 (SEQ ID NO: 145, A031500097 (SEQ ID NO: 146), A031500098 (SEQ ID NO: 147), and A031500100 (SEQ ID NO:
  • the ISVD comprises or consists of the full amino acid sequence of A0315001C02 (SEQ ID NO.: 120) or A0315026D05 (SEQ ID NO.: 121), A0315001C11 (SEQ ID NO: 122), or A0315004G01 (SEQ ID NO: 123) see Table A-2.
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9).
  • the ISVDs provided by the present technology can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence X1X2AX3G (SEQ ID NO: 533); wherein the amino acid residue X1 is selected from E or D, X2 is selected from Y or F; and the amino acid residue X 3 is selected from L or M; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence X 1 X 2 AX 3 G (SEQ ID NO: 533); wherein the amino acid residue X 1 is selected from E or D, X2 is selected from Y or F;
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 533, 534, and/or 535.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 21, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 22 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 11.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 21, a CDR2 (Kabat numbering) that is the amino acid sequence of (AINWGGGWTYYADSVKG) SEQ ID NO: 43 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 11.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 243, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 244 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 236.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 243, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 245 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 246.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 229, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 362 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 360.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 243, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 362 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 361.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315024B02 (SEQ ID NO: 16) and sequence optimized variants thereof in Table A-3 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of A0315024B02 (SEQ ID NO: 16); T028501817 (SEQ ID NO: 39), or T028501817(E1D) (SEQ ID NO: 40), A031500086 (SEQ ID NO: 135), A031500087 (SEQ ID NO: 136), A031500088 (SEQ ID NO: 137), A031500089 (SEQ ID NO: 138), A031500090 (SEQ ID NO: 139), A031500091 (SEQ ID NO: 140), A031500092 (SEQ ID NO: 141), A031500093 (SEQ ID NO: 142), A031500094 (SEQ ID NO: 143), A031500095 (SEQ ID NO: 144), A031500096 (SEQ ID NO: 145, A031500097 (SEQ ID NO: 146), A031500098 (SEQ ID NO: 147), and A031500100 (SEQ ID NO:
  • the ISVD comprises or consists of the full amino acid sequence of A0315001C02 (SEQ ID NO.: 120) or A0315026D05 (SEQ ID NO.: 121), A0315001C11 (SEQ ID NO: 122), or A0315004G01 (SEQ ID NO: 123) see Table A-2.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 16, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 16.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 39, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 39.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 40, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 40.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 120, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 120.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 121, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 121.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 135, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 135.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 136, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 136.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 137, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 137.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 138, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 138.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 139, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 139.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 140, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 140.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 141, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 141.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 142, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 142.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 143, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 143.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 144, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 144.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 145, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 145.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 146, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 146.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 147, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 147.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 148, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 148.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 149, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 149.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 122, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 122.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 123, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 123.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149, wherein the binding affinity is measured using the same method, such as, e.g, SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149
  • the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149
  • the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149, the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 16, 39, 40, 120, 121, 122, 123, 135-149, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, and CEACAM8, or other members of the CEACAM family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with dissociation constant (KD) of 10 -7 to 10 -11 moles/litre or less, and preferably 5x10 -8 to 10 -10 moles/litre or less and more preferably 1x10 -8 to 1x10 -9 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a K D of about 1.06x10 -8 moles/litre, 4.86x10 -9 moles/litre, 8.9x10 -9 moles/litre, 5.85x10 -9 moles/litre, or 6.01x10 -9 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with kon-rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 4 M -1 s -1 and 10 6 M -1 s -1 , more preferably between 10 4 M -1 s -1 and 5x10 5 M -1 s -1 , such as between 5x10 4 M -1 s -1 and 5x10 5 M -1 s -1 , even more preferably of about 1.2x10 5 M -1 s -1 , 1.6x10 5 M -1 s -1 , 1.98x10 5 M -1 s, 2.38x10 5 M -1 s , 8.68x10 4 M -1 s -1 , or 8.07x10 4 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a dissociation constant (KD) of 10 -7 to 10 -11 moles/litre or less, and preferably 10 -8 to 10 -10 moles/litre or less and more preferably 10 -8 to 1x10 -9 moles/litre, even more preferably wherein the ISVD specifically binds to cyno CEACAM5 with a K D of about 6.94x10 -9 moles/litre, 2.28x10 -9 moles/litre, 4.86x10 -9 moles/litre, 8.16x10 -9 moles/litre, or 6.47x10- 9 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a kon-rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , such as between 2x10 5 M -1 s -1 and 1x10 6 M -1 s -1 , even more preferably of about 5.9x10 5 M -1 s -1 , 9.94x10 5 M -1 s -1 , 8.62x10 5 M -1 s -1 , 3.04x10 5 M -1 s -1 , or 3.18x10 5 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD). In preferred embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM1, CEACAM6, or CEACAM8.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 5x10 -8 M or lower, or even of 10 -8 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -7 M, such as between 10 -10 M and 10 -7 M, between 10 -9 M and 5x10 -8 M or between 5x10 -9 M and 5x10 -8 M.
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 5x10 -7 M or lower, more preferably of 10 -7 M or lower, or even of 5x10 -8 M or lower, such as between 10 -7 M and 10 -10 M, between 10- 7 M and 10 -9 M, between 5x10 -8 M and 10 -9 M or between 10 -7 M and 10 -8 M, for example, as measured in a FACS internalization assay on BxPC-3 cells expressing human CEACAM5.
  • EC50 value CEACAM5-mediated internalization potency
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GX1X2X3X4X5YX6X7X8 (SEQ ID NO: 536); wherein the amino acid residue X1 is selected from R or D, the amino acid residue X2 is selected from N or T; wherein the amino acid residue X3 is selected from F or L; wherein the amino acid residue X 4 is selected from N, E, G, P, S, or T; wherein the amino acid residue X 5 is selected from S, D, or the stretch of the following amino acids: I, N and E; wherein the amino acid residue X 6 is selected from D or H; wherein the amino acid residue X7 is
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 536, 537, and/or 538.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 114, 124-127, 175-208.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 255, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 256 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 284, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 256 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 285, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 256 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 286, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 256 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 287, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 256 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 288, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 256 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 363, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 364 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 365.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 368, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 369 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 370.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 376, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 377 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 365.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 378, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 379 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 380.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315001D07 (SEQ ID NO: 114), A0315025D01 (SEQ ID NO: 124), A0315020C01 (SEQ ID NO: 125), A0315026B01 (SEQ ID NO: 126) and A0315008B03 (SEQ ID NO: 127) and sequence optimized variants thereof in Table A-7 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of A031500014 (SEQ ID NO: 175), A031500015 (SEQ ID NO: 176), A031500016 (SEQ ID NO: 177), A031500017 (SEQ ID NO: 178), A031500018 (SEQ ID NO: 179), A031500019 (SEQ ID NO: 180), A031500020 (SEQ ID NO: 181), A031500021 (SEQ ID NO: 182), A031500022 (SEQ ID NO: 183), A031500023 (SEQ ID NO: 184), A031500024 (SEQ ID NO: 185), A031500025 (SEQ ID NO: 186), A031500026 (SEQ ID NO: 187), A031500027 (SEQ ID NO: 188), A031500028 (SEQ ID NO: 189), A031500029 (SEQ ID NO: 190), A031500030 (SEQ ID NO: 191)
  • the ISVD comprises or consists of the full amino acid sequence of A0315001D07 (SEQ ID NO: 114), A0315025D01 (SEQ ID NO: 124), A0315020C01 (SEQ ID NO: 125), A0315026B01 (SEQ ID NO: 126) and A0315008B03 (SEQ ID NO: 127) (see Table A-2 and Table A-7).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9).
