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WO2025003511A1 - Double ciblage de cellules tumorales co-exprimant flt3 et cd123 par complémentation fonctionnelle de molécules de demi-corps de cycat® - Google Patents

Double ciblage de cellules tumorales co-exprimant flt3 et cd123 par complémentation fonctionnelle de molécules de demi-corps de cycat® Download PDF

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WO2025003511A1
WO2025003511A1 PCT/EP2024/068447 EP2024068447W WO2025003511A1 WO 2025003511 A1 WO2025003511 A1 WO 2025003511A1 EP 2024068447 W EP2024068447 W EP 2024068447W WO 2025003511 A1 WO2025003511 A1 WO 2025003511A1
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seq
amino acid
acid sequence
present disclosure
antibody
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Inventor
Ana OGRINC WAGNER
Andreas Bültmann
Christian Kunz
Christoph ERKEL
Felicitas STEIN
Nicole Haubst
Simon Schuster
Sebastian Jäger
Yvonne Stark
Markus Moosmeier
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Morphosys AG
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Morphosys AG
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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
    • C07K16/2809—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 against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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

Definitions

  • the present disclosure provides antibodies that specifically bind to FLT3 as well as antibodies that specifically bind to CD123.
  • the disclosure further relates to complementary pairs of CyCAT halfbody molecules comprising the FLT3 and CD123 specific antibodies of the present disclosure and their use in redirecting T-cell mediated killing of FLT3 and CD123 co-expressing tumor cells via on-cell formation of T-cell engaging trispecific antibodies.
  • the present disclosure also provides strategies for optimizing the producibility of halfbody molecules as well as the safety profile of CyCAT directed therapies by utilizing framework region mutated ⁇ CD3 variable heavy chain domains.
  • BACKGROUND FLT3 Fms-Like Tyrosine Kinase 3, CD135, FLK3, STK1
  • AML Acute Myeloid Leukemia
  • It is also the most frequently mutated gene in AML patients. Mutations resulting in constitutive activation of the receptor are associated with poor prognosis (see, e.g., Abu-Duhier et al.
  • CD123 is a tumor antigen over-expressed in a variety of hematological neoplasms. The majority of AML blasts express surface CD123. Higher expression of CD123 has been reported to be associated with poorer prognosis. CD123 is also an important marker for the identification and targeting of Leukemic stem cells (LSCs) for refractory or relapsed leukemia.
  • LSCs Leukemic stem cells
  • CD123 neutralizing antibodies Pre-clinical studies and clinical trials confirm the therapeutic utility of CD123 neutralizing antibodies, anti-CD3 ⁇ anti-CD123 bispecific antibodies or anti-CD123 chimeric antigen receptor-modified T-cell (CAR-T).
  • Co-expression of CD123 and FLT3 can be found in subpopulations of AML patient blasts. The presence of the two oncogenic markers on the surface of the same leukemic blasts provides an opportunity for the development of highly selective targeted therapy. Dual targeting of CD123 and FLT3 in form of a trispecific T-cell engaging approach has been described in published international application WO2021/089748A2 (AMGEN RES MUNICH GMBH [DE]; AMGEN INC [US]).
  • two complementary CyCAT halfbody molecules are designed, each composed of a Fab targeting moiety with specificity for either CD123 or FLT3 and either carrying a single antibody variable light chain domain (“ ⁇ CD3-VL”) or a single antibody variable heavy chain domain (“ ⁇ CD3-VH”) of a functional ⁇ CD3 Fv binding fragment (“ ⁇ CD3-Fv”).
  • ⁇ CD3-VL single antibody variable light chain domain
  • ⁇ CD3-VH single antibody variable heavy chain domain
  • ⁇ CD3-Fv a functional ⁇ CD3 Fv binding fragment
  • the two halfbody molecules and consequently the complementary ⁇ CD3-VL and ⁇ CD3-VH domain, come in close proximity and interact with each other to reconstitute the functional ⁇ CD3-Fv binding fragment.
  • the thus on-cell and on-target formed trispecific antibody is capable of engaging and stimulating cytotoxic T-cells for destruction of the cancerous cells.
  • Fully human ⁇ CD3 antibodies employed in the halfbody molecules of the present disclosure which has been selected for use in CyCAT directed therapies, has been disclosed in WO2022/063819.
  • the present disclosure provides a trispecific antibody, comprising a) a first Fab specific for CD123 ( ⁇ CD123-Fab), b) a second Fab specific for FLT3 ( ⁇ FLT3-Fab), and c) an antibody Fv fragment (Fv) specific for CD3 ( ⁇ CD3-Fv).
  • the present disclosure provides a trispecific antibody, wherein a) the ⁇ CD123-Fab binds to human CD123 and cynomolgus monkey CD123, b) the ⁇ FLT3-Fab binds to human FLT3, cynomolgus monkey FLT3 and mouse FLT3, and c) the ⁇ ⁇ CD3-Fv binds to human CD3 and cynomolgus monkey CD3.
  • the trispecific antibody according to the present disclosure is composed of a first halfbody molecule (HB1) and a second halfbody molecule (HB2), wherein a) HB1 comprises the ⁇ CD123-Fab and either the VH of the Fv specific for CD3 ( ⁇ CD3-VH) or the VL of the Fv specific for CD3 ( ⁇ CD3-VL), and b) HB2 comprises the ⁇ FLT3-Fab and either the complementary ⁇ CD3-VL or the complementary ⁇ CD3-VH, respectively, and wherein HB1 and HB2 are not linked by a covalent bond.
  • HB1 comprises the ⁇ CD123-Fab and either the VH of the Fv specific for CD3 ( ⁇ CD3-VH) or the VL of the Fv specific for CD3 ( ⁇ CD3-VL)
  • HB2 comprises the ⁇ FLT3-Fab and either the complementary ⁇ CD3-VL or the complementary ⁇ CD3-VH, respectively, and wherein HB1 and
  • the present disclosure provides a trispecific antibody, wherein a) neither the ⁇ CD3-VH by itself nor the ⁇ CD3-VL by itself is capable of binding to CD3, and/or wherein b) neither HB1 by itself nor HB2 by itself is capable of binding to CD3, and/or wherein c) neither HB1 by itself nor HB2 by itself is capable of mediating T-cell redirected killing of cells expressing CD123 and/or FLT3 on their cell surface.
  • the present disclosure provides a trispecific antibody, wherein the ⁇ CD3-VH and the ⁇ CD3-VL are capable of non-covalently associating with each other, thereby forming the ⁇ CD3-Fv.
  • the present disclosure provides a trispecific antibody, wherein the ⁇ CD3-VH present on HB1 and the complementary ⁇ CD3-VL present on HB2 or the ⁇ CD3-VL present on HB1 and the complementary ⁇ CD3-VH present on HB2, are capable of non-covalently associating with each other, thereby forming the ⁇ CD3-Fv.
  • said non-covalent association of the ⁇ CD3- VH with the ⁇ CD3-VL occurs upon binding of HB1 to CD123 and upon binding of HB2 to FLT3.
  • the present disclosure provides a trispecific antibody, wherein the C- terminus of the heavy chain of the ⁇ CD123-Fab is fused to the N-terminus of the either ⁇ CD3-VH or ⁇ CD3-VL via a first peptide linker and the C-terminus of the heavy chain of the ⁇ FLT3-Fab is fused to the N-terminus of the complementary ⁇ CD3-VL or ⁇ CD3-VH, respectively.
  • the present disclosure provides a trispecific antibody, wherein a) the C-terminus of the heavy chain of the ⁇ CD123-Fab is fused to the N-terminus of the ⁇ CD3-VH via a first peptide linker and the C-terminus of the heavy chain of the ⁇ FLT3-Fab is fused to the N-terminus of the complementary ⁇ CD3-VL, or wherein b) the C-terminus of the heavy chain of ⁇ CD123-Fab is fused to the N-terminus of the ⁇ CD3-VL via a first peptide linker and the C-terminus of the heavy chain of the ⁇ FLT3-Fab is fused to the N-terminus of the complementary ⁇ CD3-VH.
  • the present disclosure provides a trispecific antibody, wherein said first peptide linker and/or said second peptide linker comprises 1 – 40 amino acid residues. In an embodiment, the first peptide linker and/or the second peptide linker comprises the amino acid sequence GQPSG (SEQ ID NO: 42). In an embodiment, the present disclosure provides a trispecific antibody, wherein CD123 and FLT3 are expressed on the surface of the same cell. In an embodiment of the present disclosure, the trispecific antibody induces T cell mediated killing of cells expressing CD123 and FLT3 on their cell surface. In an embodiment, the trispecific antibody mediates T cell induced killing of cell expressing CD123 and FLT3 on their cell surface.
  • the trispecific antibody mediates T cell redirected killing of cell expressing CD123 and FLT3 on their cell surface.
  • the present disclosure provides a trispecific antibody, wherein the ⁇ CD3-VH comprises a V or M at position 37 (utilizing the number scheme according Kabat).
  • the present disclosure provides a trispecific antibody, wherein the ⁇ CD3-VH comprises an amino acid substitution in the framework regions, wherein said amino acid substitution reduces the antibody induced T cell mediated killing of cells having either CD123 or FLT3 on their cell surface.
  • said amino acid substitution in the ⁇ CD3- VH framework regions does not substantially inhibit the antibody induced T cell mediated killing of cells having CD123 and FLT3 on their cell surface.
  • the present disclosure provides a trispecific antibody, wherein the ⁇ CD3-VH comprises an amino acid substitution at position 37, 44 or 45 (utilizing the number scheme according Kabat), wherein said amino acid substitution reduces the antibody induced T cell mediated killing of cells having either CD123 or FLT3 on their cell surface.
  • said antibody induced T cell mediated killing of cells having either CD123 or FLT3 on their cell surface is reduced by at least 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 150- fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold as compared to the antibody induced T cell mediated killing of cells having either CD123 or FLT3 on their cell surface before said amino acid substitution of according to the present disclosure.
  • said antibody induced T cell mediated killing of cells having either CD123 or FLT3 on their cell surface is reduced between 2-fold and 100-fold, as compared to the antibody induced T cell mediated killing of cells having either CD123 or FLT3 on their cell surface before said amino acid substitution of according to the present disclosure.
  • the present disclosure provides a trispecific antibody, wherein the ⁇ CD3-VH comprises an amino acid substitution at position 37, 44 or 45 (utilizing the number scheme according Kabat), wherein said amino acid substitution increases the IC50 concentration determined for the antibody induced T-cell mediated killing of cells having either CD123 or FLT3 on their cell surface.
  • said IC50 concentration determined for the antibody induced T cell mediated killing of cells having either CD123 or FLT3 on their cell surface is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20- fold, 30-fold, 40-fold, 50-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold as compared to the IC 50 concentration determined for the trispecific antibody before said amino acid substitution of according to the present disclosure.
  • said IC50 concentration determined for the antibody induced T cell mediated killing of cells having either CD123 or FLT3 on their cell surface is increased between 2-fold and 100-fold when compared to the IC50 concentration determined for the trispecific antibody before said amino acid substitution according to the present disclosure.
  • the present disclosure provides a trispecific antibody, wherein the ⁇ CD3-VH comprises an amino acid substitution at position 37, 44 or 45 (utilizing the number scheme according Kabat), wherein said amino acid substitution increases the ratio of the IC50 concentration determined for the antibody induced T cell mediated killing of cells expressing CD123 or FLT3 on their cell surface and the IC50 concentration determined for the antibody induced T cell mediated killing of cells expressing CD123 and FLT3 on their cell surface, as compared to the ratio determined for the trispecific antibody before said amino acid substitution.
  • said ratio of the IC50 concentrations is increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold. In an embodiment of the present disclosure, said ratio of the IC50 concentrations is increased between 1.5- fold and 10-fold.
  • the amino acid substitution at position 37, 44 or 45 does not substantially inhibit the antibody induced T cell mediated killing of cells having CD123 and FLT3 on their cell surface.
  • the present disclosure provides a trispecific antibody comprising the amino acid substitution at position 37, 44 or 45 (utilizing the number scheme according Kabat) according to the present disclosure, wherein the IC50 concentration determined for the antibody induced T cell mediated killing of cells having CD123 and FLT3 on their cell surface is within 10-fold of the IC50 concentration determined for the trispecific antibody before said amino acid substitution.
  • the present disclosure provides a trispecific antibody, wherein the amino acid substitution at position 37, 44 or 45 (utilizing the number scheme according Kabat) according to the present disclosure does not substantially inhibit the binding of the ⁇ CD3-Fv to CD3.
  • the present disclosure provides a trispecific antibody comprising the amino acid substitution at position 37, 44 or 45 according to the present disclosure, wherein in presence of CD123 and FLT3, the KD determined for the trispecific antibody for binding to CD3 is within 5-fold, 4-fold, 3-fold, or 2-fold of the K D determined for said trispecific antibody before said amino acid substitution.
  • the ⁇ CD3-VH according to the present disclosure comprises a V at position 37, a G at position 44 and a L at position 45 (utilizing the number scheme according Kabat).
  • the ⁇ CD3-VH according to the present disclosure comprises a V at position 37 (utilizing the number scheme according Kabat).
  • the present disclosure provides a trispecific antibody, wherein the amino acid at position 37 of the VH of the Fv specific for CD3 is substituted for M or L, the amino acid at position 44 is substituted for A, or the amino acid at position 45 is substituted for I or F (utilizing the number scheme according Kabat).
  • said amino acid substitution in the ⁇ ⁇ CD3- VH is a V37M amino acid substitution.
  • the present disclosure provides a trispecific antibody, wherein a) the ⁇ CD123-Fab comprises an antibody variable heavy chain region (VH) comprising an heavy chain complementary determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 11, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and an antibody variable light chain region (VL) comprising a light chain complementary determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 12, and b) the ⁇ FLT3-Fab comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 27, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 18 or S
  • the ⁇ CD3-Fv comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 124, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 125 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 36, and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 126, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 39.
  • the present disclosure provides a trispecific antibody, wherein a) the ⁇ CD123-Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 13 and a VL comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 14, and b) the ⁇ FLT3-Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 30 and a VL comprising the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 31, and c) the ⁇ CD3-Fv comprises a VH comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 107 and a VL comprising the amino acid sequence of SEQ ID NO: 41.
  • the ⁇ CD3-Fv comprises a VH comprising the amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 129 and a VL comprising the amino acid sequence of SEQ ID NO: 128.
  • the present disclosure provides a trispecific antibody, wherein a) HB1 is composed of a first and second polypeptide, wherein (i) the first polypeptide comprises the light chain of the ⁇ CD123-Fab and (ii) the second polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the ⁇ CD123-Fab, the first peptide linker and either the ⁇ CD3- VH or ⁇ CD3-VL, and b) HB2 is composed of a third and fourth polypeptide, wherein (i) the third polypeptide comprises the light chain of the ⁇ FLT3-Fab, and (ii) the fourth polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the ⁇ FLT-
  • the present disclosure provides a trispecific antibody, wherein a) the first polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 108 or SEQ ID NO: 109, and wherein b) the third polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 33 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 110 or SEQ ID NO: 111.
  • the present disclosure provides a trispecific antibody, wherein a) the first polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134 or SEQ ID NO: 135, and wherein b) the third polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 33 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: 141.
  • the present disclosure provides a pharmaceutical composition comprising (a) the trispecific antibody or (b) HB1 or (c) HB2, according to the present disclosure; and a pharmaceutical acceptable carrier.
  • the present disclosure provides a kit comprising (a) the pharmaceutical composition comprising the trispecific antibody of the present disclosure or (b) the pharmaceutical composition comprising HB1 and the pharmaceutical composition comprising HB2.
  • the pharmaceutical composition according to the present disclosure comprises HB1 and HB2, and a pharmaceutical acceptable carrier.
  • the present disclosure provides (a) the trispecific antibody or (b) the complementary pair of HB1 and HB2 or (c) the pharmaceutical composition comprising HB1 and the pharmaceutical composition comprising HB2, according to the present disclosure, for use as a medicament.
  • the present disclosure provides (a) the trispecific antibody or (b) the complementary pair of HB1 and HB2 or (c) the pharmaceutical composition comprising HB1 and the pharmaceutical composition comprising HB2, according to the present disclosure, for use in the treatment of a cancer expressing CD123 and FLT3.
  • the present disclosure the pharmaceutical composition comprising HB1 and HB2 for use in the treatment of a cancer expressing CD123 and FLT3.
  • said cancer is a hematologic cancer.
  • said cancer is a leukemia.
  • said cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and hairy cell leukemia (HCL).
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • ALL acute lymphocytic leukemia
  • HCL hairy cell leukemia
  • the present disclosure provides the use as described herein, wherein the use comprises administration of an effective amount of (a) the trispecific antibody or (b) the complementary pair of HB1 and HB2, or (c) the pharmaceutical composition comprising HB1 and the pharmaceutical composition comprising HB2, according to the present disclosure.
  • the present disclosure provides the use as described herein, wherein (a) HB1 and HB2 are administered separately or (b) wherein the pharmaceutical composition comprising HB1 and the pharmaceutical composition comprising HB2 are administered separately.
  • FIG. 1 Schematic representation of a complementary pair of CyCAT halfbody molecules according to the present disclosure, each carrying a Fab with specificity for CD123 and FLT3, respectively, a peptide linker and one half an ⁇ CD3-Fv binding fragment.
  • Figure 2 Exemplary FACS based cytotoxicity assays according to Example 3 for complementary pairs of CyCAT halfbody molecules with specificity for CD123 and FLT3, respectively, according to the present disclosure (Pair 1 – 3), on the FLT3 / CD123 double-positive leukemia cancer cell lines MOLM-13 (Fig. 2A), SKM-1 (Fig. 2B), and MV4-11 (Fig. 2C) in presence of human CD8+ effector T cells.
  • FIG. 3 Exemplary FACS based cytotoxicity assays according to Example 3 for complementary pairs of CyCAT halfbody molecules (Pair 1 – 3) in comparison to corresponding single (unpaired) CyCAT halfbody molecules with specificity for CD123 and FLT3, respectively, on the FLT3 / CD123 double-positive leukemia cancer cell lines MOLM-13 (Fig. 3A), SKM-1 (Fig. 3B), and MV4-11 (Fig. 3C) in presence of human CD8+ effector T cells.
  • FIG.5A - F show binding as a function of halfbody concentration determined on the CD123 / FLT3 double positive leukaemia cell lines MOLM-13 (Fig.5A&D), MV4-11 (Fig.5C&F) and SKM-1 (Fig.5B&E).
  • Figure 6 Inhibition of IL-3 induced cell proliferation of TF-1 erythroleukemia cancer cells by the CD123 specific halfbody molecules ⁇ CD123-HB1 and ⁇ CD123-HB3.
  • FIG. 7A depicts a schematic representation of the trispecific Fab2-dsFv antibody format according to Example 9, with Fab specificities for CD123 and FLT3, respectively, and a disulfide stabilized ⁇ CD3-Fv binding fragment.
  • Fig. 7B shows exemplary FACS based cytotoxicity assays on HUVECs according to Example 9 for complementary pairs of CyCAT halfbody molecules of the present disclosure (Pair 1 – 3) in comparison to conventional bi- and trispecific antibodies with specificity for CD123 and CD3.
  • Figure 8 Exemplary FACS based cytotoxicity assays according to Example 10 in order to determine the therapeutic window for the Dual-Targeting Halfbody Pair 1 and Halfbody Pair 1 CD3mut (Fig. 8B & Fig. 8D) or Halfbody Pair 3 and Halfbody Pair 3 CD3mut (Fig. 8A & Fig. 8C) either utilizing the unmutated ⁇ CD3-VH domain or the mutated ⁇ CD3-VHV37M domain, respectively, in T cell mediated killing of MOLM-13 (Fig.8A & 8B) or SKM-1 (Fig.8C & Fig.8D) cells.
  • CD123 refers to a protein known as “Interleukin-3 receptor alpha chain” or “IL-3Ra”.
  • Human CD123 (Uniprot: P26951-1 (SP1)
  • Human FLT3 (Uniprot: P36888-1
  • CD3 refers to an antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed from the association of two of four receptor chains: CD3epsilon (CD3e), CD3delta, CD3zeta, and CD3gamma.
  • Human CD3epsilon (or human CD3e) has the amino acid sequence of UniProt P07766: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYP GSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPE DANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKP VTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 61) (signal sequence underlined, intracellular region italic, transmembrane region bold).
  • the mature extracellular domain of human CD3epsilon without signal sequence comprises the amino acid sequence of: QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIG SDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 62)
  • Cynomolgus monkey CD3epsilon (or cynomolgus CD3e or cyno CD3e) has the amino acid sequence of UniProt Q95LI5: MQSGTRWRVLGLCLLSIGVWGQDGNEEMGSITQTPYQVSISGTTVILTCSQHLG SEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLK ARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAG GRQRGQNKERPPPVPNPDYEP
  • the mature extracellular region of cynomolgus monkey CD3 epsilon without the signal sequence comprises has the amino acid sequence of: QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLF LPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIV DICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYE PIRKGQQDLYSGLNQRRI (SEQ ID NO: 64).
  • the term “about” preceding a number means plus or less 10% of said number.
  • an antigen refers to any molecule of interest that can be bound by one of the binding sites present in an antibody.
  • an antigen is a peptide, a protein or any other proteinaceous molecule.
  • an antigen may be any other organic or inorganic molecule, such as carbohydrate, fatty acid, lipid, dye or flourophor.
  • polypeptide refer to a polymer of amino acid residues and does not refer to a specific length of a product. The term applies to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including chemical synthesis.
  • CyCAT halfbody molecule refers in its broadest sense to an antigen binding molecule that specifically binds to at least one target antigen and which is composed of an antibody fragment, such as a Fab fragment and either the VH or VL of an antibody Fv fragment with specificity for CD3. Accordingly, an halfbody molecule according to the preset disclosure incorporates one half antibody Fv binding fragment with specificity for CD3.
  • WO2013/104804 JULIUS-MAXIMILIANS-UNIVERSIT ⁇ T WÜRZBURG
  • CyCAT molecule refers to a functional complex of two complementary CyCAT halfbody molecules as described herein, wherein each halfbody body molecule carries one half of an antibody Fv fragment with specific for CD3 and wherein only said complex is functional in respect to the function of said Fv fragment but not the individual CyCAT halfbody molecules by themselves.