  • the ISVDs provided by the present technology can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence X1YX2X3X4 (SEQ ID NO: 539); wherein the amino acid residue X1 is selected from D, S or the stretch of the following amino acids: I, N and E; wherein the amino acid residue X2 is selected from H or D; wherein the amino acid residue X3 is selected from L or M; and wherein the amino acid residue X 4 is selected from A or G; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of X 1 YX 2
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 539, 540, and/or 538.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 114, 124-127, 175-208.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 289, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 291 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 289, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 292 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 289, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 293 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 289, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 294 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 257.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 366, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 367 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 365.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 371, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 372 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 370.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 381, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 384 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 365.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 381, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 385 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 380.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315001D07 (SEQ ID NO: 114), A0315025D01 (SEQ ID NO: 124), A0315020C01 (SEQ ID NO: 125), A0315026B01 (SEQ ID NO: 126) and A0315008B03 (SEQ ID NO: 127) and sequence optimized variants thereof in Table A-7 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of A031500014 (SEQ ID NO: 175), A031500015 (SEQ ID NO: 176), A031500016 (SEQ ID NO: 177), A031500017 (SEQ ID NO: 178), A031500018 (SEQ ID NO: 179), A031500019 (SEQ ID NO: 180), A031500020 (SEQ ID NO: 181), A031500021 (SEQ ID NO: 182), A031500022 (SEQ ID NO: 183), A031500023 (SEQ ID NO: 184), A031500024 (SEQ ID NO: 185), A031500025 (SEQ ID NO: 186), A031500026 (SEQ ID NO: 187), A031500027 (SEQ ID NO: 188), A031500028 (SEQ ID NO: 189), A031500029 (SEQ ID NO: 190), A031500030 (SEQ ID NO: 191)
  • the ISVD comprises or consists of the full amino acid sequence of A0315001D07 (SEQ ID NO: 114), A0315025D01 (SEQ ID NO: 124), A0315020C01 (SEQ ID NO: 125), A0315026B01 (SEQ ID NO: 126) and A0315008B03 (SEQ ID NO: 127) (see Table A-2 and Table A-7).
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 114, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 114.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 175, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 175.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 176, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 176.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 177, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 177.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 178, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 178.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 179, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 179.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 180, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 180.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 181, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 181.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 182, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 182.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 183, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 183.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 184, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 184.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 185, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 185.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 186, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 186.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 187, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 187.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 188, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 188.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 189, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 189.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 190, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 190.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 191, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 191.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 192, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 192.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 193, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 193.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 194, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 194.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 195, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 195.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 196, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 196.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 197, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 197.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 198, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 198.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 199, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 199.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 200, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 200.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 201, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 201.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 202, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 202.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 203, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 203.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 204, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 204.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 205, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 205.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 206, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 206.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 207, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 207.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 208, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 208.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 124, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 124.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 125, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 125.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 126, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 126.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 127, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 127.
  • the ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above)
  • the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 114, 124-127, 175-208, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8 or other members of the CEACAM family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a dissociation constant (KD) of 10 -7 to 10 -11 moles/litre or less, and preferably 10 -7 to 10 -9 moles/litre or less and more preferably 5x10 -8 to 5x10 -9 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a K D of about 1.87x10 -8 moles/litre, 2.49x10 -8 moles/litre, 2.39x10 -8 moles/litre, 4.11x10 -8 moles/litre, or 2.67x10- 8 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a k on -rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , such as between 1x10 5 M -1 s -1 and 5x10 5 M -1 s -1 , even more preferably of about 3.96x10 5 M -1 s -1 , 2.01x10 5 M -1 s -1 , 2.19x10 5 M -1 s -1 , 1.93x10 5 M -1 s -1 , or 2.18x10 5 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a dissociation constant (KD) of 10 -6 to 10 -10 moles/litre or less, and preferably 5x10 -7 to 10 -9 moles/litre or less and more preferably 5x10 -7 to 10 -8 moles/litre, even more preferably wherein the ISVD specifically binds to cyno CEACAM5 with a K D of about 7.92x10 -7 moles/litre, 1.73x10 -7 moles/litre, 3.65x10 -8 moles/litre, 5.81x10 -8 moles/litre, 4.71x10- 8 moles/litre, or 2.72x10 -7 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a kon-rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 4 M -1 s -1 and 10 6 M -1 s -1 , more preferably between 5x10 4 M -1 s -1 and 10 6 M -1 s -1 , such as between 5x10 4 M -1 s -1 and 5x10 5 M -1 s -1 , even more preferably of about 7.98x10 4 M -1 s -1 , 8.70x10 4 M -1 s -1 , 6.61x10 4 M -1 s -1 , or 1.22x10 5 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (K D ). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with a at least 100-fold lower affinity (KD). In preferred embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM1, CEACAM6, CEACAM7 or CEACAM8.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of GX1TX2STYTX3G (SEQ ID NO: 541); wherein the amino acid residue X 1 is selected from G or L; wherein the amino acid residue X 2 is selected from V or F; and wherein the amino acid residue X 3 is selected from V or M; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 541, 542, and/or 543.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 8, 31, 32, 118-119, 130, 150-159, 171-174.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 336 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 339.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 334, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 337 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 340.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 3.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 33.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 249.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 250.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 251.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 252.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 253.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 1, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 2 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 254.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 397, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 398 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 399.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315002G06 (SEQ ID NO: 118), A0315002C10 (SEQ ID NO: 119), A0315007E07 (SEQ ID NO: 8), A0315003F04 (SEQ ID NO: 130) and sequence optimized variants thereof in Table A-6 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of A031500010 (SEQ ID NO: 171), A031500011 (SEQ ID NO: 172), A031500012 (SEQ ID NO: 173), or A031500013 (SEQ ID NO: 174) (see Table A-2, Table A-6).
  • the ISVD comprises or consists of the full amino acid sequence of A0315002G06 (SEQ ID NO: 118) or A0315002C10 (SEQ ID NO: 119) (Table A-2).
  • the ISVD comprises or consists of the full amino acid sequence of T028501789 (SEQ ID NO: 31), or T028501789 (E1D) (SEQ ID NO: 32), A031500251 (SEQ ID NO: 150), A031500252 (SEQ ID NO: 151), A031500253 (SEQ ID NO: 152), A031500254 (SEQ ID NO: 153), A031500379 (SEQ ID NO: 154), A031500382 (SEQ ID NO: 155), A031500383 (SEQ ID NO: 156), A031500385 (SEQ ID NO: 157), A031500387 (SEQ ID NO: 158), and A031500394 (SEQ ID NO: 159) (see Table A-4).
  • the ISVD comprises or consists of the full amino acid sequence of A0315007E07 (SEQ ID NO: 8) (Table A-2). In other embodiments, the ISVD comprises or consists of the full amino acid sequence of A0315003F04 (SEQ ID NO: 130) (Table A-2).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence TYTXG (SEQ ID NO: 341); wherein the amino acid residue X is selected from M or V; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence TYTXG (SEQ ID NO: 341); wherein the amino acid residue X is selected from M or V; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequences TYTXG (SEQ ID NO: 341); wherein the amino acid residue X is selected from M or V; and - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence the amino acid sequence of
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 341, 544, and/or 543.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 8, 31, 32, 118-119, 130, 150-159, 171-174.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 334 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 339.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 342, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 345 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 340.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 3.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 33.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 249.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 250.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 251.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 252.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 253.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 18 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 254.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 17, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 400 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 399.
  • ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for A0315002G06 (SEQ ID NO: 118), A0315002C10 (SEQ ID NO: 119), A0315007E07 (SEQ ID NO: 8), A0315003F04 (SEQ ID NO: 130) and sequence optimized variants thereof in Table A-6 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of A031500010 (SEQ ID NO: 171), A031500011 (SEQ ID NO: 172), A031500012 (SEQ ID NO: 173), or A031500013 (SEQ ID NO: 174) (see Table A-2, Table A-6).
  • the ISVD comprises or consists of the full amino acid sequence of A0315002G06 (SEQ ID NO: 118) or A0315002C10 (SEQ ID NO: 119) (Table A-2).
  • the ISVD comprises or consists of the full amino acid sequence of T028501789 (SEQ ID NO: 31), or T028501789 (E1D) (SEQ ID NO: 32), A031500251 (SEQ ID NO: 150), A031500252 (SEQ ID NO: 151), A031500253 (SEQ ID NO: 152), A031500254 (SEQ ID NO: 153), A031500379 (SEQ ID NO: 154), A031500382 (SEQ ID NO: 155), A031500383 (SEQ ID NO: 156), A031500385 (SEQ ID NO: 157), A031500387 (SEQ ID NO: 158), and A031500394 (SEQ ID NO: 159) (see Table A-4).
  • the ISVD comprises or consists of the full amino acid sequence of A0315007E07 (SEQ ID NO: 8) (Table A-2). In other embodiments, the ISVD comprises or consists of the full amino acid sequence of A0315003F04 (SEQ ID NO: 130) (Table A-2).
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 118, wherein the CDRs are as defined above. In one embodiment, the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 118.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 119, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 119.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 171, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 171.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 172, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 172.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 173, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 173.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 174, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 174.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 8, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 8.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 31, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 31.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 32, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 32.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 150, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 150.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 151, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 151.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 152, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 152.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 153, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 153.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 154, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 154.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 155, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 155.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 156, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 156.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 157, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 157.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 158, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 158.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 159, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 159.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 130, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 130.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID Nos: 8, 31, 32, 118-119, 130, 150-159, 171-174, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID Nos: 8, 31, 32, 118-119, 130, 150-159, 171-174, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and binds to other members of the CEACAMs family with at least 100-fold lower affinity (KD), such as a at least 400-fold lower affinity (KD).
  • KD 100-fold lower affinity
  • KD at least 400-fold lower affinity
  • the ISVD specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above)
  • the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM6, and CEACAM7.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 8, 31, 32, 118-119, 130, 150-159, 171-174, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and binds to other members of the CEACAMs family with at least 100-fold lower affinity (KD), such as a at least 400-fold lower affinity (KD).
  • KD 100-fold lower affinity
  • KD at least 400-fold lower affinity
  • the ISVD specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, and CEACAM7.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 8, 31, 32, 118-119, 130, 150-159, 171-174
  • the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 8, 31, 32, 118- 119, 130, 150-159, 171-174, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 8, 31, 32, 118-119, 130, 150-159, 171-174
  • the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID Nos: 8, 31, 32, 118- 119, 130, 150-159, 171-174, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 8, 31, 32, 118-119, 130, 150-159, 171-174, the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 8, 31, 32, 118-119, 130, 150-159, 171-174, the ISVD still specifically binds to human and cyno CEACAM5 and binds to other members of the CEACAMs family with at least 100-fold lower affinity (KD), such as a at least 400-fold lower affinity (KD).
  • KD 100-fold lower affinity
  • the ISVD specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 8, 31, 32, 118-119, 130, 150-159, 171-174, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, and CEACAM7.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a dissociation constant (KD) of 10 -7 to 10 -11 moles/litre or less, and preferably 10 -8 to 10 -11 moles/litre or less and more preferably 10 -8 to 10 -10 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a K D of about 8.83x10 -9 moles/litre, 1.44x10 -10 moles/litre, 7.90x10 -10 moles/litre, 1.34x10 -9 moles/litre or 1.24x10 -9 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with a k on -rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 5x10 6 M -1 s -1 , even more preferably of about 4.70x10 5 M -1 s -1 , or 1.91x10 6 M -1 s -1 , 1.19x10 6 M -1 s -1 , 3.17x10 5 M -1 s -1 or 7.16x10 5 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a dissociation constant (KD) of 10 -7 to 10 -10 moles/litre or less, and preferably 10 -7 to 10 -9 moles/litre or less and more preferably 5x10 -8 to 10 -9 moles/litre, even more preferably wherein the ISVD specifically binds to cyno CEACAM5 with a K D of about 3.92x10 -8 moles/litre, or 1.56x10 -9 moles/litre, 1.76x10 -8 moles/litre, 6.61x10 -9 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a k on -rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 5x10 6 M -1 s -1 , such as between 2x10 5 M -1 s -1 and 2x10 6 M -1 s -1 , even more preferably of about 2.92x10 5 M -1 s -1 , or 1.23x10 6 M -1 s -1 , 2.17x10 5 M -1 s -1 , 9.2x10 5 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In some embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and binds to other members of the CEACAMs family with at least 100-fold lower affinity (KD), such as a at least 400-fold lower affinity (KD). In preferred embodiments, the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to other members of the CEACAMs family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM1, CEACAM6, or CEACAM7.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a BxPC-3 cell line, which is a human pancreatic adenocarcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 10 -8 M or lower, or even of 10 -9 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -7 M, such as between 10- 10 M and 10 -8 M, between 10 -9 M and 5x10 -8 M or between 10 -9 M and 10 -8 M.
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 10 -7 M or lower, more preferably of 5x10 -8 M or lower, or even of 10 -8 M or lower, such as between 10 -7 M and 10 -10 M, between 10 -7 M and 10 -9 M, between 5x10 -8 M and 10 -9 M or between 10 -8 M and 10 -9 M, for example, as measured in a FACS internalization assay on BxPC-3 cells expressing human CEACAM5.
  • EC50 value CEACAM5-mediated internalization potency
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), that interacts with one or more amino acids of the CEACAM5 protein selected from K324, P325, F326, I327, T328, S329, N330, N331, S332, N333, L343, E346, D405, P406, V407, I408, L409, N410, L412, N442, P444, and A483.
  • the ISVD interacts with an epitope on CEACAM5 that comprises at least three amino acids selected K324, P325, F326, I327, T328, S329, N330, N331, S332, N333, L343, E346, D405, P406, V407, I408, L409, N410, L412, N442, P444, and A483.
  • the ISVD interacts with an epitope on CEACAM5 that comprises at least five amino acids selected from K324, P325, F326, I327, T328, S329, N330, N331, S332, N333, L343, E346, D405, P406, V407, I408, L409, N410, L412, N442, P444, and A483.
  • the ISVD interacts with an epitope on CEACAM5 that comprises at least eight amino acids selected from K324, P325, F326, I327, T328, S329, N330, N331, S332, N333, L343, E346, D405, P406, V407, I408, L409, N410, L412, N442, P444, and A483.