  • “Complementary” CyCAT halfbody molecule(s) as used herein refers to any complex or pair of two CyCAT halfbody molecules as described herein; provided that one CyCAT halfbody molecule carries at least one single ⁇ CD3-VH or ⁇ CD3-VL domain and the second CyCAT halfbody molecule carries at least the complementary unpaired ⁇ CD3- VL or ⁇ CD3-VH domain, respectively, of an ⁇ CD3 Fv fragment. Dimerization and functional complementation of the ⁇ CD3-VH and ⁇ CD3-VL domain occurs upon binding of the two complementary CyCAT halfbody molecules to their target antigen present on the surface of the same target cell.
  • binding site or “antigen binding site” or “antigen binding region” refer to a structure formed by a protein that is capable of binding or specifically binding to an antigen.
  • the binding site need not be a series of contiguous amino acids, or even amino acids in a single polypeptide chain.
  • the “binding site” is made up of a series of amino acids of a VL and a VH that interact with the antigen and that are generally, however not always, in the one or more of the CDRs in each variable region.
  • a “binding site” is or comprises or is formed by a complementary antibody variable heavy (VH) and light chain (VL) pair.
  • the VH and the V which form the binding site can be in a single polypeptide chain or in different polypeptide chains.
  • the binding site is or comprises or is formed by a VH present on a first halfbody molecule according to the present disclosure and the complementary VL is present on the second halfbody molecule according to the present disclosure, or vice versa.
  • the “binding site” has one VH and one VL.
  • the binding site comprises one or more CDRs of an antibody.
  • a binding site is derived from an antibody mimetic, such as for instance from an affibody molecule, alphabody, anticalin, avimer, DARPin, fynomer, Kunitz domain peptide, helix- turn-helix peptide, or monobody.
  • antibody molecule or “immunoglobulin” (Ig) molecule used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains, which interacts with an antigen.
  • Each heavy chain (HC) is comprised of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable domain (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FR’s arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • variable domains of the heavy and light chains contain or form a “binding site” or “antigen binding site” that selectively interacts with or binds to an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the term “antibody” includes for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies and chimeric antibodies.
  • the antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.
  • the structures and locations of immunoglobulin variable domains, e.g., CDRs may be defined using well known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., Sequences of Proteins of Immunological Interest, U.S.
  • antibody as used herein is intended to include antibody fragments, CyCAT molecules, monospecific specific antibodies as well as bispecific and multispecific antibodies.
  • antibody fragment of an antibody, as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing spatial distribution) an antigen.
  • antibody fragments include, but are not limited to, a Fab fragment (“Fab”), a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment (“Fv”) consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR).
  • Fab Fab fragment
  • F(ab)2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • Fd fragment consisting of the VH and CH1 domains
  • Fv fragment Fv fragment
  • a dAb fragment Ward et al.,
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH domains pair to form monovalent molecules (known as single chain Fv (“scFv”); see e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term “antibody fragment”.
  • Antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136).
  • Antibody fragments can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).
  • Fn3 Fibronectin type III
  • Antibody fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments which, together with complementary light chain polypeptides, form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng.8:1057-1062; and U.S. Pat. No.5,641,870).
  • “Species”, as used herein, refers to any mammal, including rodents, such as mouse or rat, and primates, such as cynomolgus monkey (Macaca fascicularis), rhesus monkey (Macaca mulatta) or humans (Homo sapiens).
  • the subject is a primate, most preferably a human.
  • EC50 refers to the concentration of an antibody or an antibody fragment or CyCAT molecule which induces a response in an assay half way between the baseline and maximum. It therefore represents the antibody concentration at which 50% of the maximal effect is observed.
  • IC50 refers to the concentration of an antibody or antibody fragment or CyCAT molecule that inhibits a response in an assay half way between the maximal response and the baseline. It represents the antibody concentration that reduces a given response by 50%.
  • inhibitors or “inhibit” or “reduction” or “reduce” or “neutralization” or “neutralize” refer to a decrease or cessation of any phenotypic characteristic (such as binding, a biological activity or function) or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic.
  • the “inhibition”, “reduction” or “neutralization” needs not to be complete as long as it is detectable using an appropriate assay.
  • by “reduce” or “inhibit” is meant the ability to cause a decrease of 20% or greater.
  • by “reduce” or “inhibit” is meant the ability to cause a decrease of 50% or greater.
  • amino ac id re sidues or “am ino acid” will be indicated either by their full name or according to the standard three-letter or o ne-letter amino acid code.
  • Natural occurring amino ac ids means the following amino a cids: Table 1: Natural occu rring amino acids
  • non-covalent association refers to molecular interactions that do not involve an interatomic bond. Noncovalent interactions involve, for example, ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals forces.
  • covalent bond refers to an interatomic bond characterized by sharing of electrons.
  • multispecific means that an antibody or halfbody molecule is able to specifically bind to two or more different antigens. Typically, a multispecific antibody or halfbody molecule comprises of two or more antigen binding sites, each of which is specific for a different antigen or epitope.
  • bispecific means that an antibody or halfbody molecule is able to specifically bind to two different antigens. Typically, a bispecific halfbody molecule comprises two antigen binding sites, each of which is specific for a different antigen or epitope.
  • trispecific means that an antibody or halfbody molecule is able to specifically bind to three different antigens.
  • a trispecific antibody or halfbody molecule comprises three antigen binding sites, each of which is specific for a different antigen or epitope.
  • an antibody, antibody fragment or halfbody molecule disclosed herein that specifically binds to a target antigen is an antibody, antibody fragment, or halfbody molecule that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other target antigens.
  • an antibody, antibody fragment or halfbody molecule specifically binds to an epitope on a protein that is conserved among the protein from different species.
  • specific binding can include, but does not require exclusive binding.
  • the antibodies, antibody fragments or halfbody molecules disclosed herein specifically bind to antigens.
  • Methods for determining whether two molecules specifically bind include, for example, a standard ELISA assay.
  • the scoring may be carried out by standard color development (e.g. secondary antibody with horseradish peroxide and tetramethyl benzidine with hydrogen peroxide).
  • the reaction in certain wells is scored by the optical density, for example, at 450 nm.
  • determination of binding specificity is performed by using not a single reference antigen, but a set of three to five unrelated antigens, such as milk powder, BSA, transferrin or the like.
  • the terms “first”, “second”, “third”, “fourth”, “fifth”, and “sixth” and “seventh”, “eight” and so on, with respect to an target antigen, halfbody, antibody, antibody fragment, Fab, Fv region, Fc region, Fc region subunit, peptide linker, spacer or polypeptide are used for distinguishing when there is more than one of each type of component. Use of these terms is not intended to confer a specific order or orientation unless explicitly so stated.
  • pharmaceutical composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “therapeutically effective amount” or “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.
  • “Administered” or “administration” includes but is not limited to delivery of a drug by an injectable form, such as, for example, an intravenous, intramuscular, intradermal or subcutaneous route or mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestible solution, capsule or tablet.
  • the administration is by an injectable form.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • treatment refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology.
  • Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • halfbody molecules according to the preset disclosure are used to delay development of a disease or to slow the progression of a disease.
  • a “human antibody” or “human antibody fragment” as used herein, includes antibodies and antibody fragments having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such sequences.
  • Human origin includes, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86).
  • immunoglobulin variable domains e.g., CDRs
  • CDRs immunoglobulin variable domains
  • the structures and locations of immunoglobulin variable domains, e.g., CDRs may be defined using well known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), eds.
  • chimeric antibody or “chimeric antibody fragment” is defined herein as an antibody which has constant antibody regions derived from, or corresponding to, sequences found in one species and variable antibody regions derived from another species.
  • the constant antibody regions are derived from, or corresponding to, sequences found in humans
  • the variable antibody regions are derived from sequences found in a non-human animal, e.g. a mouse, rat, rabbit or hamster.
  • a “humanized antibody” or “humanized antibody fragment” is defined herein as an antibody which has constant antibody regions derived from sequences of human origin and the variable antibody regions or parts thereof or only the CDRs are derived from another species. Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g.
  • isolated refers to a compound, which can be e.g. an antibody, antibody fragment or halfbody molecule, that is substantially free of other antibodies, antibody fragments or halfbody molecules having different antigenic specificities. Moreover, an isolated antibody, antibody fragment or halfbody molecule may be substantially free of other cellular material and/or chemicals. Thus, in some embodiments, the antibodies, antibody fragments or halfbody molecules provided herein are isolated antibodies, antibody fragments or halfbody molecules that have been separated from antibodies or halfbody molecules with a different specificity. An isolated antibody, antibody fragment or halfbody molecule may be a monoclonal antibody, antibody fragment or halfbody molecule.
  • An isolated antibody, antibody fragment or halfbody molecule may be a recombinant monoclonal antibody, antibody fragment or halfbody molecule.
  • An isolated antibody, antibody fragment or halfbody molecule that specifically binds to an epitope, isoform or variant of a target may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., species homologs).
  • the term “recombinant antibody”, “recombinant antibody fragment” or “recombinant halfbody molecule”, as used herein, includes all antibodies, antibody fragments or halfbody molecules according to the present disclosure that are prepared, expressed, created or segregated by means not existing in nature.
  • antibodies or halfbody molecules isolated from a host cell transformed to express the antibody or halfbody molecule antibodies selected and isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences or antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom.
  • an animal e.g., a mouse
  • such recombinant antibodies or halfbody molecules have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies or halfbody molecules can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies or halfbody molecules are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
  • a recombinant antibody or halfbody molecule may be a recombinant monoclonal antibody or a recombinant monoclonal halfbody molecule.
  • the antibodies and antibody fragment disclosed herein are isolated from the Ylanthia® antibody library as disclosed in US 13/321,564 or US 13/299,367, which both herein are incorporated by reference.
  • the term “monoclonal antibody”, “monoclonal antibody fragment” or ”monoclonal halfbody molecule” refers to an antibody, antibody fragment or halfbody molecule disclosed herein that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • Monoclonal antibodies or antibody fragments may be made by the hybridoma method as described in Kohler et a/.; Nature, 256:495 (1975) or may be isolated from phage libraries. Other methods for the preparation of clonal cell lines and monoclonal antibodies or halfbody molecule as disclosed herein expressed thereby are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5 th Ed., Ausubel et al., eds., John Wiley and Sons, New York). As used herein, the term “affinity” refers to the strength of interaction between the polypeptide and its target at a single site.
  • KD refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M).
  • KD values for antigen binding moieties like e.g. monoclonal antibodies can be determined using methods well established in the art. Methods for determining the KD of an antigen binding moiety like e.g.
  • a monoclonal antibody are SET (soluble equilibrium titration), surface plasmon resonance using a biosensor system such as a Biacore® system, or Biolayer Interferometry (BLI).
  • EMBODIMENTS Trispecific antibodies (CyCAT molecules) according to the present disclosure
  • the present disclosure provides a trispecific antibody, wherein said trispecific antibody is composed of a) a first antibody fragment specific for CD123, b) a second antibody specific for FLT3, and c) a third antibody fragment specific for CD3.
  • the first antibody fragment specific for CD123 binds to human CD123 and cynomolgus monkey CD123.
  • the second antibody fragment specific for FLT3 binds to human FLT3, cynomolgus monkey FLT3 and mouse FLT3.
  • the third antibody fragment specific for CD3 binds to human CD3 and cynomolgus monkey CD3.
  • the present disclosure provides a trispecific antibody, comprising a) a first antibody fragment specific for CD123, b) a second antibody fragment specific for FLT3, and c) a third antibody fragment specific for CD3, wherein said trispecific antibody is composed of a first halfbody molecule (HB1) and a second halfbody molecule (HB2), wherein HB1 comprises the first antibody fragment specific for CD123 and either the VH or the VL of the third antibody fragment specific for CD3, and wherein HB2 comprises the second antibody fragment specific for FLT3 and the complementary VH or the complementary VL of the third antibody fragment specific for CD3.
  • said HB1 and HB2 are a pair of halfbody molecules.
  • said HB1 and HB2 are a complementary pair halfbody molecules.
  • the present disclosure provides a pair of a first halfbody molecule (HB1) and a second halfbody molecule (HB2), wherein said HB1 comprises the first antibody fragment specific for CD123 and either the VH or the VL of the third antibody fragment specific for CD3, and wherein HB2 comprises the second antibody fragment specific for FLT3 and either the complementary VH or the complementary VL of the third antibody fragment specific for CD3.
  • HB1 comprises the first antibody fragment specific for CD123 and the VH of the third antibody fragment specific for CD3, and HB2 comprises the second antibody fragment specific for FLT3 and the complementary VL of the third antibody fragment specific for CD3.
  • HB1 comprises the first antibody fragment specific for CD123 and the VL of the third antibody fragment specific for CD3, and HB2 comprises the second antibody fragment specific for FLT3 and the complementary VH of the third antibody fragment specific for CD3.
  • HB1 and HB2 are not linked by a covalent bond.
  • HB1 and HB2 are capable of non-covalently associating with each other.
  • HB1 and HB2 are capable of non-covalently associating which each other, thereby forming a trispecific antibody according to the present disclosure.
  • the present disclosure provides a trispecific antibody, comprising a) a first antibody fragment specific for CD123, b) a second antibody fragments specific for FLT3, and c) a third antibody fragment specific for CD3, wherein said trispecific antibody is composed of a first halfbody molecule (HB1) and a second halfbody molecule (HB2), wherein HB1 comprises the first antibody fragment specific for CD123 and either the VH or the VL of the third antibody fragment specific for CD3, and wherein HB2 comprises the second antibody fragment specific for FLT3 and either the complementary VH or the complementary VL of the third antibody fragment specific for CD3, wherein HB1 and HB2 are not linked by a covalent bond and wherein said HB1 and that HB2 are capable of non-covalently associating with each other, thus forming the trispecific antibody.
  • HB1 comprises the first antibody fragment specific for CD123 and either the VH or the VL of the third antibody fragment specific for CD3
  • HB2 comprises the second antibody fragment specific for FLT3 and either the
  • the trispecific antibody according to the present disclosure is formed upon binding of HB1 to CD123 and upon binding of HB2 to FLT3. In an embodiment, the trispecific antibody according to the present disclosure is formed upon binding of HB1 to CD123 and upon binding of HB2 to FLT3, wherein CD123 and FLT3 are both expressed on the surface of the same cell.
  • the present disclosure provides a complementary pair of a first halfbody molecule (HB1) and a second halfbody molecule (HB2), wherein said HB1 comprises the first antibody fragment specific for CD123 and either the VH or the VL of the third antibody fragment specific for CD3, and wherein HB2 comprises the second antibody fragment specific for FLT3 and either the complementary VH or the complementary VL of the third antibody fragment specific for CD3, wherein HB1 and HB2 are not linked by a covalent bond and wherein said HB1 and that HB2 are capable of non-covalently associating with each other, thus forming the trispecific antibody according to the present disclosure.
  • HB1 comprises the first antibody fragment specific for CD123 and either the VH or the VL of the third antibody fragment specific for CD3
  • HB2 comprises the second antibody fragment specific for FLT3 and either the complementary VH or the complementary VL of the third antibody fragment specific for CD3, wherein HB1 and HB2 are not linked by a covalent bond and wherein said HB1 and that HB
  • said antibody fragment of the present disclosure is selected from the group consisting of a Fab fragment, a F(ab’)2 fragment, a Fv fragment, a scFv fragment, a domain antibody (dAb), a single domain heavy chain antibody, and a single domain light chain antibody.
  • said first and/or second antibody fragment is a scFv or a Fab.
  • said first antibody fragment and/or said second antibody fragment is a Fab.
  • said first antibody fragment and said second antibody fragment is a Fab.
  • said third antibody fragment is an antibody Fv fragment.
  • said non-covalent association of HB1 and HB2 occurs in solution. In some embodiments, said non-covalent association occurs on the surface of a cell. In some embodiments, said non-covalent association preferably occurs on the surface of a cell. In some embodiments, said non-covalent association occurs in presence of CD123 and FLT3. In some embodiments, said non-covalent association occurs upon binding of HB1 to CD123. In some embodiments, said non- covalent association occurs upon binding of HB1 to CD123 expressed on a cell. In some embodiments, said non-covalent association occurs upon binding of HB1 to CD123 present on the surface a cell. In some embodiments, said non-covalent association occurs upon binding of HB2 to FLT3.
  • said non-covalent association occurs upon binding of HB2 to FLT3 expressed on a cell. In some embodiments, said non-covalent association occurs upon binding of HB2 to FLT3 present on the surface of a cell. In a preferred embodiment, said non-covalent association occurs upon binding of HB1 to CD123 and upon binding of HB2 to FLT3. In a preferred embodiment, said non-covalent association occurs upon binding of HB1 to CD123 expressed or present on the surface of a cell and upon binding of HB2 to FLT3 expressed or present on the surface of a cell.
  • said non-covalent association occurs upon binding of HB1 to CD123 expressed or present on the surface of a cell and upon binding of HB2 to FLT3 expressed or present on the surface of the same cell.
  • CD123 and FLT3 are expressed or present on the same cell.
  • the presence of a cell expressing or having both, CD123 and FLT3 on its cell surface induces the non-covalent association of HB1 and HB2.
  • said non-covalent association of HB1 with HB2 occurs via dimerization of the VH or VL of the third antibody fragment specific for CD3 present of HB1 with the complementary VL or VH, respectively, of the third antibody fragment specific for CD3, present of HB2.
  • the either VH or VL of the third antibody fragment specific for CD3 present on HB1 and the complementary VL or VH, respectively, present on HB2, are capable of non-covalently associating with each other, thereby forming the third antibody fragment specific for CD3.
  • the trispecific antibody according to the present disclosure is formed upon non-covalent association of the either VH or VL present on HB1 with the complementary VL or VH, respectively, present on HB2.
  • said non-covalent association occurs in the presence of CD123 and FLT3.
  • the presence of a cell expressing or having CD123 and FLT3 on its surface induces dimerization of either the VH or VL of the third antibody fragment specific for CD3 present of HB1 with the complementary VL or VH, respectively, of the third antibody fragment specific for CD3 present of HB2.
  • the presence of a cell expressing or having CD123 and FLT3 on its surface induces the non-covalent association of either the VH or VL of the third antibody fragment specific for CD3 present of HB1 with the complementary VL or VH, respectively, of the third antibody fragment specific for CD3 present of HB2.
  • said CD123 and FLT3 are expressed or are present on the surface of the same cell.
  • the amount of trispecific antibody formed under conditions, where CD123 and FLT3 are present is higher than under conditions where CD123 and FLT3 are absent. In some embodiments, the amount of trispecific antibody formed under conditions, where CD123 and FLT3 are present, is higher than under conditions where CD123 is present but FLT3 is absent or where FLT3 is present but CD123 is absent. In some embodiments, the amount of trispecific antibody formed under conditions, where cells expressing CD123 and FLT3 on their cell surface are present, is higher than under conditions where cells expressing CD123 and FLT3 on their cell surface are absent.
  • the amount of trispecific antibody formed under conditions, where cells expressing CD123 and FLT3 on their cell surface are present is higher than under conditions where cells expressing CD123 but not FLT3 on their cell surface are present or where cells expressing FLT3 but not CD123 on their cell surface are present.
  • the amount of the third antibody fragment specific for CD3 formed under conditions, where CD123 and FLT3 are present is higher than under conditions where CD123 and FLT3 are absent.
  • the amount of the third antibody fragment specific for CD3 is formed under conditions, where CD123 and FLT3 are present is higher than under conditions where CD123 is present but FLT3 is absent or where FLT3 is present but CD123 is absent.
  • the amount of the third antibody fragment specific for CD3 formed under conditions, where cells expressing CD123 and FLT3 on their cell surface are present is higher than under conditions where cells expressing CD123 and FLT3 on their cell surface are absent. In some embodiments, the amount of the third antibody fragment specific for CD3 formed under conditions, where cells expressing CD123 and FLT3 on their cell surface are present, is higher than under conditions where cells expressing CD123 but not FLT3 on their cell surface are present or where cells expressing FLT3 but not CD123 on their cell surface are present. In an embodiment of the present disclosure, the amount of non-covalently associated HB1 and HB2 formed under conditions, where CD123 and FLT3 are present, is higher than under conditions where CD123 and FLT3 are absent.
  • the amount of non-covalently associated HB1 and HB2 formed under conditions, where CD123 and FLT3 are present is higher than under conditions where CD123 is present but FLT3 is absent or where FLT3 is present but CD123 is absent. In some embodiments, the amount of non-covalently associated HB1 and HB2 formed under conditions, where cells expressing CD123 and FLT3 on their cell surface are present, is higher than under conditions where cells expressing CD123 but not FLT3 on their cell surface are present or where cells expressing FLT3 but not CD123 on their cell surface are present.
  • the amount of trispecific antibody, and/or the amount of the third antibody fragment specific for CD3 and/or the amount of non- covalently associated HB1 and HB2, formed under conditions where CD123 and FLT3 are present is at least about 2 fold higher, 2.5 fold higher, 3 fold higher, 3.5 fold higher, 4 fold higher, 4,5 fold higher, 5 fold higher, 5,5 fold higher, 6 fold higher, 6.5 fold higher, 7 fold higher, 7.5 fold higher, 8 fold higher, 8.5 fold higher, 9 fold higher, 9.5 fold higher, 10 fold higher, 15 fold higher, 20 fold higher, 25 fold higher, 30 fold higher, 40 fold higher, 50 fold higher, 60 fold higher, 70 fold higher, 80 fold higher, 90 fold higher, 100 fold high, 150 fold higher, 200 fold higher, 300 fold higher, 400 fold higher, 500 fold higher, 600 fold higher, 700 fold higher, 800 fold higher, 900 fold higher, or 1000 fold higher, when compared to the respective amount under conditions where cells expressing CD123 but not FLT3 on their cell surface are present or where cells
  • the trispecific antibody according to the present disclosure or the pair of HB1 and HB2 according to the present disclosure has a molecular weight of less than 150 kDa, less than 145 kDa less than 140 kDa, less than 135 kDa less than 130 kDa, or less than 120 kDa. In an embodiment of the present disclosure, the trispecific antibody according to the present disclosure or the pair of HB1 and HB2 according to the present disclosure has a molecular weight of less than 250 kDa.
  • the present disclosure provides the trispecific antibody or the pair of HB1 and HB2 according to the present disclosure, wherein said trispecific antibody or said pair of HB1 and HB2 according to the present disclosure is capable of or induces T cell-mediated cell killing of cells expressing CD123 and FLT3 on their cell surface.
  • the present disclosure provides the trispecific antibody or the complementary pair of HB1 and HB2 according to the present disclosure, wherein the trispecific antibody or said pair of HB1 and HB2, induces T cell-mediated cell killing of cells expressing CD123 and FLT3 on their cell surface with an IC 50 concentration of less than about 500 pM, less than about 450 pM, less than about 400 pM, less than about 350 pM, less than about 300 pM, less than about 250 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, or less than about 10 pM.