  • the ISVD interacts with an epitope on CEACAM5 that comprises at least 10 amino acids selected from K324, P325, F326, I327, T328, S329, N330, N331, S332, N333, L343, E346, D405, P406, V407, I408, L409, N410, L412, N442, P444, and A483.
  • the ISVD interacts with an epitope on CEACAM5 that comprises following amino acids: K324, S329, N330, N331, S332, N333, E346, and D405.
  • the epitope on CEACAM5 that is specifically bound by the ISVDs of the present technology comprises the following amino acids: K324, P325, F326, I327, T328, S329, N330, N331, S332, N333, L343, E346, D405, P406, V407, I408, L409, N410, L412, N442, P444, and A483.
  • the interacting amino acids preferably have a distance of ⁇ 3.8 ⁇ , wherein the distance between the amino acids is measured e.g., in Cryogenic- electron microscopy (cryo-EM).
  • the ISVDs specifically binding to this epitope on CEACAM5 form an interaction site (paratope) on CEACAM5 only with the CDR2 and CDR3.
  • CDR2 and CDR3 of the ISVD participate in the interaction with the CEACAM5 protein.
  • CDR2 and CDR3 of the ISVD form part of the paratope (the site with which the ISVD interacts with the CEACAM5 molecule) of the ISVD.
  • the amino acids that form part of this interaction sites are selected from T52, W52a, R53, G55, S56, T57, and H58 (Kabat numbering) in CDR2.
  • the amino acids that form part of this interaction sites are R53, S56, and H58 (Kabat numbering) in CDR2. In some embodiments, the amino acids that form part of this interaction sites are T52, W52a, R53, G55, S56, T57, and H58 (Kabat numbering) in CDR2. In some embodiments, the amino acids that form part of this interaction sites are D95, I100b, T100d, T100e, Q100f, and N101 (Kabat numbering) in the CDR3.
  • the amino acids that form part of this interaction sites are D95, L96, R97, F99, G100, P100a, I100b, T100c, T100d, T100e, Q100f, I100g, and N101 (Kabat numbering) in the CDR3.
  • the amino acids that form part of this interaction sites are R53, S56, and H58 (Kabat numbering) in CDR2 and D95, I100b, T100d, T100e, Q100f, and N101 (Kabat numbering) in the CDR3.
  • the amino acids that form part of this interaction sites are T52, W52a, R53, G55, S56, T57, and H58 (Kabat numbering) in CDR2 and D95, L96, R97, F99, G100, P100a, I100b, T100c, T100d, T100e, Q100f, I100g, and N101 (Kabat numbering) in the CDR3.
  • the amino acids that form part of this interaction sites are R53, S56, or H58 (Kabat numbering) in CDR2, wherein R53 forms an interaction site ( ⁇ 3.8 ⁇ distance) with D405 in the CEACAM5 epitope, wherein S56 forms an interaction site ( ⁇ 3.8 ⁇ distance) with K324 in the CEACAM5 epitope, and/or wherein H58 forms an interaction site ( ⁇ 3.8 ⁇ distance) with E346 in the CEACAM5 epitope.
  • the amino acids that form part of this interaction sites are R53, S56, and H58 (Kabat numbering) in CDR2, wherein R53 forms an interaction site ( ⁇ 3.8 ⁇ distance) with D405 in the CEACAM5 epitope, wherein S56 forms an interaction site ( ⁇ 3.8 ⁇ distance) with K324 in the CEACAM5 epitope, and/or wherein H58 forms an interaction site ( ⁇ 3.8 ⁇ distance) with E346 in the CEACAM 5 epitope (wherein the distance between the amino acids is measured e.g., in Cryogenic-electron microscopy (cryo-EM).
  • Cryogenic-electron microscopy cryo-EM
  • the amino acids that form part of this interaction sites are D95, I100b, T100d, T100e, Q100f, or N101 (Kabat numbering) in the CDR3, wherein D95 forms an interaction site ( ⁇ 3.8 ⁇ distance) with S332 in the CEACAM5 epitope, wherein I100b forms an interaction site ( ⁇ 3.8 ⁇ distance) with S329 in the CEACAM5 epitope, wherein T100d forms an interaction site ( ⁇ 3.8 ⁇ distance) with S329 in the CEACAM epitope, wherein T100e forms an interaction site ( ⁇ 3.8 ⁇ distance) with N330 in the CEACAM5 epitope, wherein Q100f forms an interaction site ( ⁇ 3.8 ⁇ distance) with N331 in the CEACAM5 epitope, and/or wherein N101 forms an interaction site ( ⁇ 3.8 ⁇ distance) with N333 in the CEACAM 5 epitope.
  • D95 forms an interaction site ( ⁇ 3.8 ⁇ distance) with S332 in the CEACAM5 epitope
  • I100b forms an interaction site (
  • the amino acids that form part of this interaction sites are D95, I100b, T100d, T100e, Q100f, and N101 (Kabat numbering) in the CDR3, wherein D95 forms an interaction site ( ⁇ 3.8 ⁇ distance) with S332 in the CEACAM5 epitope, wherein I100b forms an interaction site ( ⁇ 3.8 ⁇ distance) with S329 in the CEACAM5 epitope, wherein T100d forms an interaction site ( ⁇ 3.8 ⁇ distance) with S329 in the CEACAM epitope, wherein T100e forms an interaction site ( ⁇ 3.8 ⁇ distance) with N330 in the CEACAM5 epitope, wherein Q100f forms an interaction site ( ⁇ 3.8 ⁇ distance) with N331 in the CEACAM5 epitope, and/or wherein N101 forms an interaction site ( ⁇ 3.8 ⁇ distance) with N333 in the CEACAM 5 epitope (wherein the distance between the amino acids is measured e.g., in Cryogenic-electron microscopy (cryo-EM
  • the amino acids that form part of this interaction sites are R53, S56, and H58 (Kabat numbering) in CDR2, and D95, I100b, T100d, T100e, Q100f, and N101 (Kabat numbering) in the CDR3, wherein R53 forms an interaction site ( ⁇ 3.8 ⁇ distance) with D405 in the CEACAM5 epitope, wherein S56 forms an interaction site ( ⁇ 3.8 ⁇ distance) with K324 in the CEACAM5 epitope, wherein H58 forms an interaction site ( ⁇ 3.8 ⁇ distance) with E346 in the CEACAM 5 epitope, wherein D95 forms an interaction site ( ⁇ 3.8 ⁇ distance) with S332 in the CEACAM5 epitope, wherein I100b forms an interaction site ( ⁇ 3.8 ⁇ distance) with S329 in the CEACAM5 epitope, wherein T100d forms an interaction site ( ⁇ 3.8 ⁇ distance) with S329 in the CEACAM5 epitope, wherein T100e forms an interaction site ( ⁇ 3.8 ⁇ distance) with D4
  • the ISVDs specifically binding to this epitope on CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence GITX1RGGSTH (SEQ ID NO: 578), wherein the amino acid residue X 1 is any amino acid independently chosen, preferably wherein the amino acid residue X 1 is W, and/or - CDR3 (AbM numbering) consists of an amino acid sequence X1LX2PFX3PITX4X5PQRIX6Y (SEQ ID NO: 581) wherein X1, X2, X3, X4, X5, and X6 are any amino acid independently chosen, preferably wherein the amino acid residue X1 is D, wherein the amino acid residue X2 is R, wherein the amino acid residue X 3 is G, wherein the amino acid residue X 4 is T, wherein the amino
  • the ISVDs specifically binding to this epitope on CEACAM5 may also be ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence GITX1RGGSTH (SEQ ID NO: 578), wherein the amino acid residue X1 is selected from W and N, preferably wherein the amino acid residue X 1 is W; and - CDR3 (AbM numbering) consists of an amino acid sequence X1LX2PFX3PITX4X5PQRIX6Y (SEQ ID NO: 581), wherein the amino acid residue X1 is selected from D and V, wherein the amino acid residue X2 is selected from R and V, wherein the amino acid residue X3 is selected from G, A, and F, wherein the amino acid residue X4 is selected from Q and T, wherein the amino acid residue X 5 is selected from T and E
  • the ISVDs specifically binding to this epitope on CEACAM5 further comprises a CDR1 (AbM Numbering) : a ) having a length of 10 amino acids; b) having an aliphatic index of 49.00 or 10.00, preferably of 10.00; c) having a grand average of hydropathicity (GRAVY) of -0.970, -0.270, -1.000 preferably -0.970; d ) comprising one positively charged and at least one negatively charged residue; e) comprising 30% or 40% hydrophobic, 10% or 20% acid, 20% or 10% basic and 40% neutral amino acids, such as comprising 30% hydrophobic, 10% acid, 20% basic and 40% neutral amino acids; or comprising 40% hydrophobic, 10% acid, 10% basic and 40% neutral amino acids; or comprising 30% hydrophobic, 20% acid, 21% basic and 40% neutral amino acids; f ) consisting of a polar neutral amino acid, a basic amino acid, a polar neutral amino acid, a hydrophobic amino acid, an acid
  • the ISVDs specifically binding to the above specific epitope on CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence GITX1RGGSTH (SEQ ID NO: 578), wherein the amino acid residue X1 is selected from W and N, preferably wherein the amino acid residue X 1 is W; and - CDR3 (AbM numbering) consists of an amino acid sequence X1LX2PFX3PITX4X5PQRIX6Y (SEQ ID NO: 581), wherein the amino acid residue X1 is selected from D and V, wherein the amino acid residue X2 is selected from R and V, wherein the amino acid residue X3 is selected from G, A, and F, wherein the amino acid residue X4 is selected from Q and T, wherein the amino acid residue X5 is selected from
  • the ISVDs specifically binding to the above specific epitope on CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTFDNX1AMG (SEQ ID NO: 593); wherein the amino acid residue X1 is selected from H, L or E; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTFDNX 1 AMG (SEQ ID NO: 593); wherein the amino acid residue X 1 is selected from H, L or E; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTFDNX1AMG (SEQ ID NO: 593); wherein the amino acid residue X1 is selected from H, L or E; and - CDR2 (AbM numbering) consists of an amino acid
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 548, 564-576. It should be noted, however, that the ISVDs specifically binding to CEACAM5 may not necessarily be limited to those binding to the above-specified epitope.
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GITX1RGGSTH (SEQ ID NO: 578), wherein the amino acid residue X1 is selected from W and N; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GITX1RGGSTH (SEQ ID NO: 578), wherein the amino acid residue X1 is selected from W and N; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GITX 1 RGGSTH (SEQ ID NO: 578), wherein the amino acid residue X 1 is selected from W and N; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence
  • any of the ISVDs described above i.e., the ISVDs binding specifically binding to CEACAM5 and which are not necessarily limited to binding to the above specific epitope on CEACAM5 and to ISVDs specifically binding to the above-specified epitope on CEACAM5.
  • the ISVDs specifically binding to CEACAM5 further comprises a CDR1 (AbM Numbering): a ) having a length of 10 amino acids; b) having an aliphatic index of 49.00 or 10.00, preferably of 10.00; c) having a grand average of hydropathicity (GRAVY) of -0.970, -0.270, -1.000 preferably -0.970; d ) comprising one positively charged and at least one negatively charged residue; e) comprising 30% or 40% hydrophobic, 10% or 20% acid, 20% or 10% basic and 40% neutral amino acids, such as comprising 30% hydrophobic, 10% acid, 20% basic and 40% neutral amino acids; or comprising 40% hydrophobic, 10% acid, 10% basic and 40% neutral amino acids; or comprising 30% hydrophobic, 20% acid, 21% basic and 40% neutral amino acids; f ) consisting of a polar neutral amino acid, a basic amino acid, a polar neutral amino acid, a hydrophobic amino acid, an acidic amino acid, a
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GITX1RGGSTH (SEQ ID NO: 578), wherein the amino acid residue X1 is selected from W and N; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GITX1RGGSTH (SEQ ID NO: 578), wherein the amino acid residue X1 is selected from W and N; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GITX 1 RGGSTH (SEQ ID NO: 578), wherein the amino acid residue X 1 is selected from W and N; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GITX1
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 579 or 580; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 579 or 580; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 579 or 580; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NOs: 582-589, b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 582-589; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 579; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 579; c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 579; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 582; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 582; f) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 582.
  • - CDR2 AbM numbering
  • - CDR2
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 579; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 579; c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 579; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 583; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 583; f) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 583.
  • - CDR2 AbM numbering
  • - CDR2
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 579; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 579; c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 579; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 584; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 584; f) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 584.
  • - CDR2 AbM numbering
  • - CDR2
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 579; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 579; c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 579; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 585; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 585; f) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 585.
  • - CDR2 AbM numbering
  • - CDR2
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 579; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 579; c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 579; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 586; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 586; f) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 586.
  • - CDR2 AbM numbering
  • - CDR2
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 579; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 579; c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 579; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 587; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 587; f) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 587.
  • - CDR2 AbM numbering
  • - CDR2
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 579; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 579; c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 579; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 588; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 588; f) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 588.
  • - CDR2 AbM numbering
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 579; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 579; c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 579; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 589; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 589; f) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 589.
  • - CDR2 AbM numbering
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 580; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 580; c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 580; and - CDR3 (AbM numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 582; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 582; f) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 582.
  • - CDR2 AbM numbering
  • - CDR2
  • the CDR2 and CDR3 sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR2 sequences of SEQ ID NOs: 579, or 580 and/or the CDR3 sequences of SEQ ID NOs: 582-589.
  • the ISVD comprise a CDR2 and CDR3 (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDR2 and CDR3 of the ISVD with the amino acid sequence selected from SEQ ID NOs: 548, 564-576.
  • the ISVD comprises a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • the ISVD comprises a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 583. In one embodiment, the ISVD comprises a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 584. In one embodiment, the ISVD comprises a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 585.
  • the ISVD comprises a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 586.
  • the ISVD comprises a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 587.
  • the ISVD comprises a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 588.
  • the ISVD comprises a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 589.
  • the ISVD comprises a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 580 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9).
  • the ISVDs provided by the present technology can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 591 or 592; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 591 or 592; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 591 or 592; and - CDR3 (Kabat numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 582-589; e) amino acid sequence
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 591; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 591; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 591; and - CDR3 (Kabat numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 582; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 582; f ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 582.
  • - CDR2 Kabat numbering
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 591; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 591; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 591; and - CDR3 (Kabat numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 583; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 583; f ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 583.
  • - CDR2 Kabat numbering
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 591; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 591; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 591; and - CDR3 (Kabat numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 584; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 584; f ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 584.
  • - CDR2 Kabat numbering
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 591; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 591; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 591; and - CDR3 (Kabat numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 585; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 585; f ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 585.
  • - CDR2 Kabat numbering
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 591; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 591; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 591; and - CDR3 (Kabat numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 586; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 586; f ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 586.