  • an IC 50 concentration of less than about 500 pM, less than about 450 pM, less than about 400 pM, less than about
  • the present disclosure provides a trispecific antibody according to the present disclosure or said complementary pair of HB1 and HB2 according to the present disclosure, wherein the trispecific antibody or said pair of HB1 and HB2 according to the present disclosure induces T cell-mediated cell killing of cells expressing CD123 and FLT3 on their cell surface with an IC50 concentration of between 10 pM and 300 pM.
  • the present disclosure provides a trispecific antibody according to the present disclosure or said complementary pair of HB1 and HB2 according to the present disclosure, wherein the trispecific antibody or said pair of HB1 and HB2 according to the present disclosure induces T cell-mediated cell killing of cells expressing CD123 and FLT3 on their cell surface with an IC50 concentration of between 1 pM and 500 pM.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB1 and HB2 according to the present disclosure, wherein the IC50 concentration for the induced T cell-mediated cell killing of cells expressing CD123 and FLT3 on their cell surface is at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 75 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold or at least 5000 lower compared to that IC50 concentration determined for the induced T cell- mediated cell killing of cells expressing CD123 or FLT3 or neither of CD123 and FLT3 on their cell surface.
  • said T cell-mediated cell killing is measured in an in vitro T cell- mediated tumor cell killing assay. In an embodiment, said T cell-mediated cell killing is measured in presence of cytotoxic T cells. In an embodiment, said T cell-mediated cell killing is measured in presence PBMCs. In an embodiment, said T cell-mediated cell killing is measured in presence of CD8+ and/CD4+ T cells. In an embodiment, said T cells or PBMCs are human cells. In an embodiment, said T cell-mediated cell killing is measured in an in vitro T cell-mediated tumor cell killing assay as described in Example 3. In an embodiment, said cells are cancer cells. In an embodiment, said cells are hematopoietic cancer cells. In an embodiment, said cells are leukemia cancer cells.
  • said cells are human cancer cells.
  • said cells are MOLM-13 cells (DSMZ: ACC 554), MV4-11 cells (ATCC CRL-9591TM), and/or SKM-1 cells (DSMZ: ACC547).
  • the present disclosure pertains to a trispecific antibody, comprising a first antibody fragment specific for CD123, a second antibody fragment specific for FLT3, and a third antibody fragment specific for CD3 as disclosed herein, wherein a) the first antibody fragment specific for CD123 comprises a VH comprising a heavy chain complementary determining region (HCDR)1 comprising the amino acid sequence of SYWIG (SEQ ID NO: 1), a HCDR2 comprising the amino acid sequence of IIYPGDSDTRYSPSFQG (SEQ ID NO: 2) or VIEPSSSDTYYAPSFQG (SEQ ID NO: 11), and a HCDR3 comprising the amino acid sequence of EHEYLYGFDV (SEQ ID NO: 3), and VL comprising a light chain complementary determining region (LCDR)1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 4), a LCDR2 comprising the amino acid sequence of AASSLQS (SEQ ID NO: 5), and a LCDR3
  • the third antibody fragment specific for CD3 comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 124, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 125 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 36, and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 126, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 39.
  • the present disclosure pertains to a trispecific antibody comprising a first antibody fragment specific for CD123, a second antibody fragment specific for FLT3 and a third antibody fragment specific for CD3 according to the present disclosure, wherein a) the first antibody fragment specific for CD123 comprises a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13, and a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 14, wherein b) the second antibody fragment specific for FLT3 comprises a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
  • the third antibody fragment specific for CD3 comprises a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 127 and SEQ ID NO: 129, and a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 128.
  • the present disclosure pertains to a trispecific antibody as disclosed herein, comprising a first halfbody molecule (HB1) according to the present disclosure and a second halfbody molecule (HB2) according to the present disclosure, wherein a) HB1 comprises a first antibody fragment specific for CD123 and either the VH or VL of a third antibody fragment specific for CD3 according to the present disclosure, wherein the first antibody fragment specific for CD123 comprises a VH comprising a heavy chain complementary determining region (HCDR)1 comprising the amino acid sequence of SYWIG (SEQ ID NO: 1), a HCDR2 comprising the amino acid sequence of IIYPGDSDTRYSPSFQG (SEQ ID NO: 2) or VIEPSSSDTYYAPSFQG (SEQ ID NO: 11), and a HCDR3 comprising the amino acid sequence of EHEYLYGFDV (SEQ ID NO: 3), and a VL comprising a light chain complementary determining region (LCDR)1 comprising the amino acid sequence of RASQSISSY
  • the present disclosure pertains to a trispecific antibody disclosed herein, comprising a first halfbody molecule (HB1) according to the present disclosure and a second halfbody molecule (HB2) according to the present disclosure, wherein a) HB1 comprises a first antibody fragment specific for CD123 and either the VH or VL of a third antibody fragment specific for CD3 according to the present disclosure, wherein the first antibody fragment specific for CD123 comprises a VH comprising a heavy chain complementary determining region (HCDR)1 comprising the amino acid sequence of SYWIG (SEQ ID NO: 1), a HCDR2 comprising the amino acid sequence of IIYPGDSDTRYSPSFQG (SEQ ID NO: 2) or VIEPSSSDTYYAPSFQG (SEQ ID NO: 11), and a HCDR3 comprising the amino acid sequence of EHEYLYGFDV (SEQ ID NO: 3), and a VL comprising a light chain complementary determining region (LCDR)1 comprising the amino acid sequence of RASQSISSY
  • the present disclosure pertains to a trispecific antibody according to the present disclosure comprising a first halfbody molecule (HB1) according to the present disclosure and a second halfbody molecule (HB2) according to the present disclosure, wherein a) HB1 comprises a first antibody fragment specific for CD123 and either the VH or VL of a third antibody fragment specific for CD3 according to the present disclosure, wherein i) the first antibody fragment specific for CD123 comprises a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 or SEQ ID NO: 13, and a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 14, and where
  • the present disclosure pertains to a trispecific antibody according to the present disclosure comprising a first halfbody molecule (HB1) according to the present disclosure and a second halfbody molecule (HB2) according to the present disclosure, wherein a) HB1 comprises a first antibody fragment specific for CD123 and either the VH or VL of a third antibody fragment specific for CD3 according to the present disclosure, wherein i) the first antibody fragment specific for CD123 comprises a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 or SEQ ID NO: 13, and a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 14, and wherein
  • said first antibody fragment is a Fab.
  • said second antibody fragment is a Fab.
  • said third antibody fragment is a Fv.
  • said first antibody fragment is a Fab, said second antibody fragment is a Fab and said third antibody fragment is a Fv.
  • the present disclosure pertains to the trispecific antibody as disclosed herein, wherein said trispecific antibody is composed of a) a first antibody fragment specific for CD123, b) a second antibody specific for FLT3, and c) a third antibody fragment specific for CD3, wherein said trispecific antibody comprises a first peptide linker and a second peptide linker, wherein the first peptide linker fuses the first antibody fragment specific for CD123 to the either VH or the VL of the third antibody fragment specific for CD3, and the second peptide linker fuses the second antibody fragment specific for FLT3 to the complementary VH or to the complementary VL, respectively, of the third antibody fragment specific for CD3.
  • the present disclosure pertains to a trispecific antibody as disclosed herein, comprising a) a first antibody fragment specific for CD123, b) a second antibody fragments specific for FLT3, and c) a third antibody fragment specific for CD3, wherein said trispecific antibody is composed of a first halfbody molecule (HB1) and a second halfbody molecule (HB2), wherein HB1 comprises the first antibody fragment specific for CD123, a first peptide linker, and either the VH or the VL of the third antibody fragment specific for CD3, wherein the first peptide linker fuses the first antibody fragment specific for CD123 to the either VH or the VL of the third antibody fragment specific for CD3, and wherein HB2 comprises the second antibody fragment specific for FLT3, a second peptide linker and either the complementary VH or the complementary VL, respectively, of the third antibody fragment specific for CD3, and wherein the second peptide linker fuses the second antibody fragment specific for FLT3 to the complementary VH or to the complementary VL of the third antibody
  • the present disclosure provides a trispecific antibody as disclosed herein, wherein a) the C-terminus of the heavy chain of the first antibody fragment specific for CD123 is fused to the N-terminus of the VH of the third antibody fragment specific for CD3 via the first peptide linker and the C-terminus of the heavy chain of the second antibody fragment specific for FLT3 is fused to the N-terminus of the complementary VL of third antibody fragment specific for CD3, or wherein b) the C-terminus of the heavy chain of first antibody fragment specific for CD123 is fused to the N-terminus of the VL of the third antibody fragment specific for CD3 via a first peptide linker and the C-terminus of the heavy chain of the second antibody fragment specific for FLT3 is fused to the N-terminus of the complementary VH of the third antibody fragment specific for CD3.
  • said first antibody fragment is a Fab
  • said second antibody fragment is a Fab
  • said third antibody fragment is a Fv.
  • said first peptide linker and said second peptide linker are identical.
  • said first peptide linker and said second peptide linker are different.
  • said first peptide linker and said second peptide linker has the same length.
  • said first peptide linker and said second peptide linker has a different length.
  • said first and/or second peptide linker is an unstructured peptide linker.
  • said first and second peptide linker is a flexible peptide linker.
  • said first and second peptide linker is a flexible unstructured peptide linker.
  • the design of the trispecific antibody and/or of HB1 and/or of HB2 according to the present disclosure can include peptide linkers that are all or partially flexible, such that the peptide linker can include a flexible peptide linker as well as one or more portions that confer less flexible structure to provide for a desired trispecific antibody and/or HB1 and/or HB2 structure.
  • said unstructured and/or flexible peptide linker does not exhibit a secondary structure.
  • said peptide linker is a stable linker.
  • said peptide linker is a non-cleavable linker, such as non-cleavable by proteases, such as matrix metalloproteinases (MMP).
  • said peptide linker is composed of only natural occurring amino acid residues.
  • said peptide linker is composed of only natural occurring amino acid residues but excluding C.
  • said first and second peptide linker is composed of the amino acid residues selected from the group: G, S, A, or T.
  • said peptide linker is composed of amino acid residues selected from the group of A, Q, D, P, H, G, S and E.
  • the first and/or second peptide linker has a length of 1 to 40 amino acid residues.
  • the peptide linker may has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.
  • the first and/or second peptide linker has a length of 5, 10, 20 or 40 amino acid residues.
  • said first and second peptide linker is composed of an amino acid sequence selected from the group but not limited thereto consisting of: (G 4 S) 3 (SEQ ID NO: 74), (GGS) 3 (SEQ ID NO: 75), GGSG (SEQ ID NO: 76), GGSGG (SEQ ID NO: 77 ), GSGSG (SEQ ID NO: 78), GSGGG (SEQ ID NO: 79), GGGSG (SEQ ID NO: 80), GSSSG (SEQ ID NO: 81), (GS)n (SEQ ID NO: 82), (G4S)n (SEQ ID NO: 83), (SG4)n (SEQ ID NO: 84), (GSGGS)n (SEQ ID NO: 85), (GGGS)n (SEQ ID NO: 86) and G4(SG4)n (SEQ ID NO: 87), wherein n is an integer between 1 and 10, typically between 2 and 4.
  • said first and second peptide linker comprises the amino acid sequence QPKAAP (SEQ ID NO: 88) or ASTKGP (SEQ ID NO: 89).
  • the first and/or second peptide peptide linker is selected from the group consisting of: GQPSG (SEQ ID NO: 42), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:90), AQPAAPAPAE (SEQ ID NO: 91), AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 92), AQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 93).
  • EPKSCGQPSG (SEQ ID NO: 94), EPKSCAQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 95), EPKSCAQPAAPAPAE (SEQ ID NO: 96), EPKSCAQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 97), EPKSCGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 98), and KTHT (SEQ ID NO: 99).
  • said first and/or second peptide peptide has a length of 5 amino acid residues.
  • the first and/or second peptide linker has the amino acid sequence of GQPSG (SEQ ID NO: 42).
  • the present disclosure provides a trispecific antibody as disclosed herein, wherein a) HB1 is composed of a first and second polypeptide, wherein the first polypeptide comprises the light chain of the first antibody fragment specific for CD123 and the second polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the first antibody fragment specific for CD123, the first peptide linker and either the VH or VL of the third antibody fragment specific for CD3, and b) HB2 is composed of a third and fourth polypeptide, wherein the third polypeptide comprises the light chain of the second antibody fragment specific for FLT3, and the fourth polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the second antibody fragment specific for FLT3, the second peptide linker, and either the complementary VL or complementary VH, respectively, of the third antibody fragment specific for CD3.
  • the present disclosure provides a trispecific antibody as disclosed herein, wherein the first polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 108 or SEQ ID NO: 109, and the third polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 33 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 110 or SEQ ID NO: 111.
  • the present disclosure provides a trispecific antibody as disclosed herein, wherein the first polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134 or SEQ ID NO: 135, and the third polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 33 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: 141.
  • the first and second antibody fragment is selected from the group consisting of a Fab fragment, a F(ab’)2 fragment, a Fv fragment, an scFv, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
  • said first antibody fragment is a Fab
  • said second antibody fragment is a Fab
  • said third antibody fragment is a Fv.
  • the present disclosure provides a trispecific antibody as disclosed herein, wherein said trispecific antibody is composed of 4 polypeptides, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46, SEQ ID NO: 108 or SEQ ID NO: 109, the third polypeptide comprises the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 33 and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 or SEQ ID NO: 50, SEQ ID NO: 110 or SEQ ID NO: 111.
  • the first polypeptide comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16
  • the second polypeptide comprises the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO:
  • the present disclosure provides a trispecific antibody as disclosed herein, wherein said trispecific antibody is composed of 4 polypeptides, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134 or SEQ ID NO: 135, the third polypeptide comprises the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 33 and the fourth polypeptide comprises the amino acid sequence of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 or SEQ ID NO: 141.
  • the first polypeptide comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16
  • the second polypeptide comprises the amino acid sequence of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO:
  • said first and second polypeptide are associated to form a first antibody fragment specific for CD123.
  • the third and fourth polypeptide are associated to form a second antibody fragment specific for FLT3.
  • the first and second polypeptide are linked to each other via a covalent bond.
  • the third and fourth polypeptide are linked to each other via a covalent bond.
  • neither said first or said second polypeptide are covalently linked to the third or fourth polypeptide.
  • the second polypeptide of HB1 and the fourth polypeptide of HB2 are capable of non-covalently associating with each other thus forming a third antibody fragment specific for CD3.
  • said third antibody fragment is an antibody Fv fragment.
  • the VH of the antibody fragment specific for CD3 is present on the second polypeptide and the VL of the antibody fragment specific for CD3 is present on the fourth polypeptide. In an embodiment, the VH of the antibody fragment specific for CD3 is present on the fourth polypeptide and the VL of the antibody fragment specific for CD3 is present on the second polypeptide.
  • CyCAT halfbody molecules according to the present disclosure The trispecific antibodies according to the present disclosure are composed of a two component system, wherein two complementary halfbody molecules interact with each other on the surface of a target cell expressing both, CD123 and FLT3.
  • Each of the two CyCAT halfbody molecules carries a Fab as a tumor targeting moiety, with specific for either CD123 and FLT3, respectively, and only one of the complementary antibody variable domains of a functional antibody Fv fragment with specificity for CD3.
  • CD123 specific CyCAT halfbody molecules the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 is part of the trispecific antibody disclosed herein.
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises an antibody fragment specific for CD123 and either the VH or VL of an antibody fragment specific for CD3.
  • said antibody fragment specific for CD123 comprises the 6 CDRs of any one of the antibodies listed in Table 5 or Table 6 of the present disclosure.
  • said antibody fragment specific for CD123 comprises a VH and a VL of any one of the antibodies listed in Table 5 or Table 6 of the present disclosure.
  • the VH of the antibody fragment specific for CD3 comprises the 3 HCDRs of any one of the antibodies listed in Table 9 or Table 40 of the present disclosure.
  • the VL of the antibody fragment specific for CD3 comprises the 3 LCDRs of any one of the antibodies listed in Table 9 or Table 40 of the present disclosure.
  • the VH of the antibody fragment specific for CD3 comprises the VH of any one of the antibodies listed in Table 9 or Table 40 of the present disclosure.
  • the VL of the antibody fragment specific for CD3 comprises the VL of any one of the antibodies listed in Table 9 or Table 40 of the present disclosure.
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises an antibody fragment specific for CD123 and either the VH or VL of an antibody Fv fragment (Fv) specific for CD3.
  • said antibody fragment specific for CD123 is a first antibody fragment according to the present disclosure.
  • said antibody fragment is a Fab.
  • said antibody fragment specific for CD3 is the third antibody fragment according to the present disclosure.
  • said antibody fragment specific for CD3 is an antibody Fv fragment.
  • said HB1 has a molecular weight of less 80 kilodalton (kDa), less than 75 kDa, less than 70 kDa, or less than 65 kDa.
  • HB1 does not comprise a Fc region. In an embodiment, said HB1 does not comprise a Fc region subunit, composed of a CH2 and CH3 immunoglobulin constant domain. In an embodiment of the present disclosure, HB1 comprises an antibody fragment specific for CD123 and the VH of a Fv specific for CD3. In an embodiment of the present disclosure, HB1 comprises an antibody fragment specific for CD123 and the VH but not the VL of a Fv specific for CD3. In an embodiment of the present disclosure, said HB1 comprises an antibody fragment specific for CD123 and the VL of a Fv specific for CD3.
  • said HB1 comprises a first antibody fragment specific for CD123 and the VL but not the VH of a Fv specific for CD3.
  • said HB1 by itself is capable of binding or binds to CD123.
  • said HB1 by itself is not capable of binding to CD3.
  • said HB1 by itself is not capable of inducing or does not induce T-cell mediated killing of a cell expressing CD123 on its cell surface.
  • said HB1 by itself is not capable of inducing or does not induce proliferation and/or activation of a T-cell expressing CD3 on its cell surface.
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises an antibody fragment specific for CD123 and either the VH or VL of a Fv specific for CD3, wherein the antibody fragment specific for CD123 and the either VH or VL of the Fv specific for CD3 are fused to each other via a peptide linker.
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises from its N-terminus to its C-terminus, an antibody fragment specific for CD123, a peptide linker, and either the VH or the VL of an antibody Fv fragment (Fv) specific for CD3.
  • said antibody fragment specific for CD123 is fused to the either VH or VL of the Fv specific for CD3 via a peptide linker.
  • the C-terminus of the antibody fragment specific for CD123 is fused to the N-terminus of the either VH or the VL of the Fv specific for CD3 via a peptide linker.
  • the C-terminus of the antibody fragment specific for CD123 is fused to the N-terminus of the VH the Fv specific for CD3 via a peptide linker.
  • the C-terminus of the antibody fragment specific for CD123 is fused to the N-terminus of the VL the Fv specific for CD3 via a peptide linker.
  • the peptide linker is or comprises any one of the peptide linkers as disclosed herein.
  • the peptide linker has a length of 1 – 40 amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 ,30 ,31, 32, 33, 34, 35, 36, 37, 38 or 39 amino acid residues.
  • the peptide linker has a length of 1 – 5 amino acid residues, such as 1, 2, 3, 4, or 5 amino acid residues.
  • the peptide linker has a length of 5 amino acid residues.
  • the peptide linker has the amino acid sequence of GQPSG (SEQ ID NO: 42).
  • CD123 specific antibodies of the present disclosure provides an antibody or antibody fragment specific for CD123.
  • the present disclosure provides an antibody or antibody fragment specific for CD123 present in the trispecific antibody or in a halfbody, such as HB1, according to the present disclosure.
  • the present disclosure pertains to an antibody or antibody fragment specific for CD123 comprising the 6 CDRs of any one of the antibodies listed in Table 5 or Table 6.
  • the present disclosure pertains to an antibody or antibody fragment specific for CD123 comprising the 6 CDRs defined by Kabat of any one of the antibodies listed in Table 5 or Table 6.
  • the present disclosure pertains to an antibody or antibody fragment specific for CD123, comprising a VH and a VL of any one of the antibodies listed in Table 5 or Table 6.
  • the antibody or antibody fragment specific for CD123 according to the present disclosure is an isolated antibody or antibody fragment.
  • said antibody or antibody fragment specific for CD123 is an human antibody or antibody fragment.
  • said antibody or antibody fragment specific for CD123 is a chimeric antibody or antibody fragment.
  • the antibody or antibody fragment specific for CD123 is a human, humanized or chimeric antibody or antibody fragment.
  • the antibody or antibody fragment specific for CD123 according to the present disclosure is a recombinant or a synthetic antibody or antibody fragment.
  • the antibody or antibody fragment specific for CD123 according to the present disclosure is a monoclonal antibody or antibody fragment.
  • said antibody fragment is selected from the group consisting of a Fab fragment, a F(ab’)2 fragment, a Fv fragment, a scFv fragment, a domain antibody (dAb), a single domain heavy chain antibody, and a single domain light chain antibody.
  • the antibody or antibody fragment specific for CD123 according to the present disclosure is of the IgG isotype. In an embodiment, the antibody or antibody fragment specific for CD123 is an IgG1.
  • the present disclosure pertains to a bispecific antibody comprising a first binding domain of an antibody or antibody fragment specific for CD123 according to the present disclosure and a second binding domain specific for CD3.
  • the present disclosure pertains to a bispecific antibody comprising a first binding domain of an antibody or antibody fragment specific for CD123 according to the present disclosure and a second binding domain of an antibody or antibody fragment specific for CD3 according to the present disclosure.
  • the antibody or antibody fragment specific for CD123 according to the present disclosure binds to human CD123.
  • said antibody or antibody fragment specific for CD123 is specific for human CD123.
  • said antibody or antibody fragment specific for CD123 according to the present disclosure is specific for human CD123 encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 51 and SEQ ID NO: 52.
  • the antibody or antibody fragment specific for CD123 according to the present disclosure is specific for a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 65.
  • the antibody or antibody fragment specific for CD123 according to the present disclosure specifically binds to a CD123 polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 65.
  • the antibody or antibody fragment specific for CD123 specifically binds to the extracellular region of human CD123.
  • said extracellular region of human CD123 comprises the amino acid sequence of SEQ ID NO 52.
  • the antibody or antibody fragment specific for CD123 according to the present disclosure is specific for human CD123 and cynomolgus monkey CD123.
  • said antibody or antibody fragment specific for CD123 specifically binds to human CD123 and cynomolgus CD123.
  • the antibody or antibody fragment specific for CD123 according to the present disclosure is specific for human CD123 and is cross-reactive to cynomolgus monkey CD123.