  • - CDR2 Kabat numbering
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 591; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 591; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 591; and - CDR3 (Kabat numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 587; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 587; f ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 587.
  • - CDR2 Kabat numbering
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 591; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 591; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 591; and - CDR3 (Kabat numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 588; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 588; f) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 588.
  • - CDR2 Kabat numbering
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 591; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 591; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 591; and - CDR3 (Kabat numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 589; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 589; f ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 589.
  • - CDR2 Kabat numbering
  • the ISVD comprises 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 592; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 592; c ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 592; and - CDR3 (Kabat numbering) consists of an amino acid sequence selected from: d) the amino acid sequence of SEQ ID NOs: 582; e) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 582; f ) amino acid sequences that have 4, 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 582.
  • - CDR2 Kabat numbering
  • the CDR2 and CDR3 sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR2 sequences of SEQ ID NOs: 591 or 592 and/or the CDR3 sequences of SEQ ID NOs: 582-589.
  • the ISVD comprise a CDR2 and CDR3 (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDR2 and CDR3 of the ISVD with the amino acid sequence selected from SEQ ID NOs: 548, 564-576.
  • the ISVD comprises a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • the ISVD comprises a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 583. In one embodiment, the ISVD comprises a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 584. In one embodiment, the ISVD comprises a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 585.
  • the ISVD comprises a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 586.
  • the ISVD comprises a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 587.
  • the ISVD comprises a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 588.
  • the ISVD comprises a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 589. In one embodiment, the ISVD comprises a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 592 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GRTFDNX1AMG (SEQ ID NO: 593); wherein the amino acid residue X 1 is selected from H, L or E; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GRTFDNX1AMG (SEQ ID NO: 593); wherein the amino acid residue X1 is selected from H, L or E; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GRTFDNX 1 AMG (SEQ ID NO: 593); wherein the amino acid residue X 1 is selected from H, L or E; d) and - CDR2 (AbM numbering) consists of an amino acid sequence selected from:
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID Nos: 593, 578, and/or 581.
  • the ISVD comprise CDRs (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 548, 564-576.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 594, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 594, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 583.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 594, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 584.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 594, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 585.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 594, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 586.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 594, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 587.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 594, CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 588.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 594, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 589.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 594, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 580 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 595, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • the ISVD comprises a CDR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 596, a CDR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 579 and a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • CDR1 AbM numbering
  • CDR2 AbM numbering
  • CDR3 AbM numbering
  • Specific examples of such ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for T023200134 (SEQ ID NO: 548) and sequence optimized variants thereof in Table A-17 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of T028500003 (SEQ ID NO: 564), T028500004 (SEQ ID NO: 565), T028500005 (SEQ ID NO: 566), T028500591 (SEQ ID NO: 567), T0285000588 (SEQ ID NO: 568), T028500582 (SEQ ID NO: 569), T028500593 (SEQ ID NO: 570), T028500592 (SEQ ID NO: 571), T028500587 (SEQ ID NO: 572), T028500581 (SEQ ID NO: 573), T028500590 (SEQ ID NO: 574), T028500594 (SEQ ID NO: 575), or T028500583 (SEQ ID NO: 576) (see Table A-2, Table A-17).
  • the SEQ ID NOs for the CDR sequences referred to above are based on the CDR definition according to the AbM definition (see Table A-8). It is noted that the SEQ ID NOs for the CDR sequences defined according to the Kabat definition can likewise be used (see Table A-9). Accordingly, the ISVDs provided by the present technology, specifically binding to CEACAM5 as described above using the AbM definition, can be also described using the Kabat definition.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence NX1AMG (SEQ ID NO: 597); wherein the amino acid residue X 1 is selected from H, L or E; b ) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence NX 1 AMG (SEQ ID NO: 597); wherein the amino acid residue X 1 is selected from H, L or E; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence NX1AMG (SEQ ID NO: 597); wherein the amino acid residue X1 is selected from H, L or E; and - CDR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence NX1
  • the CDR sequences have at least 90% amino acid sequence identity, more preferably at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the CDR sequences of SEQ ID NOs: 597, 590, and/or 581.
  • the ISVD comprise CDRs (Kabat numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDRs of the ISVD with the amino acid sequence selected from SEQ ID NOs: 548, 564-576.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 598, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 598, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 583.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 598, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 584.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 598, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 585.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 598, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 586.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 598, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 587.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 598, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 588.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 598, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 589.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 598, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 592 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 599, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • the ISVD comprises a CDR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 600, a CDR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 591 and a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 582.
  • CDR1 Kabat numbering
  • CDR2 Kabat numbering
  • CDR3 Kabat numbering
  • Specific examples of such ISVDs that specifically bind to CEACAM5 have one or more, or all, framework regions as indicated for T023200134 (SEQ ID NO: 548) and sequence optimized variants thereof in Table A-17 (in addition to the CDRs as defined above).
  • the ISVD comprises or consists of the full amino acid sequence of T028500003 (SEQ ID NO: 564), T028500004 (SEQ ID NO: 565), T028500005 (SEQ ID NO: 566), T028500591 (SEQ ID NO: 567), T0285000588 (SEQ ID NO: 568), T028500582 (SEQ ID NO: 569), T028500593 (SEQ ID NO: 570), T028500592 (SEQ ID NO: 571), T028500587 (SEQ ID NO: 572), T028500581 (SEQ ID NO: 573), T028500590 (SEQ ID NO: 574), T028500594 (SEQ ID NO: 575), or T028500583 (SEQ ID NO: 576) (see Table A-2, Table A-17).
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 548, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 548.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 564, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 564.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 565, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 565.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 566, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 556.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 567, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 567.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 568, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 568.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 569, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 569.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 570, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 570.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 571, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 571.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 572, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 572.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 573, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 573.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 574, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 574.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 575, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 575.
  • the ISVD specifically binding to human CEACAM5 may have a sequence identity of more than 90%, such as more than 95% or even more than 99%, with SEQ ID NO: 576, wherein the CDRs are as defined above.
  • the ISVD specifically binding to CEACAM5 comprises or consists of the amino acid sequence of SEQ ID NO: 576.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 548, 564-576, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 548, 564-576, wherein the binding affinity is measured using the same method, such as, e.g, SPR.
  • the ISVD specifically binding to CEACAM5 when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD). In another embodiment, when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when such an ISVD specifically binding to CEACAM5 has 3, 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 548, 564-576, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 548, 564-576, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD when the CDRs of such an ISVD specifically binding to CEACAM5 have at least 90% amino acid sequence identity, at least 95% amino acid sequence identity, such as 99% amino acid sequence identity or more, with a corresponding reference CDR sequence (above), the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM1, CEACAM6, CEACAM7 and CEACAM8, or other members of the CEACAM family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 548, 564-576, the ISVD has at least (essentially) the same binding affinity to human CEACAM5 compared to one of the VHHs with SEQ ID NOs: 548, 564-576, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 548, 564-576, the ISVD has at least (essentially) the same binding affinity to cyno CEACAM5 compared to one of the VHHs with SEQ ID NOs: 548, 564-576, wherein the binding affinity is measured using the same method, such as, e.g., SPR.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 548, 564-576, the ISVD still specifically binds to human CEACAM5 and cyno CEACAM5 with less than 10-fold difference in affinity (KD).
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 548, 564-576, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to other members of the CEACAMs family.