  • the antibody or antibody fragment specific for CD123 is specific for human CD123 and cross-reactively binds to cynomolgus monkey CD123. In an embodiment, the antibody or antibody fragment specific for CD123 specifically binds to human CD123 and cross- reactively binds to cynomolgus CD123. In an embodiment, said cynomolgus CD123 is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 53 and SEQ ID NO: 54. In an embodiment, said cynomolgus CD123 comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 53 and SEQ ID NO: 54.
  • the antibody or antibody fragment specific for CD123 specifically binds to the extracellular region of cynomolgus CD123.
  • said extracellular region of cynomolgus CD123 comprises the amino acid sequence of SEQ ID NO: 54.
  • the antibody or antibody fragment specific for CD123 specifically binds to a cynomolgus CD123 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 54, and SEQ ID NO: 66.
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises an antibody fragment specific for CD123 according to the present disclosure and either the VH or VL of an antibody Fv fragment specific for CD3, wherein HB1 and/or the antibody fragment specific for CD123 has a monovalent affinity for a human CD123 polypeptide comprising the amino acid sequence of SEQ ID NO: 65, with a K D of about 0.50 nM or less, such as about 0.45 nM or less, about 0.40 nM or less, about 0.35 nM or less, about 0.30 nM or less, about 0.25 nM or less, about 0.20 nM or less, about 0.15 nM or less, about 0.10 nM or less, about 0.05 nM or less, about 0.04 nM, about 0.03 nM or less, about 0.02 nM or less, or about 0.01 nM or less.
  • HB1 comprises an antibody fragment specific for CD123 according to the present disclosure and either the VH or V
  • HB1 and/or the antibody fragment specific for CD123 binds to a human CD123 polypeptide comprising the amino acid sequence of SEQ ID NO: 65, with a K D of about 0.50 nM or less, such as about 0.45 nM or less, about 0.40 nM or less, about 0.35 nM or less, about 0.30 nM or less, about 0.25 nM or less, about 0.20 nM or less, about 0.15 nM or less, about 0.10 nM or less, about 0.05 nM or less, about 0.04 nM, about 0.03 nM or less, about 0.02 nM or less, or about 0.01 nM or less.
  • HB1 and/or the antibody fragment specific for CD123 has a monovalent affinity to a cynomolgus monkey CD123 polypeptide comprising the amino acid sequence of SEQ ID NO: 66, with a KD of about 1 nM or less, such that about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or about 0.1 nM less, about 0.05 nM or less, about 0.04 nM, about 0.03 nM or less, about 0.02 nM or less, or about 0.01 nM or less.
  • HB1 and/or the antibody fragment specific for CD123 binds to a cynomolgus monkey CD123 polypeptide comprising the amino acid sequence of SEQ ID NO: 66, with a KD of about 1 nM or less, such that about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or about 0.1 nM less, about 0.05 nM or less, about 0.04 nM, about 0.03 nM or less, about 0.02 nM or less, or about 0.01 nM or less.
  • HB1 and/or the antibody fragment specific for CD123 binds to a human CD123 polypeptide comprising the amino acid sequence of SEQ ID NO: 65 and to a cynomolgus monkey CD123 peptide comprising the amino acid sequence of SEQ ID NO: 66 with a monovalent affinity of about 1 nM or less, such that about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or about 0.1 nM less, about 0.05 nM or less, about 0.04 nM, about 0.03 nM or less, about 0.02 nM or less, or about 0.01 nM or less.
  • said antibody fragment specific for CD123 is a Fab.
  • said monovalent affinity or KD is determined for a Fab.
  • said monovalent affinity or K D is determined by an in vitro biolayer interferometry binding assay (BLI) at 25°C.
  • the monovalent affinity or KD is determined by an in vitro bio-layer interferometry binding assay (BLI) as described herein in Example 4.
  • the antibody or antibody fragment specific for CD123 comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 11, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a variable light chain region (VL) comprising a light chain complementary determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 12.
  • VH heavy chain variable region
  • HCDR heavy chain complementary determining region
  • LCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 11
  • a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3
  • VL variable light chain region
  • the antibody or antibody fragment specific for CD123 comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6.
  • the antibody or antibody fragment specific for CD123 comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5 and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 12.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 comprises a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 comprises a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 14.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 comprises (a) a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 or SEQ ID NO: 13 and (b) a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 14.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 comprises a VH comprising (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 13 and (b) a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 14.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 comprises a VH and a VL, wherein said VH and said VL are selected from the group consisting of: (a) a VH comprising the amino acid sequence of SEQ ID NO: 7 and the VL comprising the amino acid sequence of SEQ ID NO: 8 and (b) a VH comprising the amino acid sequence of SEQ ID NO: 13 and the VL comprising the amino acid sequence of SEQ ID NO: 14.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 and a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 comprises a VH comprising the amino acid sequence of SEQ ID NO: 13 and a VL comprising the amino acid sequence of SEQ ID NO: 14.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 comprises (a) an antibody heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 9 and an antibody light chain (LC) comprising the amino acid sequence of SEQ ID NO: 10 or (b) an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and an antibody light comprising the amino acid sequence of SEQ ID NO: 16.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 is a Fab.
  • said antibody heavy chain is a Fab heavy chain.
  • said antibody light chain is a Fab light chain.
  • HB1 and/or the antibody fragment specific for CD123 according to the present disclosure binds to CD123 expressed on cells with an EC50 concentration of 10 nM or less, such as 9 nM or less, 8 nM or less, 7 nM or less, 6 nM less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM
  • said antibody fragment specific for CD123 is a Fab.
  • said CD123 is human CD123.
  • said cells are human cells.
  • said cells are selected from the group consisting of MOLM-13, MV4-11, and SKM-1 cells.
  • said EC50 concentration is determined by an FACS assay as described herein in Example 7.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 binds to CD123 present or expressed on the surface of a cell.
  • the present disclosure provides an HB1, wherein the first antibody fragment specific for CD123 binds to a cell having CD123 on its cell surface.
  • said cell is a hematopoietic cell. In an embodiment, said cell is a leukocyte. In an embodiment, said cell is a myeloblast cells. In an embodiment, said cell is a malignant cell. In an embodiment, said cell is a cell of a hematological malignancy. In an embodiment, said cell is a cancer cell. In some embodiments, said cell is a tumor cell. In an embodiment, said cancer is a hematological tumor. In an embodiment, said cancer is a hematological cancer. In an embodiment, said cancer is a leukemia.
  • said cancer is Acute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML), Chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Chronic myelogenous leukemia (CML), Acute monocytic leukemia (AMoL)
  • HB1 comprises a first antibody fragment specific for CD123 and either the VH or VL of an Fv specific for CD3, wherein a) the first antibody fragment specific for CD123 comprises a VH comprising a heavy chain complementary determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 1, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 11, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a VL comprising a light chain complementary determining region (LCDR)1 comprising
  • the VH of the Fv specific for CD3 comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO:124, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 125 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 36, and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 126, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 39.
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises a first antibody fragment specific for CD123 and either the VH or VL of an Fv specific for CD3, wherein a) the first antibody fragment specific for CD123 comprises a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 or SEQ ID NO: 13 and a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 14, and wherein b) the VH of the Fv specific for CD3 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
  • the VH of the Fv specific for CD3 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 129
  • the VL of the Fv specific for CD3 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 128.
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises a first antibody fragment specific for CD123 and either the VH or VL of an Fv specific for CD3, wherein a) the first antibody fragment specific for CD123 comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 13 and a VL comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 14, and wherein b) the VH of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 107 or the VL of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID SEQ ID NO: 41.
  • HB1 comprises a first antibody fragment specific for CD123 and either the VH or VL of an Fv specific for CD3
  • a) the first antibody fragment specific for CD123 comprises a VH comprising the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 13 and a VL comprising the amino acid
  • the VH of the Fv specific for CD3 comprises an amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 129 or the VL of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 128.
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises a Fab specific for CD123 and either the VH or VL of an Fv specific for CD3, wherein the Fab specific for CD123 comprises a Fab heavy chain comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15 and a Fab light chain comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16, and wherein either VH of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 107 or the VL of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 41.
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises a Fab specific for CD123 and either the VH or VL of an Fv specific for CD3, wherein the Fab specific for CD123 comprises a Fab heavy chain comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15 and a Fab light chain comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 16, and wherein the either VH of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 129 or the VL of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 128.
  • HB1 comprises a Fab specific for CD123 and either the VH or VL of an Fv specific for CD3
  • the Fab specific for CD123 comprises a Fab heavy chain comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15 and a Fab light chain comprising the amino acid sequence of SEQ ID
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises a first antibody fragment specific for CD123 and either the VH or VL of an antibody Fv fragment specific for CD3, wherein said HB1 is composed of a first and second polypeptide, wherein the first polypeptide comprises a light chain of a Fab specific for CD123 and the second polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the Fab specific for CD123, a peptide linker and either the VH or VL of the antibody Fv region specific for CD3, wherein the heavy and light chain of the Fab form the first antibody fragment specific for CD123.
  • HB1 comprises a first antibody fragment specific for CD123 and either the VH or VL of an antibody Fv fragment specific for CD3, wherein said HB1 is composed of a first and second polypeptide, wherein the first polypeptide comprises a light chain of a Fab specific for CD123 and the second polypeptide comprises from its N-terminus to its C
  • the present disclosure provides a first halfbody molecule (HB1), wherein said HB1 comprises a Fab specific for CD123 and either the VH or VL of an antibody Fv fragment specific for CD3, wherein said HB1 is composed of a first and second polypeptide, wherein the first polypeptide comprises the light chain of Fab specific for CD123 and the second polypeptide comprises from its N-terminus to its C- terminus, the heavy chain of the Fab, a peptide linker and either the VH or VL of the antibody Fv region specific for CD3.
  • HB1 comprises a Fab specific for CD123 and either the VH or VL of an antibody Fv fragment specific for CD3, wherein said HB1 is composed of a first and second polypeptide, wherein the first polypeptide comprises the light chain of Fab specific for CD123 and the second polypeptide comprises from its N-terminus to its C- terminus, the heavy chain of the Fab, a peptide linker and either the VH or VL of the antibody Fv region
  • the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 16 and wherein the second polypeptide of HB1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 108 and SEQ ID NO: 109.
  • SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 108 and SEQ ID NO: 109 amino acid sequence selected from the group consisting of
  • the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 16 and wherein the second polypeptide of HB1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 108 and SEQ ID NO: 109.
  • HB1 first halfbody molecule
  • the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 16 and wherein the second polypeptide of HB1 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134 and SEQ ID NO: 135.
  • the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 16 and wherein the second polypeptide of HB1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134 and SEQ ID NO: 135.
  • the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises the amino acid sequence SEQ ID NO: 10 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 43.
  • the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises the amino acid sequence SEQ ID NO: 10 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 44. In an embodiment, the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises the amino acid sequence SEQ ID NO: 10 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 108.
  • the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises the amino acid sequence SEQ ID NO: 10 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 130, SEQ ID NO: 131, or SEQ ID NO: 132, In an embodiment, the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises the amino acid sequence SEQ ID NO: 16 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 45.
  • HB1 first halfbody molecule
  • the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises the amino acid sequence SEQ ID NO: 16 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 46. In an embodiment, the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises the amino acid sequence SEQ ID NO: 16 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 109.
  • the present disclosure provides a first halfbody molecule (HB1), wherein the first polypeptide of HB1 comprises the amino acid sequence SEQ ID NO: 16 and the second polypeptide of HB1 comprises the amino acid sequence of SEQ ID NO: 133, SEQ ID NO: 134 or SEQ ID NO: 135.
  • FLT3 specific CyCAT halfbody molecules In an embodiment, the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 is part of the trispecific antibody according to the present disclosure.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises an antibody fragment specific for FLT3 and either the VH or VL of an antibody fragment specific for CD3.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises an antibody fragment specific for FLT3 and either the VH or VL of an antibody Fv fragment (Fv) specific for CD3.
  • said antibody fragment specific for FLT3 comprises the 6 CDRs of any one of the antibodies listed in Table 7 or Table 8 of the present disclosure.
  • said antibody fragment specific for FLT3 comprises a VH and a VL of any one of the antibodies listed in Table 7 or Table 8 of the present disclosure.
  • the VH of the antibody fragment specific for CD3 comprises the 3 HCDRs of any one of the antibodies listed in Table 9 or Table 40 of the present disclosure.
  • the VL of the antibody fragment specific for CD3 comprises the 3 LCDRs of any one of the antibodies listed in Table 9 or Table 40 of the present disclosure.
  • the VH of the antibody fragment specific for CD3 comprises the VH of any one of the antibodies listed in Table 9 or Table 40 of the present disclosure.
  • the VL of the antibody fragment specific for CD3 comprises the VL of any one of the antibodies listed in Table 9 or Table 40 of the present disclosure.
  • said antibody fragment specific for FLT3 is a second antibody fragment according to the present disclosure.
  • said antibody fragment specific for FLT3 is a Fab.
  • said antibody fragment specific for CD3 is a third antibody fragment according to the present disclosure.
  • said antibody fragment specific for CD3 is an antibody Fv fragment.
  • HB2 has a molecular weight of less 80 kilodalton (kDa), less than 75 kDa, less than 70 kDa, or less than 65 kDa.
  • said HB2 does not comprise a Fc region.
  • said HB2 does not comprise a Fc region subunit, composed of a CH2 and CH3 immunoglobulin constant domain.
  • HB2 comprises an antibody fragment specific for FLT3 and the VH of an antibody Fv fragment specific for CD3.
  • HB2 comprises an antibody fragment specific for FLT3 and the VH but not the VL of an antibody Fv fragment specific for CD3. In an embodiment of the present disclosure, HB2 comprises an antibody fragment specific for FLT3 and the VL of an antibody Fv fragment specific for CD3. In an embodiment of the present disclosure, HB2 comprises an antibody fragment specific for FLT3 and the VL but not the VH of an antibody Fv fragment specific for CD3. In an embodiment of the present disclosure, HB2 by itself is capable of binding or binds to FLT3. In an embodiment, HB2 by itself is not capable of binding or does not bind to CD3.
  • said HB2 by itself is not capable of inducing or does not induce T-cell mediated killing of a cell expressing FLT3 on its cell surface.
  • said HB2 by itself is not capable of inducing or does not induce proliferation and/or activation of a T-cell expressing CD3 on its cell surface.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises an antibody fragment specific for FLT3 and either the VH or VL of a Fv specific for CD3, wherein the antibody fragment specific for FLT3 and the either VH or VL of the Fv specific for CD3 are linked to each other via a peptide linker.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises from its N-terminus to its C-terminus, an antibody fragment specific for FLT3, a peptide linker, and either the VH or VL of an antibody Fv fragment (Fv) specific for CD3.
  • the antibody fragment specific for FLT3 is linked to the either VH or VL of the Fv specific for CD3 via a peptide linker.
  • the C-terminus of the antibody fragment specific for FLT3 is fused to the N-terminus of the either VH or the VL of the Fv specific for CD3 via a peptide linker.
  • the peptide linker is or comprises any one of the peptide linkers as disclosed herein.
  • the second peptide linker has a length of 1 – 40 amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30 , 31, 32, 33, 34, 35, 36, 37, 38 or 39 amino acid residues.
  • the peptide linker has a length of 1 – 5 amino acid residues, such as 1, 2, 3, 4, or 5 amino acid residues.
  • the peptide linker has a length of 5 amino acid residues, In a preferred embodiment, the peptide linker has the amino acid sequence of GQPSG (SEQ ID NO: 42).
  • FLT3 specific antibodies of the present disclosure provides an antibody or antibody fragment specific for FLT3.
  • the present disclosure pertains to an antibody or antibody fragment specific for FLT3 comprising the 6 CDRs of any one of the antibodies listed in Table 7 or Table 8.
  • the present disclosure pertains to an antibody or antibody fragment specific for FLT3 comprising the 6 CDRs defined by Kabat of any one of the antibodies listed in Table 7 or Table 8.
  • the disclosure pertains to an antibody or antibody fragment specific for FLT3, comprising a VH and a VL of any one of the antibodies listed in Table 7 or Table 8.
  • the antibody or antibody fragment specific for FLT3 according to the present disclosure is an isolated antibody or antibody fragment.
  • said antibody or antibody fragment specific for FLT3 is a human antibody or antibody fragment.
  • said antibody or antibody fragment specific for FLT3 is a chimeric antibody or antibody fragment.
  • the antibody or antibody fragment specific for FLT3 is a human, humanized or chimeric antibody or antibody fragment.
  • the antibody or antibody fragment specific for FLT3 according to the present disclosure is a recombinant or synthetic antibody or antibody fragment.
  • said antibody or antibody fragment specific for FLT3 is a monoclonal antibody or antibody fragment.
  • said antibody fragment is selected from the group consisting of a Fab fragment, a F(ab’) 2 fragment, a Fv fragment, a scFv fragment, a domain antibody (dAb), a single domain heavy chain antibody, and a single domain light chain antibody.
  • the antibody or antibody fragment specific for CD123 according to the present disclosure is of the IgG isotype.
  • the antibody or antibody fragment specific for FLT3 is an IgG1.
  • the present disclosure pertains to a bispecific antibody comprising a first binding domain of an antibody or antibody fragment specific for FLT3 according to the present disclosure and a second binding domain of an antibody or antibody fragment specific for CD3 according to the present disclosure.
  • the present disclosure provides an antibody or antibody fragment specific for FLT3 according to the present disclosure, present in the trispecific antibody or in HB2 of the present disclosure.
  • the present disclosure provides a trispecific antibody or HB2 according to the present disclosure, comprising the antibody or antibody fragment specific for FLT3 according to the present disclosure.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises an antibody fragment specific for FLT3 according to the present disclosure and either the VH or VL of a Fv specific for CD3.
  • said antibody or antibody fragment specific for FLT3 binds to human FLT3.
  • the antibody or antibody fragment specific for FLT3 is specific for human FLT3.
  • the antibody or antibody fragment specific for FLT3 is specific for human FLT3 encoded an amino acid sequence selected from the group consisting of SEQ ID NO: 55 and SEQ ID NO: 56.
  • the antibody or antibody fragment specific for FLT3 is specific for a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 67.
  • the antibody or antibody fragment specific for FLT3 according to the present disclosure specifically binds to a FLT3 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 67.
  • the antibody or antibody fragment specific for FLT3 specifically binds to the extracellular region of human FLT3.
  • said extracellular region of human FLT3 comprises the amino acid sequence of SEQ ID NO 56.
  • the antibody or antibody fragment specific for FLT3 according to the present disclosure is specific for human FLT3 and cynomolgus monkey FLT3. In an embodiment, the antibody or antibody fragment specific for FLT3 according to the present disclosure is specific for human FLT3, cynomolgus monkey FLT3 and mouse FLT3. In an embodiment, said antibody or antibody fragment specific for FLT3 specifically binds to human FLT3 and cynomolgus FLT3. In an embodiment, said antibody or antibody fragment specific for FLT3 specifically binds to human FLT3, cynomolgus FLT3 and mouse FLT3.
  • the antibody or antibody fragment specific for FLT3 according to the present disclosure is specific for human FLT3 and is cross-reactive to cynomolgus monkey FLT3. In an embodiment, the antibody or antibody fragment specific for FLT3 according to the present disclosure is specific for human FLT3 and is cross-reactive to cynomolgus monkey FLT3 and mouse FLT3. In an embodiment, the antibody or antibody fragment specific for FLT3 according to the present disclosure is specific for human FLT3 and cross-reactively binds to cynomolgus monkey FLT3.
  • the antibody or antibody fragment specific for FLT3 is specific for human FLT3 and cross-reactively binds to cynomolgus monkey FLT3 and mouse FLT3.
  • said cynomolgus FLT3 is encoded by an amino acid sequence selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 58.
  • said cynomolgus FLT3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 57 and SEQ ID NO: 58.
  • said mouse FLT3 is encoded an amino acid sequence selected from the group consisting of SEQ ID NO: 59 and SEQ ID NO: 60.
  • said mouse FLT3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 59 and SEQ ID NO: 60.
  • the second antibody fragment specific for FLT3 specifically binds to the extracellular region of cynomolgus FLT3.
  • said extracellular region of cynomolgus FLT3 comprises the amino acid sequence of SEQ ID NO 58.
  • the second antibody fragment specific for FLT3 specifically binds to the extracellular region of mouse FLT3.
  • said extracellular region of mouse FLT3 comprises the amino acid sequence of SEQ ID NO 60.
  • the antibody or antibody fragment specific for FLT3 specifically binds to a cynomolgus FLT3 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 68.
  • the antibody or antibody fragment specific for FLT3 specifically binds to a mouse FLT3 peptide having polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 69.
  • said antibody or antibody fragment is a Fab.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises a antibody fragment specific for FLT3 according to the present disclosure and either the VH or VL of an antibody Fv fragment specific for CD3, wherein HB2 and/or said antibody fragment specific for FLT3 has a monovalent affinity for a human FLT3 polypeptide comprising the amino acid sequence of SEQ ID NO: 67, with a dissociation rate constant (KD) of about 0.50 nM or less, such as about 0.45 nM or less, about 0.40 nM or less, about 0.35 nM or less, about 0.30 nM or less, about 0.25 nM or less, about 0.20 nM or less, about 0.15 nM or less, about 0.10 nM or less, about 0.05 nM or less, about 0.04 nM, about 0.03 nM or less, about 0.02 nM or less, or about 0.01 nM or less.
  • KD dissociation rate constant
  • HB2 and/or the antibody fragment specific for FLT3 binds to a human FLT3 polypeptide comprising the amino acid sequence of SEQ ID NO: 67, with a KD of about 0.50 nM or less, such as about 0.45 nM or less, about 0.40 nM or less, about 0.35 nM or less, about 0.30 nM or less, about 0.25 nM or less, about 0.20 nM or less, about 0.15 nM or less, about 0.10 nM or less, about 0.05 nM or less, about 0.04 nM, about 0.03 nM or less, about 0.02 nM or less, or about 0.01 nM or less.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises a antibody fragment specific for FLT3 according to the present disclosure and either the VH or VL of an antibody Fv fragment specific for CD3, wherein HB2 and/or the second antibody fragment specific for FLT3, has a monovalent affinity to a cynomolgus FLT3 polypeptide comprising the amino acid sequence of SEQ ID NO: 68, with a KD of about 1 nM or less, such that about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or about 0.1 nM less, about 0.05 nM or less, about 0.04 nM, about 0.03 nM or less, about 0.02 nM or less, or about 0.01 nM or less.
  • HB2 comprises
  • HB2 and/or the antibody fragment specific for FLT3 binds to a cynomolgus FLT3 polypeptide comprising the amino acid sequence of SEQ ID NO: 68, with a KD of about 1 nM or less, such that about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or less, about 0.1 nM or less, about 0.05 nM or less, about 0.04 nM or less, about 0.03 nM or less, about 0.02 nM or less, or about 0.01 nM or less.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises a antibody fragment specific for FLT3 according to the present disclosure and either the VH or VL of an antibody Fv fragment specific for CD3, wherein HB2 and/or the antibody fragment specific for FLT3, has a monovalent affinity to a mouse FLT3 polypeptide comprising the amino acid sequence of SEQ ID NO: 69, with a KD of about 50 nM or less, such that about 45 nM or less, about 40 nM or less, about 35 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 5 nM less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less.
  • HB2 comprises a antibody fragment specific for FLT3 according to the present disclosure and either the VH or VL of an antibody Fv
  • HB2 and/or the antibody fragment specific for FLT3 binds to a mouse FLT3 polypeptide comprising the amino acid sequence of SEQ ID NO: 69, with a KD of about 50 nM or less, such that about 45 nM or less, about 40 nM or less, about 35 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 5 nM less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises a antibody fragment specific for FLT3 according to the present disclosure and either the VH or VL of an antibody Fv fragment specific for CD3, wherein HB2 and/or the antibody fragment specific for FLT3 has a monovalent affinity to a human FLT3 polypeptide comprising the amino acid sequence of SEQ ID NO: 67, with a KD of about 0.5 nM or less, and a monovalent affinity to a cynomolgus FLT3 polypeptide comprising the amino acid sequence of SEQ ID NO: 68 with a K D of about 1 nM or less, and a monovalent affinity to a mouse FLT3 polypeptide comprising the amino acid sequence of SEQ ID NO: 69, with a KD of about 50 nM or less.
  • HB2 comprises a antibody fragment specific for FLT3 according to the present disclosure and either the VH or VL of an antibody Fv fragment specific for CD3, wherein HB
  • said antibody fragment specific for FLT3 is a Fab. In an embodiment, said antibody fragment specific for FLT3 is a second antibody fragment or a second Fab according to the present disclosure.
  • said monovalent affinity or KD is determined for a Fab. In an embodiment, the monovalent affinity or KD is determined by an in vitro biolayer interferometry binding assay (BLI) at 25°C. In an embodiment, the monovalent affinity or KD is determined by an in vitro bio-layer interferometry binding assay (BLI) as described herein in Example 4.
  • the antibody or antibody fragment specific for FLT3 comprises a heavy chain variable region (VH) comprising a heavy chain complementary determining region (HCDR)1 comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 27 , a HCDR2 comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 28, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 19, and a variable light chain region (VL) comprising a light chain complementary determining region (LCDR)1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 29.
  • VH heavy chain variable region
  • HCDR heavy chain complementary determining region
  • LCDR2 comprising the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 28
  • a HCDR3 comprising the amino acid sequence of SEQ ID NO: 19
  • the antibody or antibody fragment specific for FLT3 comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 22.
  • the antibody or antibody fragment specific for FLT3 comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 27, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 28, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 29.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 comprises a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 23 and SEQ ID NO: 30.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 comprises a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 31.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 comprises a VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 23 or SEQ ID NO: 30 and a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 31.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 comprises a VH comprising an amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 30 and a VL comprising an amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 31.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 comprises a VH and a VL, wherein said VH and said VL are selected from the group consisting of (a) a VH comprising the amino acid sequence of SEQ ID NO: 23 and the VL comprising the amino acid sequence of SEQ ID NO: 24 and (b) a VH comprising the amino acid sequence of SEQ ID NO: 30 and the VL comprising the amino acid sequence of SEQ ID NO: 31.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 comprises a VH comprising the amino acid sequence of SEQ ID NO: 23 and a VL comprising the amino acid sequence of SEQ ID NO: 24.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 comprises a VH comprising the amino acid sequence of SEQ ID NO: 30 and a VL comprising the amino acid sequence of SEQ ID NO: 31.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 comprises (a) an antibody heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 25 and an antibody light chain (LC) comprising the amino acid sequence of SEQ ID NO: 26 or (b) an antibody heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and an antibody light comprising the amino acid sequence of SEQ ID NO: 33.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 is a Fab.
  • said antibody heavy chain is a Fab heavy chain.
  • said antibody light chain is a Fab light chain.
  • HB2 and/or the antibody fragment specific for FLT3 according to the present disclosure binds to FLT3 expressed on cells with an EC50 concentration of 300 nM or less, such as 250 nM or less, 200 nM or less, 150 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 65 nM or less, 60 nM or less, 55 nM or less, 50 nM less, 45 nM or less, 40 nM or less, 35 nM or less, 30 nM or less, 25 nM or less, 20 nM or less, 15 nM, 10 nM or less, 9 nM or less, 8 nM or
  • said antibody fragment specific for FLT3 is a Fab.
  • said FLT3 is human FLT3.
  • said cells are human cells.
  • said cells are selected from the group consisting of MOLM-13, MV4-11, and SKM-1 cells.
  • said EC50 concentration is determined by an FACS assay as described herein in Example 7.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 binds to FLT3 present or expressed on the surface of a cell.
  • the present disclosure provides an HB2, wherein the second antibody fragment specific for FLT3 binds to a cell having FLT3 on its cell surface.
  • said cell is a hematopoietic cell.
  • said cell is a leukocyte.
  • said cell is a myeloblast cells.
  • said cell is a malignant cell.
  • said cell is a cell of a hematological malignancy.
  • said cell is a cancer cell.
  • said cell is a tumor cell.
  • said cancer is a hematological tumor.
  • said cancer is a hematological cancer.
  • said cancer is a leukemia.
  • said cancer is Acute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML), Chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Chronic myelogenous leukemia (CML), Acute monocytic leukemia (AMoL)
  • ALL Acute lymphoblastic leukemia
  • AML Acute myelogenous leukemia
  • CLL Chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CML Chronic myelogenous leukemia
  • Acute monocytic leukemia Acute monocytic leukemia
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises a second antibody fragment specific for FLT3 and either the VH or VL of an Fv specific for CD3, wherein a) the second antibody fragment specific for FLT3 comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ
  • the VH of the Fv specific for CD3 comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 124, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 125 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 36, and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 126, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 39.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises a second antibody fragment specific for FLT3 and either the VH or VL of an Fv specific for CD3, wherein a) the second antibody fragment specific for FLT3 comprises VH comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 23 or SEQ ID NO: 30, and a VL comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 31 and wherein b) either the VH of the Fv specific for CD3 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • the VH of the Fv specific for CD3 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 129
  • the VL of the Fv specific for CD3 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 128.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises a second antibody fragment specific for FLT3 and either the VH or VL of an Fv specific for CD3, wherein a) the first antibody fragment specific for FLT3 comprises a VH comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 30, and a VL comprising the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 31, and wherein b) either the VH of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 107 or the VL of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 41.
  • HB2 comprises a second antibody fragment specific for FLT3 and either the VH or VL of an Fv specific for CD3
  • the first antibody fragment specific for FLT3 comprises a VH comprising the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 30, and a VL comprising the amino acid sequence
  • the VH of the Fv specific for CD3 comprises an amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 129 or the VL of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 128.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises a Fab specific for FLT3 and either the VH or VL of an Fv specific for CD3, wherein a) the Fab specific for FLT3 comprises a Fab heavy chain comprising the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 32 and a Fab light chain comprising the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 33, and wherein b) either the VH of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 107 or the VL of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 41.
  • the VH of the Fv specific for CD3 comprises an amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 129 or the VL of the Fv specific for CD3 comprises the amino acid sequence of SEQ ID NO: 128.
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises a second antibody fragment specific for FLT3 and either the VH or VL of a Fv specific for CD3, wherein said HB2 is composed of a third and fourth polypeptide, wherein the third polypeptide comprises a light chain of the Fab specific for FLT3 and the fourth polypeptide comprises from its N-terminus to its C- terminus, the heavy chain of the Fab specific for FLT3, a peptide linker and either the VH or VL of the Fv specific for CD3, wherein the heavy chain and light chain of the Fab form the second antibody fragment specific for FLT3.
  • HB2 comprises a second antibody fragment specific for FLT3 and either the VH or VL of a Fv
  • the present disclosure provides a second halfbody molecule (HB2), wherein said HB2 comprises a Fab specific for FLT3 and either the VH or VL of a Fv specific for CD3, wherein said HB2 is composed of a third and fourth polypeptide, wherein the third polypeptide comprises the light chain of a Fab specific for FLT3 and the fourth polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the Fab, a peptide linker and either the VH or VL of the antibody Fv region specific for CD3.
  • HB2 comprises a Fab specific for FLT3 and either the VH or VL of a Fv specific for CD3
  • the third polypeptide comprises the light chain of a Fab specific for FLT3
  • the fourth polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the Fab, a peptide linker and either the VH or VL of the antibody Fv region specific for CD3.
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 26 and SEQ ID NO: 33 and wherein the fourth polypeptide of HB2 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 110 and SEQ ID NO: 111.
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 26 and SEQ ID NO: 33 and wherein the fourth polypeptide of HB2 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 141.
  • SEQ ID NO: 136 SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 141.
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26 and SEQ ID NO: 33 and wherein the fourth polypeptide of HB2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 110 and SEQ ID NO: 111.
  • HB2 second halfbody molecule
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 26 and SEQ ID NO: 33 and wherein the fourth polypeptide of HB2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140 and SEQ ID NO: 141.
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises the amino acid sequence SEQ ID NO: 26 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 47.
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises the amino acid sequence SEQ ID NO: 26 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 48.
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises the amino acid sequence SEQ ID NO: 26 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 110.
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises the amino acid sequence SEQ ID NO: 26 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 138
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises the amino acid sequence SEQ ID NO: 33 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 49.
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises the amino acid sequence SEQ ID NO: 33 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 50.
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises the amino acid sequence SEQ ID NO: 33 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 111.
  • the present disclosure provides a second halfbody molecule (HB2), wherein the third polypeptide of HB2 comprises the amino acid sequence SEQ ID NO: 33 and the fourth polypeptide of HB2 comprises the amino acid sequence of SEQ ID NO: 139, SEQ ID NO: 140, or SEQ ID NO: 141.
  • CD3 specific antibodies of the present disclosure provides a trispecific antibody with specificity for CD123, FLT3 and CD3 as disclosed herein.
  • the present disclosure provides a trispecific antibody comprising a first antibody fragment specific for CD123, a second antibody fragment specific for FLT3, and a third antibody fragment specific for CD3 as disclosed herein.
  • the present disclosure provides a trispecific antibody comprising a first antibody fragment specific for CD123, a second antibody specific for FLT3, and a third antibody fragment specific for CD3 as disclosed herein, wherein said trispecific antibody is composed of a first halfbody molecule (HB1) and a second halfbody molecule (HB2), wherein HB1 comprises the first antibody fragment specific for CD123 and either the VH or the VL of the third antibody fragment specific for CD3, and wherein HB2 comprises the second antibody fragment specific for FLT3 and either the complementary VH or the complementary VL of the third antibody fragment specific for CD3.
  • HB1 comprises the first antibody fragment specific for CD123 and either the VH or the VL of the third antibody fragment specific for CD3
  • HB2 comprises the second antibody fragment specific for FLT3 and either the complementary VH or the complementary VL of the third antibody fragment specific for CD3.
  • the present disclosure provides a trispecific antibody comprising a first antibody fragment specific for CD123, a second antibody specific for FLT3, and a third antibody fragment specific for CD3 as disclosed herein, wherein said trispecific antibody is composed of a first halfbody molecule (HB1) and a second halfbody molecule (HB2), wherein HB1 comprises the first antibody fragment specific for CD123 and either the VH or the VL of the third antibody fragment specific for CD3, and wherein HB2 comprises the second antibody fragment specific for FLT3 and either the complementary VH or the complementary VL of the third antibody fragment specific for CD3.
  • the present disclosure also pertains to an antibody or antibody fragment specific for CD3.
  • said antibody fragment specific for CD3 is an antibody Fv fragment.
  • said antibody fragment specific for CD3 is the third antibody Fv fragment present in the trispecific antibody according to the present disclosure.
  • the third antibody fragment according to the present disclosure is an antibody Fv fragment.
  • said antibody fragment specific for CD3 is composed or consists of an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL).
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • the antibody or antibody fragment specific for CD3 or the VH or the VL of the Fv specific for CD3 originates from or is any one of the CD3 specific antibodies disclosed in International Application No. PCT/EP2021/076052, which disclosure is incorporated herein in its entirety.
  • the antibody or antibody fragment specific for CD3 or the VH and the VL of the Fv specific for CD3 comprise germline protein sequences of the VH and VL pairs VH3-07/V ⁇ 1-47.
  • the framework regions of the antibody or antibody fragment specific for CD3 or of the VH or the VL of the Fv specific for CD3 according to the present disclosure comprise germline protein sequences of the VH and VL pairs VH3- 07/V ⁇ 1-47.
  • the VH of the antibody fragment specific for CD3 according to the present disclosure is of the VH3 human germline gene family.
  • the framework regions of the VH of the antibody fragment specific for CD3 is encoded by the human VH3-07 human germline gene.
  • the VL of the antibody fragment specific for CD3 ( ⁇ CD3-VL) is of the V ⁇ 1 human germline gene family.
  • the framework regions of the VL of the antibody fragment specific for CD3 are encoded by the V ⁇ 1-47 human germline gene.
  • the VH of the antibody fragment specific for CD3 according to the present disclosure comprises VH3 human germline protein sequences.
  • the framework regions of the VH of the antibody fragment specific for CD3 comprises VH3-07 human germline protein sequences.
  • the VL of the antibody fragment specific for CD3 comprises V ⁇ ⁇ ⁇ human germline protein sequences.
  • the framework regions of the VL of the antibody fragment specific for CD3 comprises V ⁇ 1-47 human germline protein sequences.
  • the VH of the antibody fragment specific for CD3 according to the present disclosure comprises a V or M at position 37 (utilizing the nomenclature according Kabat).
  • the VH of the antibody fragment specific for CD3 according to the present disclosure comprises a M at position 37 (utilizing the nomenclature according Kabat).
  • the VH of the antibody fragment specific for CD3 according to the present disclosure comprises a V at position 37 (utilizing the nomenclature according Kabat).
  • the VH of the antibody fragment specific for CD3 according to the present disclosure comprises an amino acid substitution in framework regions 1, 2, 3 or 4.
  • the ⁇ CD3-VH domain according to the present disclosure comprises not more than one amino acid substitution.
  • said amino acid substitution is a V37M amino acid substitution. (utilizing the nomenclature according Kabat).
  • V at position 37 is replaced for a M (V37M).
  • the M at position 37 inhibits the association of the ⁇ CD123-Fab light chain with the ⁇ CD3-VH present on HB1 during recombinant production of HB1.
  • the M at position 37 inhibits the association of the ⁇ FLT3-Fab light chain with the ⁇ CD3-VH present of HB2 during recombinant production of HB2. In some embodiments, the M at position 37 (utilizing the nomenclature according Kabat) reduces the amount of ⁇ CD123-Fab light chain associated with the ⁇ CD3-VH during recombinant production of HB1. In some embodiments, the M at position 37 (utilizing the nomenclature according Kabat) reduces the amount of ⁇ FLT3-Fab light chain associated with the ⁇ CD3-VH during recombinant production of HB2.
  • said M at position 37 reduces the amount of detectable ⁇ CD123-Fab light chain after production of HB1. In some embodiments, the M at position (utilizing the nomenclature according Kabat) reduces the amount of detectable ⁇ FLT3-Fab light chain after production of HB2. In an embodiment, said reduction is determined under non-reducing conditions. In an embodiment, said reduction is determined by SDS chromatography under non-reducing conditions. In an embodiment, said SDS chromatography is CE-SDS.
  • CE-SDS as used herein refers to capillary electrophoresis sodium dodecyl sulfate.
  • the amount of detectable ⁇ CD123-Fab light chain or ⁇ FLT3-Fab light chain is below 10%, such as 9%, 8%, 7%, %, 5%, 4%, 3%, 2% or 1%. In some embodiments, the amount of detectable ⁇ CD123-Fab light chain or ⁇ FLT3-Fab light chain under non-reducing conditions is reduced by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95%.
  • said reduction is compared to the amount of detectable ⁇ CD123-Fab light chain or ⁇ FLT3-Fab light chain as compared to the amount detected for the corresponding halfbody molecule before said amino acid substitution according to the present disclosure.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB1 and HB2 according to the present disclosure, wherein in presence of cells expressing either CD123 or FLT3 or neither of CD123 and FLT3 on their cell surface, said M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH: a) reduces the dimerization of ⁇ CD3-VH present on HB1 with ⁇ CD3-VL present on HB2 or of ⁇ CD3-VH present on HB-2 with ⁇ CD3-VL present on HB-1, and/or b) reduces the dimerization of HB1 and HB2; and/or c) reduces the formation of the aCD3-Fv; and/or
  • said reduction is compared to the trispecific antibody or the complementary pair of HB1 and HB2 according to the present disclosure before said amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB1 and HB2 comprising a M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH, wherein the IC50 concentration determined for the antibody or pair of HB1 and HB2 induced T cell-mediated killing of cells expressing either CD123 or FLT3 on their cell surface is at least 2-fold, 5-fold, 10- fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, 300- fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold or at least 5000-fold higher as compared to the IC50
  • the present disclosure provides a trispecific antibody or a complementary pair of HB1 and HB2 according to the present disclosure, wherein the M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH does not substantially inhibit the dimerization of ⁇ CD3-VH present on HB1 with ⁇ CD3-VL present on HB2 or of ⁇ CD3-VH present on HB2 with ⁇ CD3-VL present on HB1 in presence of a cells expressing CD123 and FLT3 on their cell surface.
  • the M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH does not substantially inhibit the formation of ⁇ CD3-Fv in presence of a cell expressing CD123 and FLT3 on their cell surface. In some embodiments, the M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH according to the present does not substantially inhibit the binding of the ⁇ CD3-Fv to CD3 in presence of cells expressing CD123 and FLT3 on their cell surface.
  • the M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH does not substantially inhibit the dimerization of HB1 with HB2 in presence of a cells expressing CD123 and FLT3 on their cell surface. In some embodiments, the M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH does not substantially inhibit the formation of the T cell engaging trispecific antibody in presence of cells expressing CD123 and FLT3 on their cell surface.
  • the M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH does not substantially inhibit the activity of the trispecific antibody or of the complementary pair of HB1 and HB2 according to the present disclosure to induce T cell mediated killing of cells expressing CD123 and FLT3 on their cell surface.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB1 and HB2 according to the present disclosure comprising a M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH, wherein the IC50 concentration determined for the antibody induced T cell mediated killing of cells expressing CD123 and FLT3 on their cell surface is within 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 15 fold, or 20 fold of the IC50 concentration determined for the trispecific antibody or complementary pair of HB1 and HB2 before the amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB1 and HB2 according to the present disclosure comprising a M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH, wherein the IC50 concentration determined for the antibody induced T cell mediated killing of cells expressing CD123 and FLT3 on their cell surface is substantially the same as the IC 50 concentration determined for the trispecific antibody or complementary pair of HB1 and HB2 before the amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB1 and HB2 according to the present disclosure comprising a M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH, where in presence of CD123 and FLT3, the KD determined for CD3 binding is within 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 15 fold, or 20 fold of the KD determined for CD3 binding of the trispecific antibody or of the complementary pair of HB1 and HB2 before the amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB1 and HB2 according to the present disclosure comprising a M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH, wherein in presence CD123 and FLT3, the KD determined for CD3 binding is substantially the same as the K D determined for CD3 binding of the trispecific antibody or of the complementary pair of HB1 and HB2 before the amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the VH of the antibody fragment specific for CD3 is present on HB1 and the VL of the antibody fragment specific for CD3 is present on HB2.
  • the VL of the antibody fragment specific for CD3 is present on HB1 and the VH of the antibody fragment specific for CD3 is present on HB2. In an embodiment of the present disclosure, said VH of the antibody fragment specific for CD3 by itself is not able to bind to CD3. In an embodiment, said VL of the antibody fragment specific for CD3 by itself is not able to bind to CD3. In an embodiment of the present disclosure, neither the VH of the Fv specific for CD3 by itself nor the VL of the Fv specific for CD3 by itself is able to bind to CD3. In an embodiment of the present disclosure, the VH and VL of the antibody fragment specific for CD3 are not linked by a covalent bond.
  • the VH of the antibody fragment specific for CD3 present on HB1 and the VL of the antibody specific present on HB2 are not linked by a covalent bond.
  • said VH of the antibody fragment specific for CD3 present on HB2 and said VL of the antibody fragment specific for CD3 present on HB1 are not linked by a covalent bond.
  • the VH and the VL of the antibody fragment specific for CD3 are complementary to each other.
  • the VH and the VL of the antibody fragment specific for CD3 are capable of dimerizing with each other. In an embodiment, said dimerization results in the formation of the Fv specific for CD3.
  • said VH and said VL are capable of dimerizing with each other thus forming the Fv specific for CD3.
  • said VH and said VL of the antibody fragment specific for CD3 is a dimerization domain.
  • said VH and said VL of the antibody fragment specific for CD3 comprises a dimerization interface.
  • said VH and said VL are capable of non-covalently associating with each other.
  • said non-covalent association results in the formation of the Fv specific for CD3.
  • said VH and said VL are capable of non- covalently associating with each other thus forming the Fv specific for CD3.
  • said dimerization and/or said non-covalent association occurs in solution.
  • the third antibody fragment or the Fv specific for CD3 comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 35 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 36, and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 37, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 39.
  • the third antibody fragment or the Fv specific for CD3 comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:124, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 125 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 36, and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 126, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 39.
  • the VH of the antibody fragment specific for CD3 comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 35 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 36.
  • the VH of the antibody fragment specific for CD3 comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 124, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 125 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 36.
  • the VL of the antibody fragment specific for CD3 comprises a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 37, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 39.
  • the VL of the antibody fragment specific for CD3 comprises a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 126, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 39.
  • the third antibody fragment or the Fv specific for CD3 comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 40 and SEQ ID NO: 107 and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41.
  • VH heavy chain variable region
  • VL light chain variable region
  • the third antibody fragment or the Fv specific for CD3 comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 127 or SEQ ID NO: 129 and a light chain variable region (VL) comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 128.
  • VH heavy chain variable region
  • VL light chain variable region
  • the third antibody fragment or the Fv specific for CD3 comprises a heavy chain variable region (VH) comprising a framework amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 40 and SEQ ID NO: 107 and a light chain variable region (VL) comprising a framework amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41.