  • the ISVD specifically binding to human CEACAM5 has a sequence identity of more than 90%, such as more than 95% or more than 99%, with one of the VHHs with SEQ ID NOs: 548, 564-576, the ISVD still specifically binds to human and cyno CEACAM5 and does not bind to CEACAM6 or other members of the CEACAM family.
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with dissociation constant (KD) of 10 -7 to 10 -11 moles/litre or less, and preferably 1x10 -7 to 10 -10 moles/litre or less such as more preferably 1x10 -8 to 1x10 -10 moles/litre, even more preferably wherein the ISVD specifically binds to human CEACAM5 with a KD of about 1.20x10 -9 moles/litre, 2.82x10 -9 moles/litre, 6.51x10 -9 moles/litre, 1.27x10 -8 moles/litre, 2.44x10 -8 moles/litre, 3.07 x10 -8 moles/litre, 3.17 x10 -8 moles/litre, 7.62 x10 -8 moles/litre, 9.28 x10- 8 moles/litre, 1.59 x10 -7 moles/litre, or 1.97 x10 -7 moles/litre, as determined by SPR.
  • KD dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5 with k on -rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 4 M -1 s -1 and 10 6 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , such as between 1x10 5 M -1 s -1 and 5x10 5 M -1 s -1 , even more preferably of about 4.3x10 5 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a dissociation constant (K D ) of 10 -7 to 10 -11 moles/litre or less, and preferably 10 -7 to 10 -10 moles/litre or less and more preferably 5x10 -8 to 10 -9 moles/litre, even more preferably wherein the ISVD specifically binds to cyno CEACAM5 with a KD of about 1.90x10 -9 moles/litre, 3.06x10 -9 moles/litre, 6.07x10 -9 moles/litre, 8.06x10 -9 moles/litre, 3.16x10 -8 moles/litre, 3.71x10 -8 moles/litre, 1.65x10 -8 moles/litre, 7.04x10 -8 moles/litre, 1.16x10 -8 moles/litre, 1.55x10 -8 moles/litre, or 9.68x10 -8 moles/litre, as determined by SPR.
  • K D dissociation constant
  • the immunoglobulin single variable domains of the present technology specifically bind to cyno CEACAM5 with a kon-rate of between 10 4 M -1 s -1 to about 10 7 M -1 s -1 , preferably between 10 5 M -1 s -1 and 10 7 M -1 s -1 , more preferably between 10 5 M -1 s -1 and 10 6 M -1 s -1 , as determined by SPR.
  • the immunoglobulin single variable domains of the present technology specifically bind to human and cyno CEACAM5 and do not bind to human and cyno CEACAM1, CEACAM6, CEACAM7 and CEACAM8.
  • a preferred assay for measuring binding and internalization of the ISVD (or polypeptide comprising the ISVD) to CEACAM5 exposed on a cell surface is a FACS assay.
  • the ISVDs or polypeptides of the present invention can be applied to cancer cell lines overexpressing CEACAM5, such as a LS174T cell line, which is a human colonic carcinoma cell line.
  • VHH bound to human CEACAM5 can be detected with a fluorophore- labelled anti-VHH antibody and the cells can be analyzed with a flow cytometer to evaluate the binding properties of ISVDs or polypeptides of the present invention.
  • the internalization can be measured using a Live-Cell Analysis Systems capable of recording fluorescence and bright field images, such as the Incucyte instrument (Sartorius).
  • the immunoglobulin single variable domains of the present technology specifically bind to human CEACAM5.
  • the immunoglobulin single variable domains of the present technology may have EC50 values of 10 -7 M or lower, more preferably of 5x10 -8 M or lower, or even of 10 -8 M or lower.
  • the immunoglobulin single variable domains of the present technology may have EC50 values between 10 -11 M and 10 -7 M, such as between 10 -10 M and 10 -7 M, between 10 -9 M and 5x10 -8 M or between 10 -9 M and 5x10 -9 M, such as 1.98x10 -9 M,
  • the immunoglobulin single variable domains of the present technology has an CEACAM5-mediated internalization potency (EC50 value) of 5x10 -7 M or lower, more preferably of 10 -7 M or lower, or even of 5x10 -8 M or lower, such as between 10 -7 M and 10 -10 M, between 10- 7 M and 10 -9 M, between 5x10 -8 M and 10 -9 M or between 5x10 -8 M and 10 -8 M, for example, as measured in a fluorescence based internalization assay on LS174T cells expressing human CEACAM5.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR3 (AbM numbering) consists of the amino acid sequence GDHRGPWYN (SEQ ID NO: 610).
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR3 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 610; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 610; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 610.
  • FR1 to FR4 framework regions
  • CDR1 to CDR3 complementarity determining regions
  • the ISVDs specifically binding to CEACAM5 may further comprise a CDR1 (AbM numbering): a ) having a length of 10 amino acids (AbM numbering); b) having an aliphatic index of 0.00; c) having a grand average of hydropathicity (GRAVY) of -0.080; d) comprising one negatively charged residue; e) comprising 30% hydrophobic, 10% acidic and 60% neutral amino acids; f) consisting of a polar neutral amino acid, a hydrophobic amino acid, a polar neutral amino acid, a hydrophobic amino acid, three polar neutral amino acids, an acidic amino acid, a hydrophobic amino acid, and a polar neutral amino acid; and/or g ) comprising at least one, preferably all of the amino acids G, F, T, S, Y, D, or M.
  • a CDR1 AbM numbering
  • the ISVDs specifically binding to CEACAM5 may further comprise a CDR2 (AbM numbering): a ) having a length of 10 amino acids (AbM numbering); b) having an aliphatic index of 39.00; c) having a grand average of hydropathicity (GRAVY) of -0.810; d) comprising one positively charged residue; e) comprising 10% hydrophobic, 10% basic and 80% neutral amino acids; f) consisting of a polar neutral amino acid, a hydrophobic amino acid, a polar neutral amino acid, a basic amino acid, a polar neutral amino acid, six polar neutral amino acid, preferably in that order; and/or g ) comprising at least one, preferably all of the amino acids T, I, N, R, G, S or T.
  • a CDR2 AbM numbering
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR3 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 610; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 610; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 610; and - CDR1 (AbM numbering): a) has a length of 10 amino acids; b) has an aliphatic index of 0.00; c) has a grand average of hydropathicity (GRAVY) of -0.080; d) comprises one negatively charged residue; e) comprises 30% hydrophobic, 10% acidic and 60% neutral amino acids; f) consisting of a polar neutral
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR3 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 610; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 610; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 610; and - CDR2 (AbM numbering): a) has a length of 10 amino acids (AbM numbering); b) having an aliphatic index of 39.00; c) having a grand average of hydropathicity (GRAVY) of -0.810; d) comprises one positively charged residue; e) comprises 10% hydrophobic, 10% basic and 80% neutral amino acids; f) consists of
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR3 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 610; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 610; c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 610; - CDR1 (AbM numbering): a) has a length of 10 amino acids; b) has an aliphatic index of 0.00; c) has a grand average of hydropathicity (GRAVY) of -0.080; d) comprises one negatively charged residue; e) comprises 30% hydrophobic, 10% acidic and 60% neutral amino acids; f) consists of a polar neutral amino acid sequence selected
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR3 (AbM numbering) consists of the amino acid sequence GDHRGPWYN (SEQ ID NO: 610); and - CDR1 (AbM numbering): a) has a length of 10 amino acids; b) has an aliphatic index of 0.00; c) has a grand average of hydropathicity (GRAVY) of -0.080; d) comprises one negatively charged residue; e) comprises 30% hydrophobic, 10% acidic and 60% neutral amino acids; f) consists of a polar neutral amino acid, a hydrophobic amino acid, a polar neutral amino acid, a hydrophobic amino acid, three polar neutral amino acids, an acidic amino acid, a hydrophobic amino acid, and a polar neutral amino acid; and/or g )
  • the ISVDs comprise a CDR3 (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDR3 of the ISVD with the amino acid sequence selected from SEQ ID NOs: 547, 549-563.
  • CDR3 AbM numbering
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - FR1 (AbM numbering) consists of the amino acid sequence EVQLVESGGGXVQPGGSLRLSCAAS (SEQ ID NO: 647); wherein the amino acid residue X is selected from V and L.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - FR1 (AbM numbering) consists of the amino acid sequence EVQLVESGGGXVQPGGSLRLSCAAS (SEQ ID NO: 647); wherein the amino acid residue X is V.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - FR1 (AbM numbering) consists of the amino acid sequence EVQLVESGGGXVQPGGSLRLSCAAS (SEQ ID NO: 647); wherein the amino acid residue X is L.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - FR2 (AbM numbering) consists of the amino acid sequence WVRQX1PGKGX2EWVS (SEQ ID NO: 641); wherein the amino acid residue X 1 is selected from A and P; and wherein the amino acid residue X 2 is selected from I and L.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - FR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence WVRQPPGKGIEWVS (SEQ ID NO: 625); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 625; or c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 625.
  • FR2 AbM numbering
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein FR2 (AbM numbering) consists of an amino acid sequence selected from: a ) the amino acid sequence WVRQAPGKGIEWVS (SEQ ID NO: 626); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 626; or c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 626.
  • FR2 AbM numbering
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein FR2 (AbM numbering) consists of an amino acid sequence selected from: a ) the amino acid sequence WVRQPPGKGLEWVS (SEQ ID NO: 627); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 627; or c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 627.
  • FR2 AbM numbering
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - FR3 (AbM numbering) consists of the amino acid sequence YX 1 X 2 X 3 VX 4 GRFTISRDNAX 5 NTLYLQMNSLX 6 X 7 EDTAX 8 YYCTT (SEQ ID NO: 651); wherein the amino acid residue X 1 is selected from R and A, wherein the amino acid residue X 2 is selected from V and D, wherein the amino acid residue X 3 is selected from P and S, wherein the amino acid residue X 4 is selected from E and K, wherein the amino acid residue X 5 is selected from E and K, wherein the amino acid residue X 6 is selected from K and R, wherein the amino acid residue X 7 is selected from T, S and P, and wherein the amino acid residue X 8 is selected from V and
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR3 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence of SEQ ID NO: 610; b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 610; or c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 610; and - FR2 (AbM numbering) consists of an amino acid sequence selected from: I.
  • amino acid sequence WVRQPPGKGIEWVS SEQ ID NO: 625
  • amino acid sequence WVRQAPGKGIEWVS SEQ ID NO: 626
  • the ISVD comprises a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 610 and a FR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 625. In one embodiment, the ISVD comprises a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 610 and a FR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 626. In one embodiment, the ISVD comprises a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 610 and a FR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 627.
  • the ISVDs comprise a FR1, FR2, FR3 and FR4 and a CDR3 (AbM numbering) with an amino acid sequence that has at least 70% amino acid sequence identity, preferably at least 80% amino acid sequence identity, more preferably at least 90% amino acid sequence identity, such as 95% amino acid sequence identity or 99% amino acid sequence identity or more, or even essentially 100% amino acid sequence identity with the amino acid sequences of the CDR3 and FR1, FR2, FR3, and FR4 of the ISVD with the amino acid sequence selected from SEQ ID NOs: 547, 549- 563.
  • the ISVD comprises a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 610, a FR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 4, a FR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 625, a FR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 628, and a FR4 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 640.
  • CDR3 AbM numbering
  • FR1 AbM numbering
  • FR2 AbM numbering
  • FR3 AbM numbering
  • FR4 AbM numbering
  • the ISVD comprises a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 610, a FR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 34, a FR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 625, a FR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 629, and a FR4 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 640.
  • CDR3 AbM numbering
  • FR1 AbM numbering
  • FR2 AbM numbering
  • FR3 AbM numbering
  • FR4 AbM numbering
  • the ISVD comprises a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 610, a FR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 34, a FR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 625, a FR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 630, and a FR4 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 640.
  • CDR3 AbM numbering
  • FR1 AbM numbering
  • FR2 AbM numbering
  • FR3 AbM numbering
  • FR4 AbM numbering
  • the ISVD comprises a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 610, a FR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 34, a FR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 625, a FR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 629, and a FR4 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 7.
  • the ISVD comprises a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 610, a FR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 34, a FR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 626, a FR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 631, and a FR4 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 640.
  • CDR3 AbM numbering
  • FR1 AbM numbering
  • FR2 AbM numbering
  • FR3 AbM numbering
  • FR4 AbM numbering
  • the ISVD comprises a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 610, a FR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 34, a FR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 626, a FR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 632, and a FR4 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 640.
  • CDR3 AbM numbering
  • FR1 AbM numbering
  • FR2 AbM numbering
  • FR3 AbM numbering
  • FR4 AbM numbering
  • the ISVD comprises a CDR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 610, a FR1 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 34, a FR2 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 625, a FR3 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 633, and a FR4 (AbM numbering) that is the amino acid sequence of SEQ ID NO: 640.
  • CDR3 AbM numbering
  • FR1 AbM numbering
  • FR2 AbM numbering
  • FR3 AbM numbering
  • FR4 AbM numbering
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - FR1 (Kabat numbering) consists of the amino acid sequence EVQLVESGGGXVQPGGSLRLSCAASGFTFS (SEQ ID NO: 650); wherein the amino acid residue X is V.
  • the ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein FR2 (Kabat numbering) consists of an amino acid sequence selected from: a) the amino acid sequence WVRQPPGKGLEWVS (SEQ ID NO: 627); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence of SEQ ID NO: 627; or c) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence of SEQ ID NO: 627.
  • FR2 Kabat numbering
  • the ISVD comprises a CDR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 610, a FR1 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 624, a FR2 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 625, a FR3 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 645, and a FR4 (Kabat numbering) that is the amino acid sequence of SEQ ID NO: 640.
  • ISVDs specifically binding to CEACAM5 are ISVDs that comprise 4 framework regions (FR1 to FR4, respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein: - CDR1 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence GFTFSSYDMS (SEQ ID NO: 611); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence GFTFSSYDMS (SEQ ID NO: 611); c ) amino acid sequences that have 3, 2, or 1 amino acid difference with the amino acid sequence GFTFSSYDMS (SEQ ID NO: 611); and - CDR2 (AbM numbering) consists of an amino acid sequence selected from: a) the amino acid sequence TINRGGSSTS (SEQ ID NO: 612); b) amino acid sequences that have at least 80% amino acid identity with the amino acid sequence TINRGGSSTS (SEQ ID NO: 612); c

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Abstract

La présente technologie concerne des polypeptides qui se lient à CEACAM5. La présente technologie concerne également des acides nucléiques, des vecteurs et des compositions.
PCT/EP2024/088111 2023-12-22 2024-12-20 Domaines variables uniques d'immunoglobuline ciblant ceacam5 Pending WO2025133252A1 (fr)

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