  • VH heavy chain variable region
  • VL light chain variable region
  • the third antibody fragment or the Fv specific for CD3 comprises a heavy chain variable region (VH) comprising a framework amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 127 or SEQ ID NO: 129 and a light chain variable region (VL) comprising a framework amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 128.
  • VH heavy chain variable region
  • VL light chain variable region
  • the third antibody fragment or the Fv specific for CD3 according to the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 107 and a VL comprising the amino acid sequence of SEQ ID NO: 41.
  • the third antibody fragment or the Fv specific for CD3 according to the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 129 and a VL comprising the amino acid sequence of SEQ ID NO: 128.
  • the VH of the antibody fragment specific for CD3 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 107. In an embodiment of the present disclosure, the VH of the antibody fragment specific for CD3 comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 129.
  • the VH of the antibody fragment specific for CD3 comprises a framework region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 107.
  • the VH of the antibody fragment specific for CD3 comprises a framework region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 127 or SEQ ID NO: 129.
  • the VH of antibody fragment specific for CD3 comprises the amino acid sequence of SEQ ID NO: 40. In an embodiment of the present disclosure, the VH of antibody fragment specific for CD3 comprises the amino acid sequence of SEQ ID NO: 107. In an embodiment of the present disclosure, the VH of antibody fragment specific for CD3 comprises the amino acid sequence of SEQ ID NO: 127. In an embodiment of the present disclosure, the VH of antibody fragment specific for CD3 comprises the amino acid sequence of SEQ ID NO: 129.
  • the VL of the antibody fragment specific for CD3 comprises the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41.
  • the VL of the antibody fragment specific for CD3 comprises a framework region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41.
  • the VL of the antibody fragment specific for CD3 comprises the amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 128.
  • the VL of the antibody fragment specific for CD3 comprises a framework region amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the framework region amino acid sequence of SEQ ID NO: 128.
  • the VL of the antibody fragment specific for CD3 comprises the amino acid sequence of SEQ ID NO: 41.
  • the VL of the antibody fragment specific for CD3 comprises the amino acid sequence of SEQ ID NO: 128.
  • said VH of the antibody or antibody fragment specific for CD3 is ⁇ CD3-VH according to the present disclosure.
  • said VL of the antibody or antibody fragment specific for CD3 is the ⁇ CD3-VL according to the present disclosure.
  • said antibody or antibody fragment specific for CD3 is the third antibody fragment in the trispecific antibody according to the present disclosure.
  • said antibody or antibody fragment specific for CD3 is the ⁇ CD3-Fv according to the present disclosure.
  • the antibody fragment specific for CD3 according to the present disclosure binds to human CD3.
  • said antibody fragment specific for CD3 binds to human CD3 and cynomolgus monkey CD3. In an embodiment, said antibody fragment specific for CD3 binds to human CD3 and cross-reactively binds to cynomolgus CD3. In an embodiment, said binding is specific. In an embodiment, the antibody fragment specific for CD3 according to the present disclosure binds to human cells expressing human CD3. In an embodiment, said antibody fragment specific for CD3 according to the present disclosure binds to cynomolgus monkey cells expressing cynomolgus monkey CD3.
  • said antibody fragment specific for CD3 binds to human cells expressing human CD3 and to cynomolgus monkey cells expressing cynomolgus monkey CD3.
  • said antibody fragment specific for CD3 according to the present disclosure binds to immune cells expressing CD3.
  • said antibody fragment specific for CD3 according to the present disclosure binds to T cells expressing CD3.
  • said antibody fragment specific for CD3 according to the present disclosure binds to cytotoxic T-cells expressing CD3.
  • said CD3 is CD3epsilon.
  • said CD3 is human CD3epsilon.
  • said human CD3epsilon comprises the amino acid sequence of SEQ ID NO: 61 or SEQ ID NO: 62.
  • said CD3 is cynomolgus CD3epsilon.
  • said cynomolgus CD3epsilon comprises the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64.
  • said antibody fragment specific for CD3 binds to a human CD3 epsilon polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 70 and SEQ ID NO: 71.
  • said antibody fragment specific for CD3 according to the present binds to a cynomolgus CD3 epsilon polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 72 and SEQ ID NO: 73.
  • said CD3 is CD3epsilon/CD3delta.
  • said CD3 is composed of CD3epsilon and CD3delta.
  • said CD3 is human CD3epsilon/CD3delta.
  • said human CD3epsilon/CD3delta comprises the amino acid sequence of SEQ ID NO: 100.
  • said CD3 is cynomolgus CD3epsilon/CD3delta.
  • said cynomolgus CD3epsilon/CD3delta comprises the amino acid sequence of SEQ ID NO: 101.
  • said antibody fragment specific for CD3 binds to a human CD3epsilon/CD3delta polypeptide comprising the amino acid sequence of SEQ ID NO: 100.
  • said antibody fragment specific for CD3 according to the present binds to a cynomolgus CD3epsilon/CD3delta polypeptide comprising an amino acid sequence of SEQ ID NO: 101.
  • the antibody fragment specific for CD3 according to the present disclosure binds to the extracellular region of human CD3epsilon.
  • the third antibody fragment or the Fv specific for CD3 according to the present disclosure binds to the extracellular region human and cynomolgus CD3 epsilon.
  • said extracellular region of human CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 62.
  • said extracellular region of cynomolgus CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 64.
  • the antibody fragment specific for CD3 specifically binds to a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 100 and SEQ ID NO: 101.
  • the present disclosure provides a trispecific antibody according to the present disclosure or an antibody fragment specific for CD3 according to the present disclosure, wherein said trispecific antibody or said antibody fragment specific for CD3 has a monovalent affinity for a human CD3epsilon polypeptide comprising the amino acid sequence of SEQ ID NO: 71, with a dissociation rate constant (KD) of about 10 nM or less, such as about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less.
  • KD dissociation rate constant
  • the present disclosure provides a trispecific antibody according to the present disclosure or an antibody fragment specific for CD3 according to the present disclosure, wherein said trispecific antibody or said antibody fragment specific for CD3 has a monovalent affinity for a human CD3epsilon/CD3delta polypeptide comprising the amino acid sequence of SEQ ID NO: 100, with a dissociation rate constant (K D ) of about 30 nM or less, such as about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less.
  • K D dissociation rate constant
  • the present disclosure provides a trispecific antibody according to the present disclosure or an antibody fragment specific for CD3 according to the present disclosure, wherein said trispecific antibody or said antibody fragment specific for CD3 has a monovalent affinity for a cynomolgus monkey CD3epsilon/CD3delta polypeptide comprising the amino acid sequence of SEQ ID NO: 101, with a dissociation rate constant (KD) of about 30 nM or less, such as about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less.
  • KD dissociation rate constant
  • said antibody fragment specific for CD3 according to the present disclosure is the third antibody fragment or the Fv fragment according to the present disclosure.
  • said monovalent affinity or K D is determined for a Fv fragment.
  • the monovalent affinity or K D is determined by an in vitro biolayer interferometry binding assay (BLI) at 25°C.
  • the monovalent affinity or KD is determined by an in vitro bio-layer interferometry binding assay (BLI) as described herein in Example 5.
  • the antibody or antibody fragment specific for CD3 according to the present disclosure is an isolated antibody or antibody fragment.
  • said antibody or antibody fragment specific for CD3 is a human antibody or antibody fragment.
  • said antibody or antibody fragment specific for CD3 is a recombinant antibody or antibody fragment. In an embodiment, said antibody or antibody fragment specific for CD3 is a synthetic antibody or antibody fragment. In an embodiment, said antibody or antibody fragment specific for CD3 is a chimeric antibody or antibody fragment. In an embodiment, said antibody or antibody fragment specific for CD3 is a monoclonal antibody or antibody fragment. Nucleic acids In an embodiment, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for CD123 according to the present disclosure.
  • the present disclosure provides a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the antibodies or antibody fragments specific for CD123 according to the present disclosure as disclosed in Table 5 or Table 6.
  • the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for FLT3 according to the present disclosure.
  • the present disclosure provides a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the antibodies or antibody fragments specific for FLT3 according to the present disclosure as disclosed in Table 7 or Table 8.
  • the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an halfbody molecule according to the present disclosure. In an embodiment, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an halfbody molecule according to the present disclosure as disclosed in Tables 11 – 18.
  • the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for CD123 according to the present disclosure, or an antibody or antibody fragment specific for FLT3 according to the present disclosure, or an HB1 according to the present disclosure, or an HB2 according to the present disclosure, or an trispecific antibody according to the present disclosure.
  • the present disclosure provides a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for CD123 according to the present disclosure, an antibody or antibody fragment specific for FLT3 according to the present disclosure, an HB1 according to the present disclosure, an HB2 according to the present disclosure, or an trispecific antibody according to the present disclosure.
  • the host cell according to the present disclosure is able to express an antibody or antibody fragment specific for CD123 according to the present disclosure, an antibody or antibody fragment specific for FLT3 according to the present disclosure, an HB1 according to the present disclosure, an HB2 according to the present disclosure or an trispecific antibody according to the present disclosure, encoded by the vector composition or the nucleic acid composition according to the present disclosure.
  • the host cell is an isolated host cell.
  • said host cell is a mammalian cell.
  • said mammalian cell is a human cell.
  • the present disclosure refers to a method of producing an antibody or antibody fragment specific for CD123 according to the present disclosure, or an antibody or antibody fragment specific for FLT3 according to the present disclosure, or an HB1 according to the present disclosure, or an HB2 according to the present disclosure, or an trispecific antibody according to the present disclosure.
  • a method of producing an antibody or antibody fragment specific for CD123 according to the present disclosure, or an antibody or antibody fragment specific for FLT3 according to the present disclosure, or an HB1 or an HB2 according to the present disclosure, or an trispecific antibody according to the present disclosure comprises culturing a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for CD123, an antibody or antibody fragment specific for FLT3, or an HB1, or an HB2, or an trispecific antibody according to the present disclosure, under conditions suitable for expression of the antibody or antibody fragment specific for CD123 according, or the antibody or antibody fragment specific for FLT3, or HB1 or HB2, or the trispecific antibody according to the present disclosure, and isolating the same from the host cell or host cell culture medium.
  • the present disclosure provides a method for inducing lysis of a cancer cell expressing CD123 and FLT3, comprising contacting said cancer cell in the presence of a cytotoxic T cell with a complementary pair of HB1 and HB2 or with the trispecific antibody, according to the present disclosure.
  • the present disclosure provides a method for inhibition of proliferation of a cancer cell expressing CD123 and FLT3, comprising contacting said cancer cell in the presence of a cytotoxic T cell with a complementary pair of HB1 and HB2 or with the trispecific antibody, according to the present disclosure.
  • the present disclosure provides a method for killing a cancer cell expressing CD123 and FLT3, comprising contacting said cancer cell, in the presence of a cytotoxic T cell, with a complementary pair of HB1 and HB2 or with the trispecific antibody, according to the present disclosure.
  • the present disclosure provides a method for killing a cancer cell expressing AG1 and AG2 on its cell surface, comprising contacting said cancer cell, in the presence of a cytotoxic T cell, with a complementary pair of HB1 and HB2 or with the trispecific antibody, according to the present disclosure.
  • the present disclosure provides a method for inducing T cell proliferation in the presence of a cancer cell expressing CD123 and FLT3, comprising contacting said cancer cell, in the presence of a T-cell, with a complementary pair of HB1 and HB2, or with the trispecific antibody according to the present disclosure.
  • the present disclosure provides a method for stimulating a primary T cell response in the presence of cancer cells expressing CD123 and FLT3, comprising contacting said cancer cells in the presence of said T cell with a complementary pair of HB1 and HB2 or with the trispecific antibody, according to the present disclosure.
  • the present disclosure provides a method for re-directing cytotoxic activity of a T cell to a cancer cell expressing CD123 and FLT3, comprising contacting said cancer cell, in the presence of said T cell, with a complementary pair of HB1 and HB2 or with the trispecific antibody, according to the present disclosure.
  • the present disclosure provides a method for re-directing cytotoxic activity of a T cell to a cancer cell expressing AG1 and AG2, comprising contacting said cancer cell, in the presence of said T cell, with a complementary pair of HB1 and HB2 or with the trispecific antibody, according to the present disclosure.
  • the present disclosure provides a method for the treatment of cancer, preferably a hematological cancer, more preferably a leukemia, more preferably AML.
  • the trispecific antibody or the complementary pair of HB1 and HB2, according to the present disclosure may be used in therapeutic methods.
  • the present disclosure provides a complementary pair HB1 and HB2 according to the present disclosure or the trispecific antibody according to the present disclosure for the treatment of a disease.
  • the present disclosure provides a complementary pair HB1 and HB2 or the trispecific antibody, according to the present disclosure, for use in the treatment of a disease.
  • the present disclosure provides a complementary pair of HB1 and HB2 according or the trispecific antibody, according to the present disclosure, for use in the treatment of a disease in an subject in need thereof.
  • the present disclosure provides the use of a complementary pair of HB1 and HB2 or of the trispecific antibody, according to the present disclosure, for the manufacture of a medicament.
  • the present disclosure provides a complementary pair of HB1 and HB2 or the trispecific antibody, according to the present disclosure, for use as a medicament.
  • the present disclosure provides a complementary pair of HB1 and HB2, or the trispecific antibody according to the present disclosure, for use as a medicament for the treatment of a disease in an subject in need thereof.
  • the complementary pair of HB1 and HB2 or the trispecific antibody according to the present disclosure are for use in the treatment of a patient who is suffering from a cancer or for use in diagnosis in a patient who is suffering from a cancer.
  • the complementary pair of HB1 and HB2 or the trispecific antibody, according to the present disclosure may be used in the prevention and/or treatment of diseases, which are mediated by biological pathways in which CD123 and FLT3 are involved.
  • the present disclosure provides the use of a complementary pair of HB1 and HB2 or the trispecific antibody, according to the present disclosure, for the treatment of cancer, comprising cancer cells expressing CD123 and FLT3, in a subject, comprising: (a) selecting a subject who is afflicted with a cancer, (b) collecting one or more biological samples from the subject, (c) identifying the CD123 and FLT3 expressing cancer cells in the one or more samples; and (d) administering to the subject an effective amount of a complementary pair of HB1 and HB2 according to the present disclosure or the trispecific antibody according to the present disclosure.
  • said disease is associated with the undesired presence of CD123.
  • said disease is associated with the undesired presence of FLT3. In an embodiment, said disease is associated with the undesired presence of CD123 and FLT3.
  • the present disclosure provides a complementary pair of HB1 and HB2 or of the trispecific antibody, according to the present disclosure, for use in a method of treating a subject having a disease, comprising administering to the subject a therapeutically effective amount of HB1 and HB2 according to the present disclosure. In an embodiment, said method or use according further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent.
  • the subject in need of treatment is typically a mammal, more specifically a human.
  • HB1 and HB2 or the trispecific antibody would be formulated, dosed, and administered in a way consistent with good medical practice.
  • the complementary pair of HB1 and HB2 according to the present disclosure are administered separately.
  • said HB1 and HB2 are administered consecutively.
  • HB1 and HB2 are administered one after the other.
  • the present disclosure provides a method for induction of tumor regression in a patient who has cancer, comprising administering to said subject, a therapeutically effective amount of a complementary pair of HB1 and HB2 or of the trispecific antibody according to the present disclosure.
  • the present disclosure provides a method for improving survival of a subject who has cancer, comprising administering to said subject, a therapeutically effective amount of HB1 and HB2 or of the trispecific antibody, according to the present disclosure.
  • the present disclosure provides a method for eliciting, stimulating or inducing an immune response in a subject who has cancer, comprising administering to said subject, a therapeutically effective amount of HB1 and HB2 or of the trispecific antibody according to the present disclosure.
  • the present disclosure provides a method for enhancing or inducing anti-cancer immunity in a subject who has cancer, comprising administering to said subject, a therapeutically effective amount of HB1 and HB2 or of the trispecific antibody, according to the present disclosure.
  • the disease to be treated is a proliferative disease.
  • the disease is a cancer or a tumor.
  • said disease is cancer.
  • said cancer is a cancer expressing FLT3.
  • said cancer is a cancer expressing CD123.
  • said cancer is a cancer expressing FLT3 and CD123.
  • said cancer is a hematologic cancer.
  • said cancer is a leukemia.
  • said cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and hairy cell leukemia (HCL).
  • the present disclosure provides a pharmaceutical composition comprising the trispecific antibody according to the present disclosure, and a pharmaceutically acceptable carrier.
  • the present disclosure provides a pharmaceutical composition comprising HB1 and HB2 according to the present disclosure, said pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • HB1 and HB2 may be formulated or comprised in a separate pharmaceutical composition.
  • the present disclosure provides a pharmaceutical composition comprising an halfbody molecule according to the present disclosure and at least one pharmaceutically acceptable carrier.
  • the present disclosure provides a first pharmaceutical composition comprising HB1 according to the present disclosure and at least one pharmaceutically acceptable carrier.
  • the present disclosure provides a second pharmaceutical composition comprising HB2 according to the present disclosure and at least one pharmaceutically acceptable carrier.
  • the pharmaceutical compositions may further comprise at least one other pharmaceutically active compound according to the present disclosure.
  • the pharmaceutical compositions according to the present disclosure can be used in the diagnosis, prevention and/or treatment of diseases associated with the presence of CD123 and/or FLT3.
  • the present disclosure provides (a) a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2, according to the present disclosure, or (b) a pharmaceutical composition comprising the trispecific antibody according to the present disclosure, suited for prophylactic, therapeutic and/or diagnostic use in a mammal, more particular in a human.
  • the present disclosure provides (a) a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2, according to the present disclosure, or (b) a pharmaceutical composition comprising the trispecific antibody according to the present disclosure, for use in the prevention and/or treatment of a disease associated with the undesired presence of CD123 and FLT3.
  • the present disclosure provides (a) a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2, according to the present disclosure, or (b) a pharmaceutical composition comprising the trispecific antibody according to the present disclosure, for the use as a medicament.
  • the present disclosure provides (a) a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2, according to the present disclosure, or (b) a pharmaceutical composition comprising the trispecific antibody according to the present disclosure for use in the prevention and/or treatment of a disease.
  • the present disclosure provides a method for the treatment of a disease in a subject in need thereof using (a) a pharmaceutical composition comprising HB1 and a pharmaceutical composition comprising HB2 according to the present disclosure or (b) a pharmaceutical composition comprising the trispecific antibody according to the present disclosure.
  • the disease is a proliferative disease.
  • the disease is a cancer or a tumor.
  • said disease is cancer.
  • said cancer is a cancer expressing FLT3. In an embodiment, said cancer is a cancer expressing CD123. In an embodiment, said cancer is a cancer expressing FLT3 and CD123. In an embodiment, said cancer is a hematologic cancer. In an embodiment, said cancer is a leukemia. In an embodiment, said cancer is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and hairy cell leukemia (HCL).
  • Pharmaceutical compositions according to the present disclosure may comprise a therapeutically effective amount of (a) HB1 and HB2 according to the present disclosure or (b) the trispecific antibody according to the present disclosure, dissolved in a pharmaceutically acceptable carrier.
  • the present disclosure provides a kit comprising HB1 and HB2 according to the present disclosure or the trispecific antibody according to the present disclosure.
  • the present disclosure provides a kit comprising a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2, according to the present disclosure.
  • the present disclosure provides a kit comprising the trispecific antibody or a pharmaceutical composition comprising the trispecific antibody, according to the present disclosure.
  • the appropriate dosage of HB1 and HB2 or of the trispecific antibody according to the present disclosure needs to ensure effective amounts for the purpose intended and will depend on the type of disease to be treated, the route of administration, the body weight of the subject, the severity and course of the disease, whether the HB1 and HB2 or the trispecific antibody are administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the subject's clinical history and response to the pair of complementary halfbody molecules, and the discretion of the attending physician.
  • Effective dosages and schedules for administering pharmaceutical compositions comprising HB1 and HB2 or the trispecific antibody according to the present disclosure may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly.
  • the present disclosure provides HB1 and HB2 according the present disclosure or the trispecific antibody according to the present disclosure, wherein said HB1 and HB2 or said trispecific antibody are administered at a dose sufficient to achieve a therapeutically effective serum level.
  • the administration of HB1 and HB2 according to the present disclosure encompass separate administration, in which case, administration of HB1 occurs prior to, simultaneously, and/or following, administration of the HB2 or vice versa.
  • HB1 and HB2 or the trispecific antibody according to the present disclosure are administered intravenously.
  • HB1 and HB2 are administered subcutaneously.
  • the trispecific antibody is administered subcutaneously.
  • Combination Therapies HB1 and HB2 according to the present disclosure or the trispecific antibody according to the present disclosure may be administered in combination with one or more other therapeutic agents.
  • “Therapeutic agent” encompasses any agent administered to treat a symptom or disease in a subject in need of such treatment.
  • an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers.
  • Such other therapeutic agents are suitably present in combination in amounts that are effective for the purpose intended.
  • Combination therapies encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of HB1 and HB2 according to the present disclosure or of the trispecific antibody according to the present disclosure can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent.
  • HB1 and HB2 according to the present disclosure or the trispecific antibody according to the present disclosure can also be used in combination with radiation therapy.
  • CyCAT halfbody molecules with improved production and heterodimerization properties An observation that may be made during recombinant production of halfbody molecules comprising a Fab targeting moiety and an single ⁇ CD3-VH domain is Fab light-chain mispairing, wherein during recombinant production the Fab light chain (or more specifically, the VL of the Fab light chain) pairs with the unpaired ⁇ CD3-VH resulting in a newly formed but inactive antibody Fv fragment, which is inactive in respect of target and CD3 binding.
  • Such produced halfbody composition may be strongly heterogeneous and less functional when used in CyCAT directed therapies and thus can therefore pose significant challenges for clinical development.
  • complementary halfbody molecules Another observation which may occur during or after administration of complementary halfbody molecules to a patient is residual dimerization or complex formation of complementary halfbodies even in the absence of target cells expressing both target antigens of interest on their cell surface.
  • Such “unwanted heterodimerization” of halfbody molecules may result in on-target as well as off-target T-cell activation caused by the target independent complementation of the ⁇ CD3-Fv fragment.
  • Target independent heterodimerization as well as light-chain mispairing are likely caused by the general intrinsic low affinity binding of VH/VL domains via hydrophobic interactions of amino acid residues forming the antibody VH/VL interface.
  • VH/VL interface amino acid residues generally tend to be close to the CDRs or make interactions with CDR residues, and any modification of such residues may not only impact VH/VL interactions, but also might impact antigen binding interactions by being close to the antigen-antibody interface.
  • the present invention utilizes single amino acid substitutions (“Interface Mutation”) in the antibody VH/VL interface region (i.e. in any of the four VH framework regions) of the ⁇ CD3-VH domain according to the present disclosure.
  • Interface Mutation as used in the context of the present disclosure is selected in such way that it weakens the interface affinity between any VL domain (i.e.
  • ⁇ CD123 Fab-VL, ⁇ FLT3-Fab-VL, or ⁇ CD3-VL domain Fab-VL, ⁇ FLT3-Fab-VL, or ⁇ CD3-VL domain
  • ⁇ CD3-VHmut mutated ⁇ CD3-VH domain
  • the weakened interface affinity between the mutated ⁇ CD3-VH and ⁇ CD3-VL domain remains strong enough to enable on-cell reconstitution of the functional ⁇ CD3-Fv fragment.
  • an “Interface Mutation” as used in the context of the present disclosure may comprise any amino acid substitution at any position in the VH framework regions of the ⁇ CD3-VH domain that is sufficient to provide the reduced antibody VH/VL interface affinity as described herein.
  • appropriate Interface Mutations were identified through amino acid scanning by sequentially substituting each amino acid residue present at positions forming the VH/VL interface region of the ⁇ CD3-VH with the natural occurring amino acid residues except for cysteine.
  • Halfbody molecules utilizing single amino acid substituted ⁇ CD3-VH domains are then produced and functionally tested as described in the present examples.
  • the present disclosure provides a halfbody molecule (HB) comprising a Fab fragment (Fab) specific for an antigen (AG1) and the VH ( ⁇ CD3- VH) but not the VL ( ⁇ CD3-VL) of a Fv fragment specific for CD3 ( ⁇ CD3-Fv), wherein the ⁇ CD3-VH domain of said halfbody molecule comprises an amino acid substitution in the framework regions, wherein said amino acid substitution i) inhibits the association of the Fab light chain with the ⁇ CD3-VH present on HB during recombinant production of said HB, and/or ii) reduces the amount of Fab light chain associated with the ⁇ CD3-VH during recombinant production of said HB, and/or iii) reduces the amount of detectable Fab light chain after production of said HB.
  • Fab Fab fragment
  • AG1 antigen
  • VH ⁇ CD3- VH
  • VL ⁇ CD3-VL
  • said reduction is determined by SDS chromatography under non- reducing conditions.
  • said SDS chromatography is CE-SDS.
  • the amount of detectable Fab light chain is below 10%, such as 9%, 8%, 7%, %, 5%, 4%, 3%, 2% or 1%.
  • the amount of detectable Fab light chain under non-reducing conditions is reduced by more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% as compared to the amount of detectable Fab light chain determined before said amino acid substitution in ⁇ CD3-VH of the present disclosure.
  • the present disclosure provides a method for reducing the amount of associated Fab light chain with the ⁇ CD3-VH present of a halfbody during recombinant production of said halfbody, which method comprises the step of substituting one amino acid in the framework regions of the ⁇ CD3-VH with a natural occurring amino acid except cysteine.
  • the present disclosure provides a method for reducing the amount of Fab light chain detectable after recombinant production of the halfbody, which method comprises the step of substituting one amino acid in the framework regions of the ⁇ CD3-VH with a natural occurring amino acid except cysteine.
  • the present disclosure provides a method for producing a halfbody according to the present disclosure, said method comprises the steps of substituting one amino acid in the framework regions of the ⁇ CD3-VH present of said halfbody with a natural occurring amino acid except cysteine and producing said halfbody molecule comprising said amino acid substitution, wherein said amino acid substitution reduces the amount of associated Fab light chain with the ⁇ CD3-VH present of that halfbody.
  • said method further comprises the step of analyzing the amount of detectable Fab light chain after production of said halfbody.
  • said method further comprises the step of (i) producing the halfbody body molecule before said amino acid substitution, (ii) analyzing the amount of detectable Fab light chain and (iii) comparing the amount of detectable Fab light chain before and after the amino acid substitution in the ⁇ CD3-VH.
  • said Fab light chain is detected or said amount of Fab light chain is determined by CE-SDS under non-reducing conditions.
  • said reduction is determined by SDS chromatography under non- reducing conditions.
  • said SDS chromatography is CE-SDS.
  • the amount of detectable Fab light chain under reducing conditions is below 10%, such as 9%, 8%, 7%, %, 5%, 4%, 3%, 2% or 1%.
  • said halfbody is produced as a pharmaceutical composition.
  • said halfbody comprises a Fab fragment (Fab) specific for an antigen (AG1) composed of a Fab light and heavy chain and the VH ( ⁇ CD3-VH) but not the VL ( ⁇ CD3-VL) of a Fv fragment specific for CD3 ( ⁇ CD3-Fv).
  • the present disclosure provides a pair of complementary halfbody molecules or a trispecific antibody composed of said complementary pair of halfbody molecules, wherein a) the first halfbody molecule (HB1) comprises i. a first antibody fragment (Fab1) specific for a first antigen (AG1) ii. either a first VH ( ⁇ CD3-VH1) or first VL ( ⁇ CD3-VL1) of a Fv fragment specific for CD3 ( ⁇ CD3-Fv), and b) the second halfbody molecule (HB2) comprising i. a second antibody fragment (Fab2) specific for a second antigen (AG2), ii.
  • a first halfbody molecule comprises i. a first antibody fragment (Fab1) specific for a first antigen (AG1) ii. either a first VH ( ⁇ CD3-VH1) or first VL ( ⁇ CD3-VL1) of a Fv fragment specific for CD3 ( ⁇ CD3-Fv), and b) the second halfbody molecule (HB2) comprising
  • ⁇ CD3-VH2 complementary VL ( ⁇ CD3- VL2) of the ⁇ CD3-Fv, wherein HB1 and HB2 are not linked via a covalent bond
  • ⁇ CD3-VH1 or ⁇ CD3-VH2 comprises an amino acid substitution in the framework regions with a natural occurring amino acid except cysteine, and wherein in presence of either AG1 or AG2 or in absence of AG1 and AG2, the presence of said an amino acid substitution, i. inhibits the dimerization of ⁇ CD3-VH1 with ⁇ CD3-VL2 or of ⁇ CD3-VL1 with ⁇ CD3-VH2, respectively, and/or ii.
  • said inhibition is compared to the trispecific antibody or the complementary pair of HB1 and HB2 according to the present disclosure before said amino acid substitution.
  • the present disclosure provides a complementary pair of halfbody molecules or a trispecific antibody composed of said complementary pair of halfbody molecules, wherein in presence of AG1 and AG2, said amino acid substitution in ⁇ CD3- VH1 or ⁇ CD3-VH2 i. does not substantially inhibit the dimerization of ⁇ CD3-VH1 with ⁇ CD3-VL2 or of ⁇ CD3-VL1 with ⁇ CD3-VH2, ii. does not substantially inhibit the dimerization of HB1 with HB2, iii. does not substantially inhibit the formation of the ⁇ CD3-Fv, iv. does not substantially inhibit the binding of the ⁇ CD3-Fv to CD3, v.
  • T-cell engaging trispecific antibody molecule does not substantially inhibit the formation of the T-cell engaging trispecific antibody molecule, and/or vi. does not substantially inhibit the activity of the trispecific antibody molecule to mediate T-cell redirected killing of cells having AG1 and AG2 on their cell surface.
  • said inhibition is compared to trispecific antibody or the complementary pair of HB1 and HB2 before said amino acid substitution.
  • the present disclosure provides a complementary pair of halfbody molecules or a trispecific antibody composed of said complementary pair of halfbody molecules, wherein after said amino acid substitution in the ⁇ CD3-VH1 or ⁇ CD3-VH2, the IC 50 concentration determined for the T cell-mediated killing of cells having either AG1 or AG2 on their cell surface is at least 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 75 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, or at least 1000 fold higher compared to that IC 50 concentration determined for the complementary pair of HB1 and HB2 or of the trispecific antibody composed of said complementary pair of halfbody molecules before said amino acid substitution.
  • the present disclosure provides a complementary pair of halfbody molecules or a trispecific antibody composed of said complementary pair of halfbody molecules, wherein after said amino acid substitution in the ⁇ CD3-VH1 or ⁇ CD3-VH2, the IC50 concentration determined for the T cell-mediated killing of cells having AG1 and AG2 on their cell surface is within 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 15 fold, or 20 fold of that IC50 concentration determined for the complementary pair of halfbody molecules or the trispecific antibody before said amino acid substitution.
  • the present disclosure provides a complementary pair of halfbody molecules or a trispecific antibody composed of said complementary pair of halfbody molecules, wherein after said amino acid substitution in the ⁇ CD3-VH1 or ⁇ CD3-VH2, and wherein in presence of AG1 and AG2 the CD3 binding affinity determined for the ⁇ CD3-Fv is within 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 15 fold, or 20 fold of the binding affinity determined for the ⁇ CD3-Fv before said amino acid substitution.
  • the CD3 binding affinity determined for the ⁇ CD3-Fv in presence of AG1 and AG2 is substantially the same as compared to the CD3 binding affinity determined for the ⁇ CD3-Fv before said amino acid substitution.
  • the present disclosure pertains to a method for improving the therapeutic window for a complementary pair of halfbody molecules according to the present disclosure, said method comprises substituting one amino acid in the framework regions of the aCD3-VH1 or aCD3-VH2, respectively, with a natural occurring amino acid except cysteine, wherein the amino acid substitution, increases the ratio of the IC50 (AG1 or AG2) / IC50 (AG1 and AG2) as compared to the IC50 (AG1 or AG2) / IC50 (AG1 and AG2) before said amino acid substitution, wherein said ratio of the IC 50 (AG1 or AG2) / IC50 (AG1 and AG2) is defined as the ratio of the IC 50 concentration determined for the antibody or halfbody pair induced T-cell mediated killing of cells expressing either AG1 or AG2 on their cell surface and the IC50 concentration determined for the antibody or halfbody pair induced T-cell mediated killing of cells expressing AG1 and AG2 on their cell surface, or wherein the amino acid substitution increases the
  • said increase in the ratio of IC50 (AG1 or AG2) / IC50 (AG1 and AG2) is at least 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 75 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, or at least 1000 fold higher.
  • said increase in the IC50 (AG1 or AG2) is at least 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 75 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, or at least 1000 fold higher.
  • said retain of the IC50 is within 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5.5 fold, 6 fold, 6.5 fold, 7 fold, 7.5 fold, 8 fold, 8.5 fold, 9 fold, 9.5 fold, 10 fold, 15 fold, or 20 fold.
  • said antibody or halfbody pair induced T-cell mediated killing is measured in an in vitro T cell-mediated tumor cell killing assay.
  • said T cell-mediated cell killing is measured in presence of cytotoxic T cells.
  • said T cell-mediated cell killing is measured in presence PBMCs.
  • said T cell-mediated cell killing is measured in presence of CD8+ and/CD4+ T cells.
  • said T cells or PBMCs are human cells.
  • said T cell-mediated cell killing is measured in an in vitro T cell-mediated tumor cell killing assay as described in Example 3.
  • said ⁇ CD3-VH, ⁇ CD3-VH1, or ⁇ CD3-VH2 comprises not more than one amino acid substitution.
  • said ⁇ CD3-VH, ⁇ CD3-VH1, ⁇ CD3-VH2, ⁇ CD3-VL, ⁇ CD3-VL1, or ⁇ CD3-VL2 originates from, is a derivative of, or is any one of the CD3 specific antibodies selected from the group of: muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34 or any humanized variants of SP34, I2C, H2C, TR-66 or X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII- 87, 12F6, T3/RW2- 8C8, T3/RW2-4B6, OKT3D,
  • the ⁇ CD3-VH, ⁇ CD3-VH1, ⁇ CD3-VH2, ⁇ CD3-VL, ⁇ CD3-VL1, or ⁇ CD3-VL2 originates from or is any one of the variable domains of the CD3 specific antibodies disclosed in International Application No. PCT/EP2021/076052, which disclosure is incorporated herein in its entirety.
  • the ⁇ CD3-VH, ⁇ CD3-VH1, ⁇ CD3-VH2, ⁇ CD3-VL, ⁇ CD3-VL1, or ⁇ CD3-VL2 comprise any one of the amino acid sequences shown in Tables 9 and Table 40 of the present specification.
  • the framework regions of ⁇ CD3-VH, ⁇ CD3-VH1, ⁇ CD3-VH2, ⁇ CD3-VL, ⁇ CD3-VL1, or ⁇ CD3-VL2 comprise germline protein sequences or human consensus. framework regions.
  • the framework regions of ⁇ CD3-VH, ⁇ CD3-VH1, ⁇ CD3-VH2, ⁇ CD3-VL, ⁇ CD3-VL1, or ⁇ CD3-VL2 according to the present disclosure comprise germline protein sequences of the VH and VL pairs as disclosed in International Application No. PCT/EP2011/070473; which disclosure is incorporated herein in its entirety.
  • germline amino acid sequence means the amino acid sequence of a variable region of antibody or functional fragment thereof encoded by a germline gene
  • gene sequence or “germline gene” means the nucleic acid sequence of a germline gene encoding a variable region of an antibody or functional fragment thereof.
  • Antibody germline amino acid sequences encoded by the germline genes or germline gene sequences are for example disclosed in the following publications: for VH: Tomlinson et al., (1992), J. Mol. Biol. 227, 776-798; Matsuda et al. (1998), J Exp Med 188(11):2151-62; and LeFranc MP (2001), Exp Clin Immunogenet. 18(2):100-16.
  • the ⁇ CD3-VH according to the present disclosure is of the VH1, VH3 or VH5 human germline gene family.
  • the ⁇ CD3-VH comprises VH1, VH3 or VH5 human germline protein sequences.
  • the framework regions of the ⁇ CD3-VH are encoded by the human VH1-18, VH1-46, VH1-69, VH3-07, VH3-15, VH3-21, VH3-23, VH3-53, VH3-74, or VH5-51 human germline gene.
  • the framework regions of the ⁇ CD3-VH comprises VH1-18, VH1-46, VH1-69, VH3-07, VH3- 15, VH3-21, VH3-23, VH3-53, VH3-74, or VH5-51 human germline protein sequences.
  • the ⁇ CD3-VL according to the present disclosure is of the V ⁇ ⁇ , V ⁇ 2 or V ⁇ 3 human germline gene family.
  • the ⁇ CD3-VL comprises V ⁇ ⁇ , V ⁇ 2 or V ⁇ 3 human germline protein sequences.
  • the framework regions of the ⁇ CD3-VL are encoded by the V ⁇ 1-40, V ⁇ 1-47, V ⁇ 1-51, V ⁇ 2-11, V ⁇ 2-23, or V ⁇ 3-01 human germline gene.
  • the framework regions of the ⁇ CD3-VL comprises V ⁇ 1-40, V ⁇ 1-47, V ⁇ 1-51, V ⁇ 2-11, V ⁇ 2-23, or V ⁇ 3-01 human germline protein sequences.
  • the amino acid substitution in the ⁇ CD3-VH according to the present disclosure is located within any one of antibody framework regions 1, 2, 3 or 4 of the ⁇ CD3-VH. In some embodiments, said amino acid substitution is located in framework region 1 of the ⁇ CD3-VH. In some embodiments, said amino acid substitution is located in framework region 2 of the ⁇ CD3-VH. In some embodiments, said amino acid substitution is located in framework region 3 of the ⁇ CD3-VH. In some embodiments, said amino acid substitution is located in framework region 4 of the ⁇ CD3-VH. Accordingly, in some embodiments, the amino acid substitution according to the present disclosure is the interface region of the ⁇ CD3-VH.
  • the “interface” or interface region” of the ⁇ CD3-VH domain disclosed herein includes those contact amino acid residues in the ⁇ CD3-VH domain which interact or would contact one or more contact amino acid residues in the “interface” or “interface region” of a complementary VL domain.
  • the amino acid substitution is at position 35, 37, 39, 44, 45, 47, 91, 93, 95, 100, or 103 of the ⁇ CD3-VH (utilizing the nomenclature according Kabat).
  • said amino acid substitution is at position 37, 44 or 45 of the ⁇ CD3-VH (utilizing the nomenclature according Kabat).
  • said amino acid substitution is at position V37, G44, or L45 (utilizing the nomenclature according Kabat). In an embodiment, said amino acid substitution is a V37M, V37L, G44A, L45I or L45F amino acid substitution (utilizing the nomenclature according Kabat). In some embodiments, in the ⁇ CD3-VH of an halfbody molecule according to the present disclosure, V at position 37 is replaced with a M (V37M) or L (V37L), G at position 44 is replaced with A (G44A) or L at position 45 is replaced with F (L45F) or I (L45I) (utilizing the nomenclature according Kabat).
  • the ⁇ CD3-VH according to the present disclosure comprises a V, M or L at position 37 (utilizing the nomenclature according Kabat). In some embodiments, the ⁇ CD3-VH according to the present disclosure comprises a G or A at position 44 (utilizing the nomenclature according Kabat). In some embodiments, the ⁇ CD3-VH according to the present disclosure comprises a L, F, or I at position 45 (utilizing the nomenclature according Kabat). In some embodiments, the ⁇ CD3-VH according to the present disclosure comprises the amino acid sequence of SEQ ID NO: 107 or 129. In some embodiments, the ⁇ CD3-VL according to the present disclosure comprises the amino acid sequence of SEQ ID NO: 41 or 128.
  • HB1 and HB2 are capable of forming a heterodimer with each other, wherein said formation of a heterodimer of HB1 and HB2 occurs via dimerization of ⁇ CD3-VH1 with ⁇ CD3-VL2 or of ⁇ CD3-VL1 with ⁇ CD3-VH2.
  • said formation of a heterodimer occurs on the surface of a cell expressing AG1 and AG2 on its cell surface.
  • said dimerization of ⁇ CD3-VH1 with ⁇ CD3-VL2 or of ⁇ CD3-VL1 with ⁇ CD3-VH2 results in the formation of the ⁇ CD3-Fv.
  • said heterodimerization of HB1 and HB2 results in the formation of the T-cell engaging trispecific antibody.
  • HB1, HB2, ⁇ CD3-VH1, ⁇ CD3-VH2, ⁇ CD3- VL1 and/or ⁇ CD3-VL2 by themselves are not capable of binding to CD3.
  • AG1 and AG2 are different antigens.
  • AG1 and AG2 are present or expressed on the surface of the same cell.
  • the combination of AG1 and AG2 is present or expressed on the surface of the same cell.
  • said cell is a cancer cell or a tumor cell.
  • AG1 and/or AG2 is a cancer associated antigen.
  • Fab1 and ⁇ CD3-VH1 or Fab1 and ⁇ CD3-VL1 are linked via a peptide linker.
  • Fab2 and ⁇ CD3-VH2 or Fab2 and ⁇ CD3-VL2 are linked via a peptide linker.
  • said peptide linker is any one of the peptide linkers disclosed in the present specification.
  • Table 4 Amino acid sequences of recombinant human and cynomolgus CD3epsilon and human and cynomolgus CD3 epsilon/delta extracellular domain antigens.
  • Antibody Sequences Table 7: Antibody sequences of the FLT3 specific antibody ⁇ FLT3-Fab1
  • Table 9 Antibody sequences of the human CD3 specific antibody ⁇ CD3-Fv used in the CyCAT halfbody molecules in the present examples.
  • Table 10 Sequences of the first or second peptide linker used in the CyCAT halfbody molecules according to the present disclosure fusing the ⁇ CD123-Fab or the ⁇ FLT3-Fab to the ⁇ CD3-VH or ⁇ CD3-VL domains.
  • CyCAT Halfbody Sequences Table 11 Sequences of the two polypeptides forming the ⁇ CD123 halfbody molecule ⁇ CD123- HB1 according to the present disclosure comprising the Fab portion of ⁇ CD123-Fab1 fused to the VH of the CD3 specific antibody ⁇ CD3-Fv.
  • Table 12 Sequences of the two polypeptides forming the ⁇ CD123 halfbody molecule ⁇ CD123- HB2 comprising the Fab portion of the CD123 specific antibody ⁇ CD123-Fab1 fused to the VL domain of the CD3 specific antibody ⁇ CD3-Fv.
  • Table 13 Sequences of the two polypeptides forming the CyCAT halfbody molecule ⁇ CD123- HB3 comprising the Fab portion of the CD123 specific antibody ⁇ CD123-Fab2 fused to the VL domain of the CD3 specific antibody ⁇ CD3-Fv.
  • Table 14 Sequences of the two polypeptides forming the CyCAT halfbody molecule ⁇ CD123- HB4 comprising the Fab portion of the CD123 specific antibody ⁇ CD123-Fab2 fused to the VH domain of the CD3 specific antibody ⁇ CD3-Fv.
  • Table 15 Sequences of the two polypeptides forming the CyCAT halfbody molecule ⁇ FLT3-HB1 comprising the Fab portion of the FLT3 specific antibody ⁇ FLT3-Fab1 fused to the VH
  • Table 16 Sequences of the two polypeptides forming the CyCAT halfbody molecule ⁇ FLT3-HB2 comprising the Fab portion of the FLT3 specific antibody ⁇ FLT3-Fab1 fused to the VL
  • Table 17 Sequences of the two polypeptides forming the halfbody molecule or CyCAT halfbody ⁇ FLT3-HB3 molecule according to the present disclosure comprising the Fab portion of the FLT3 specific antibody ⁇ FLT3-Fab2 fused to the VL domain of the CD specific antibody ⁇ CD3-Fv.
  • Table 18 Sequences of the two polypeptides forming the CyCAT halfbody molecule ⁇ FLT3-HB4 according to the present disclosure comprising the Fab portion of the FLT3 specific WORKING EXAMPLES
  • Example 1 Identification of fully human ⁇ CD123 and fully human ⁇ FLT3 specific antibodies from the MorphoSys Ylanthia ® library For antibody generation, the MorphoSys Ylanthia® library was used to select Fab fragments with specificity for human CD123 and human FLT3, respectively.
  • the MorphoSys Ylanthia® library (Tiller et al. mAbs 5:3, 1–26; May/June (2013) and U.S. Patent No.
  • each panning strategy comprised at least 3 individual rounds of panning against the respective antigens including soluble human and cynomolgus monkey CD123; and/or using cells with endogenous or recombinant expression of human CD123. Thereafter several thousand clones were rescreened for binding to human and cynomolgus CD123 as well as to the counter target CD116 (GM-CSF receptor). Antibody clones of interest were subjected for VH/VL sequencing.
  • phage derived from maturation libraries were subjected to at least three rounds of maturation pannings. Panning stringency was increased by lowering antigen concentration and/or cell count of target antigen expressing cells in each panning round (Low et al.1996). In addition to antigen reduction, off-rate selections were performed (Hawkins et al. 1992) using excesses of unbiotinylated target antigens as competitor to further increase selection stringency. All strategies were combined with prolonged washing steps.
  • CyCAT halfbody molecules with Fab specificity for CD123 were characterized for ELISA binding to human and cynomolgus CD123 and to the counter target human CD116, binding to recombinant cell lines with expression of human or cynomolgus CD123 and/or to the MOLM-13, MV4-11 and SKM-1 leukaemia cell lines with endogenous expression of human CD123, and finally for affinity on human and cynomolgus CD123 antigens via solution equilibrium titration (SET) and/or Octet/SPR.
  • SET solution equilibrium titration
  • CyCAT halfbody molecules with Fab specificity for FLT3 were characterized for ELISA binding to human, cynomolgus and mouse FLT3 and to the counter targets human cKIT, human PDGFRA, human CSF1R; cell binding to MOLM-13, MV4-11 and/or SKM-1 leukaemia cell lines with endogenous expression of human FLT3; and for their affinity on human and cynomolgus FLT3 via solution equilibrium titration (SET) and/or Octet/SPR. Furthermore, complementary pairs of CyCAT halfbody molecules for each FLT3 specific antibody were tested for their ability to mediate T-cell redirected killing of the FLT3 expressing leukaemia cancer lines MOLM-13 and SKM-1 cells.
  • each CyCAT halfbody molecule is composed of two polypeptides chains providing the Fab fragment with specificity for CD123 or FLT3, respectively, and either the VL or the VH of an ⁇ CD3 antibody.
  • CyCAT halfbody polypeptides according to the present disclosure described herein are set forth in Tables: 11 – 20.
  • Table 19 SEQ ID NOs. of the polypeptides forming the ⁇ CD123 and ⁇ FLT3 CyCAT halfbody molecules according to the present disclosure.
  • Table 20 Preferred complementary pairs of ⁇ CD123 and ⁇ FLT3 halfbody molecules and associated SEQ ID Nos. forming the trispecific antibodies of the present disclosure. Cloning of CyCAT halfbody molecules with specificity for CD123 or FLT3.
  • nucleic acid sequences or desired gene segments were generated by PCR using appropriate templates or were gene synthesized as linear DNA fragments with appropriate flanking regions (e.g. suitable restriction enzyme recognition sites, linker sequences) in-house or by an external provider.
  • flanking regions e.g. suitable restriction enzyme recognition sites, linker sequences
  • the nucleic acid sequences or gene segments flanked by singular restriction endonuclease cleavage sites were cloned into respective mammalian expression vectors using standard molecular biology methods.
  • CyCAT molecules For expression of CyCAT halfbody molecules, exponentially growing eukaryotic HEK293-6E cells were transfected with a mammalian expression vector encoding all components of a CyCAT molecule, resulting in a 1:1 heterodimer of two polypeptides forming (a) the Fab heavy chain of the ⁇ CD123- or of the ⁇ FLT3 antibody, respectively, fused to either the VH or VL of the ⁇ CD3 Fv fragment via a peptide linker, and (b) the corresponding Fab light chain of the ⁇ CD123 or the ⁇ FLT3 antibody, respectively.
  • Example 3 T cell mediated killing of cancer cells induced by functional complementation of anti-CD123 and anti-FLT3 CyCAT halfbody molecules
  • Complementary pairs of CyCAT halfbody molecules with specificity for CD123 and FLT3 were allowed for on-target functional complementation to trispecific antibodies with specificity for CD123, FLT3 and CD3.
  • FACS-based cytotoxicity assay with stimulated human CD8+ effector T cells The potency of functionally complemented pairs of anti-CD123 and anti-FLT3 CyCAT halfbody molecules according to the present disclosure in redirecting stimulated human CD8+ effector T cells against the CD123 and FLT3 double positive human AML cell lines SKM-1, MOLM-13 and MV4-11 was measured in a 48 hour FACS-based cytotoxicity assay. Tested pairs of halfbody molecules are depicted in Table 20. Isolation of peripheral mononuclear blood cells 500 mL whole blood was collected from healthy donors. Equal amount of PBS supplemented with 2% fetal bovine serum (Sigma, #F7524) was added and gently mixed.
  • PBMC containing supernatant was transferred into appropriate number of 50 mL conical tubes and filled with PBS up to 45 mL each. Cells were washed twice in PBS containing 2% fetal bovine serum and counted. Isolation of CD8 positive T cells PBMC suspensions were transferred into 50 mL conical tubes and centrifuged at 300x g for 8 minutes at room temperature.
  • Target cell labeling For the analysis of cell lysis in flow cytometry assays, the fluorescent membrane dye CellTrace Violet (Invitrogen, #C34557A) was used to label human CD123 and FLT3 double positive MOLM-13, MV4-11 or SKM-1 as target cells and distinguish them from effector cells. Briefly, cells were harvested, washed once with PBS and adjusted to 1x10 6 cells/mL in PBS and the membrane dye Cell Trace Violet (4 ⁇ /10 6 cells).
  • CellTrace Violet Invitrogen, #C34557A
  • This assay was designed to quantify the lysis of human CD123 and FLT3 double- positive MOLM-13, MV4-11 or SKM-1 target cells in the presence of serial dilutions of pre-mixed complementary pairs of CD123 and FLT3 specific CyCAT halfbody molecules. Equal volumes of Cell Trace Violet-labeled target cells and CD8+ effector T cells were mixed, resulting in an E:T cell ratio of 5:1.
  • Zombie Green is an amine-reactive fluorescent dye that is non-permanent to viable cells, but permanent to cells with compromised membranes, thus dead cells become identifiable by fluorescent emission.
  • APC/Cyanine7 anti-human CD8 Antibody Biolegend #344714, clone SK1 was added to label CD8+ effector T- cells. Samples were measured by flow cytometry on a NovoCyte instrument and analyzed by NovoExpress software (both from Agilent). Target cells were identified as Cell Trace Violet-positive cells.
  • CD8+ effector T-cells were identified as APC/Cyanine7- positive cells. Zombie Green-negative target cells were classified as living target cells.
  • n number of events Using GraphPad Prism 5 software (Graph Pad Software, San Diego), the percentage of cytotoxicity was plotted against the corresponding final mixed pairs of CyCAT halfbody concentrations. Dose response curves were analyzed with the four parametric logistic regression models for evaluation of sigmoid dose response curves with fixed hill slope and EC50 values were calculated. Results The results of the FACS-based cytotoxicity assay for the 3 pairs of CyCAT halfbody molecules carrying the newly identified human CD123 and human FLT3 specific antibodies according of the present disclosure are summarized in Table 22 and Figure 2.
  • Table 22 Results of the cytotoxicity assays for dual targeting of CD123 and FLT3 double positive leukaemia cell lines MOLM-13, MV4-11 and SKM-1 with complementary pairs of CyCAT halfbody molecules with specificity for CD123 and FLT3 via on cell formation of functional T-cell engaging trispecific antibodies.
  • Example 4 KD determination on human and cynomolgus monkey CD123 and human, cynomolgus monkey and mouse FLT3 by Biolayer Interferometry (BLI) Method For KD determinations, unpaired CD123 specific CyCAT halfbody molecules and FLT3 specific CyCAT halfbody molecules were used. Affinity determination by determining kinetic rate constants was performed on an Octet HTX (FortéBIO, Sartorius AG) instrument as described below.
  • CyCAT halfbody molecules diluted in assay buffer D-PBS, 0.05 % (v/v) PS 20, 0.1 % (w/v) BSA
  • assay buffer D-PBS, 0.05 % (v/v) PS 20, 0.1 % (w/v) BSA
  • human CD123 antigens SEQ ID NO: 65
  • cynomolgus monkey CD123 antigen SEQ ID NO: 66
  • assay buffer 2n serial dilution with seven concentrations in total starting with a concentration of 30 nM (human CD123) and 200 nM (cynomolgus monkey CD123).
  • the starting concentrations were 50 nM human FLT3 (SEQ ID NO: 67), 150 nM (cynomolgus monkey FLT3 (SEQ ID NO: 68) and 700 nM mouse FLT3 (SEQ ID NO: 69).
  • a blank sample with assay buffer was included for referencing, i.e. correcting for dissociation of captured antibody.
  • the association phase was recorded for 420 s, followed by a dissociation phase of 900 s (FLT3) or 1200 s (CD123).
  • FLT3 dissociation phase of 900 s
  • CD123 1200 s
  • a summary of the determined affinities on human, cynomolgus and mouse FLT3 for ⁇ FLT3-Fab1 and ⁇ FLT3-Fab2 is provided in Table 24 and reveal monovalent affinities for the newly identified FLT3 specific antibodies in the sub-nanomolar range on human and cynomolgus FLT3 antigens. Affinities on mouse FLT3 were determined in the double digit nanomolar range.
  • Method Streptavidin-coated biosensors (FortéBIO, Sartorius AG) were loaded with a mixture of anti-human Fab kappa/lambda biotin conjugates (CaptureSelect, Thermo Scientific) in immobilization buffer (PBS, 0.05% (v/v) Tween 20) resulting in a high-density anti- human Fab kappa/lambda sensor surface.
  • unpaired CyCAT halfbodies were diluted in assay buffer (DPBS, 0.05% (v/v) Tween-20, 0.1% (w/v) BSA) and sequentially injected until a loading threshold value of approx.
  • each CyCAT group was comprised of nine animals (three animals per each T-cell donor), while control groups (vehicle and positive control) were comprised of only six animals (two animals per each T-cell donor).
  • the respective CyCAT halfbodies were pre-mixed (1:1 vol) and incubated for 30min at RT.
  • Compound administration was performed intravenously Q2Dx6 (vehicle and CyCAT halfbody molecules) or BIWx3 (positive control) with an application volume of 5mL/kg for all animals. Tumor and body weight measurements were carried out two to three times per week and animal health status was checked daily. Results The results of the animal studies are shown in Figure 4.
  • Example 7 Cancer cell binding of ⁇ CD123 and ⁇ FLT3 CyCAT halfbody molecules
  • FACS buffer including FCS and azide to prevent receptor internalization. Cell suspensions were transferred into microtiter plates.
  • CyCAT halfbodies were titrated separately in low binding 96 well plates and transferred to cells, followed by subsequent incubation of plates at 4°C for 1 hour. Sample volume and cell- number are adjusted to plate type used. Following incubation, cells were spun down and washed with FACS buffer. Fluorophore-conjugated secondary reagents were used for detection of bound antibodies. Plates were measured using the Intellicyt HTFC/iQue System and data was analyzed using proprietary software or FlowJo. Results Results of the cell binding experiments are shown in Table 26 and Figure 5.
  • TF-1 suspension cells were grown in RPMI-1640 + 20% FCS+ 2ng/mL GM-CSF at 37°C, 5% CO2 and humidity. Cells were 3x washed in 20 mL HBSS (Gibco), after centrifugation cells were resuspend in medium w/o GM-CSF but supplemented with 0.1 nM IL-3. 1x104 TF-1 cells were seeded/well of a 96 well plate (white flat, costar). ⁇ CD123 halfbodies were titrated separately in a low binding PP plate (96 well) and transferred to TF-1 cells in IL-3 containing medium in the assay plate.
  • Reference bispecific antibody XmAb14045 Main chain 1: scFv-Fc EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKG RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGDSYVSWFAYWGQGTLVTVSSGKPGSGKPGSG KPGSGKPGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQQKPGKSPRGLIGGTNKRAPG VPARFSGSLLGGKAALTISGAQPEDEADYYCALWYSNHWVFGGGTKLTVLEPKSSDKTHTCPPCPAPPV AGPSVFLFPPKPKDTLMISRTPE
  • pan T cells were freshly prepared from human whole blood and resuspend in CellToxGreen (Promega) containing HUVEC assay medium (w/o phenol red). Pan T cells were used at an effector to target cell ratio of 6:1. CyCAT halfbodies were titrated separately in a low binding PP plate (96 well), Refmabs were as well diluted in a low binding PP plate (96 well). Diluted compounds were stored at 4°C until pan T cells were ready. Titrated complementary CyCAT halfbodies were combined on the cells in the assay plate, pan T cells were added at the given ratio and the assay plate was incubated for 72hrs at 37°C, 5% CO 2 and humidity.
  • cytotoxicity was determined by measuring fluorescence intensity at Tecan reader F500 (Ex 485/20 nm, Em 530/25 nm, fluorescence bottom, gain optimal), after calculation of signal over background, GraphPad Prism was used to determined EC50. Supernatants were transferred to separate 96 well plate and stored at -20°C for read out of cytokine release (ELISA). Results Results of the cytotoxicity assay is shown in Fig. 7B. As expected, conventional bi- or trispecific antibodies with specificity for CD3 and CD123 mediated strong T-cell redirected cell killing of normal HUVECs.
  • Example 10 FTL3 and CD123 specific CyCAT halfbodies with improved production and heterodimerization properties ⁇ FLT3 specific halfbody molecules ⁇ FLT3-HB1 and ⁇ FLT3-HB4 as well as CD123 specific halfbody molecules ⁇ CD123-HB1 and ⁇ CD123-HB4 were modified by introducing a V37M single point mutation into the framework region 2 of the ⁇ CD3-VH domain (unmutated ⁇ CD3-VH (SEQ ID NO: 40); mutated ⁇ CD3-VHV37M (SEQ ID NO: 107)).
  • the variant halfbody molecules were mammalian produced in parallel to their counterpart halfbodies utilizing the unmutated ⁇ CD3-VH.
  • CyCAT halfbody polypeptides A summary of the amino acid sequences of the produced CyCAT halfbody polypeptides according to Example 10 are set forth in Tables 28 – 32.
  • various control halfbodies comprising the Fab fragment of Trastuzumab (HERCEPTIN® / anti-HER2), Cetuximab (Erbitux® / anti-EGFR) or MOR03207 (anti- lysozyme (in-house) were generated utilizing either the unmutated ⁇ CD3-VH (SEQ ID NO: 40) or the mutated ⁇ CD3-VHV37M (SEQ ID NO: 107).
  • Control halfbody sequences are summarized in Table 33.
  • Table 35 CD3 binding kinetics (BLI) of halfbody pairs utilizing the unmutated ⁇ CD3-VH or the mutated ⁇ CD3-VH V37M domain, respectively T cell mediated killing of cancer cells induced by functional complementation of complementary halfbody molecules utilizing a mutated ⁇ CD3-VHV37M domain Cytotoxicity assays were conducted as described in Example 3. The results of the FACS-based cytotoxicity assay for the tested pairs of halfbody molecules are summarized in Table 36 and shown in Figure 8.
  • Table 36 Results of the cytotoxicity assays for Dual-Targeting of CD123/FLT3 double positive MOLM-13 or SKM-1 cells with complementary pairs of halfbody molecules with specificity for CD123 and FLT3 utilizing either the unmutated ⁇ CD3-VH or mutated ⁇ CD3-VHV37M domain, respectively Therapeutic or assay window determined for halfbody pairs in killing of cancer cells Due to the intrinsic property of VH and VL antibody variable domains to associate with each other, a residual functional complementation of complementary halfbody molecules may also occur in solution in the absence of one or both target antigens of interest.
  • Such “unwanted heteroassociation” may lead to undesired T cell activation and relies, amongst other factors, on the local concentration of the complementary halfbodies molecules and the interface affinity between the interacting variable domains.
  • a “Mono-Targeting” control cytotoxicity assay was performed in parallel to the “Dual- Targeting” approach.
  • the Fab specificity for the halfbody molecule carrying the ⁇ CD3-VL domain was altered to an irrelevant antigen (i.e. lysozyme) not expressed on the surface of the target cell.
  • any cell killing activity determined in such Mono-Targeting cytotoxicity assay can be linked to preformed halfbody complexes with functionally complemented ⁇ CD3-Fv fragments.
  • the ratio of the cell killing activity determined for the Mono-Targeting and the cell killing activity determined for the Dual-Targeting approach defines the “Assay Window” or “Therapeutic Window” for a given halfbody pair.
  • a larger Assay Window indicates that a given halfbody pair can be mixed or can be present at higher local concentrations without forming significant amounts of undesired functional complexes and therefore provides a particular safe combination of such halfbody molecules.
  • Results Cytotoxicity assays were conducted as described in Example 3.
  • Table 37 Results of the cytotoxicity assays for Mono- and Dual-Targeting halfbody pairs on MOLM-13 cells
  • Table 38 Results of the cytotoxicity assays for Mono- and Dual-Targeting halfbody pairs on SKM-1 cells
  • Table 39 Results of the cytotoxicity assays for Mono- and Dual-Targeting control halfbody pairs with specificity for EGFR, HER2 or Lysozyme, respectively, on EGFR/HER2 double positive SKOV-3 cells.
  • Example 11 FTL3 and CD123 specific CyCAT halfbodies with improved production and heterodimerization properties utilizing high affinity ⁇ CD3 variable domains.
  • Example 10 was repeated with halfbody molecules comprising identical Fab fragments and linkers as described before but utilizing ⁇ CD3 variable domains obtained from a high affinity ⁇ CD3 antibody (“ ⁇ CD3high”) as described in WO2022/063819. Amino acid sequences of this high affinity antibody are summarized in Table 40.
  • halfbody molecules comprising a V37M mutated ⁇ CD3-VHhigh domain of this antibody (“ ⁇ CD3high-mut” or “ ⁇ CD3high-Fvmut) were produced and further characterized as described below. Sequences of produce halfbodies of Example 11 are summarized in Table 41.
  • Table 40 Amino acid sequences of the high affinity ⁇ antibody and its VH-V37M mutated variant ⁇ CD3high-mut
  • Table 41 SEQ ID NOs of produced halfbody molecules according to Example 11 Results Quality Control Production of halfbody molecules was performed as described in Example 2. The results of the quality control are summarized in Table 42. Similar as observed for the low-affinity CD3 antibody utilized in Example 10, halfbody molecules utilizing the mutated high affinity ⁇ CD3-VHV37M domain revealed a strongly reduced Fab light chain (LC) appearance after production when compared to the chain (LC) appearance determined for their counterpart halfbody molecule utilizing the unmutated high affinity ⁇ CD3-VHhigh domain (Table 42, last column).
  • LC Fab light chain
  • Table 43 CD3 binding kinetics (BLI) of CyCAT halfbody pairs comprising the V37M mutated ⁇ CD3high-VHV37M domain ** dissociation approaching assay limit T cell mediated killing of cancer cells induced by functional complementation of halfbody molecules utilizing a mutated high affinity ⁇ CD3high-VHV37M domain Cytotoxicity assays were conducted as described in Example 3. The results of the FACS-based cytotoxicity assay for the tested pairs of CyCAT halfbody molecules are summarized in Table 44.
  • Table 44 Results of the cytotoxicity assays on CD123/FLT3 double positive cell lines MOLM-13 and SKM-1 with complementary pairs of for Dual-Targeting halfbody molecules with specificity for CD123 and FLT3, respectively utilizing the unmutated ⁇ CD3high-VH or mutated ⁇ CD3high-VHV37M domain Assay window determined for complementary halfbody pairs in killing of cancer cells The Assay Window for each halfbody pair was determined as described in Example 10. Cytotoxicity assays were conducted as described in Example 3. The results of the FACS-based cytotoxicity assays are summarized in Tables 45 - 47.
  • Table 45 Results of the cytotoxicity assays for Mono- and Dual-Targeting halfbody pairs on SKM-3 cells
  • Table 46 Results of the cytotoxicity assays for Mono- and Dual-Targeting halfbody pairs on MOLM-13 cells
  • Table 47 Results of the cytotoxicity assays for Mono- and Dual-Targeting control halfbody pairs on EGFR/HER2 double positive SKOV3 cells

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Abstract

La présente invention concerne de nouveaux anticorps humains qui se lient spécifiquement à CD123 ainsi que de nouveaux anticorps humains qui se lient spécifiquement à FLT3. La présente invention concerne en outre des paires complémentaires de molécules de demi-corps CyCAT® englobant les nouveaux anticorps spécifiques de FLT3 et CD123 selon l'invention et leur utilisation dans la redirection de la destruction médiée par les lymphocytes T de cellules cancéreuses de leucémie co-exprimant FLT3 et CD123, par la formation sur cellule d'anticorps trispécifiques activant des lymphocytes T. La présente invention concerne également des mesures pour optimiser la productibilité de molécules de demi-corps ainsi que le profil de sécurité de thérapies dirigées par CyCAT.
PCT/EP2024/068447 2023-06-30 2024-07-01 Double ciblage de cellules tumorales co-exprimant flt3 et cd123 par complémentation fonctionnelle de molécules de demi-corps de cycat® Pending WO2025003511A1 (fr)

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Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO2001005950A2 (fr) 1999-07-20 2001-01-25 Morphosys Ag Nouveaux procedes permettant la presentation de (poly)peptides/proteines sur des particules bacteriophages au moyen de liaisons disulfure
US6703199B1 (en) 1997-06-12 2004-03-09 Research Corporation Technologies, Inc. Artificial antibody polypeptides
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
WO2013104804A2 (fr) 2012-01-13 2013-07-18 Julius-Maximilians-Universität Würzburg Complémentation fonctionnelle bipartite induite par un antigène double
US8728981B2 (en) 2010-11-19 2014-05-20 Morphosys Ag Collection and methods for its use
WO2016201065A1 (fr) * 2015-06-12 2016-12-15 Seattle Genetics, Inc. Anticorps anti-cd123 et conjugués de ceux-ci
WO2017021362A1 (fr) * 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps pour flt3 et cd3
WO2017210443A1 (fr) 2016-06-01 2017-12-07 Xencor, Inc. Anticorps bispécifiques qui lient cd123 et cd3
WO2021089748A2 (fr) 2019-11-06 2021-05-14 Amgen Research (Munich) Gmbh Molécules de liaison à des antigènes à cibles multiples destinées à être utilisées dans des maladies prolifératives
WO2022063819A1 (fr) 2020-09-24 2022-03-31 Morphosys Ag Nouveaux anticorps humains se liant au cd3 epsilon humain

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6982321B2 (en) 1986-03-27 2006-01-03 Medical Research Council Altered antibodies
US5821337A (en) 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US6703199B1 (en) 1997-06-12 2004-03-09 Research Corporation Technologies, Inc. Artificial antibody polypeptides
US7087409B2 (en) 1997-12-05 2006-08-08 The Scripps Research Institute Humanization of murine antibody
WO2001005950A2 (fr) 1999-07-20 2001-01-25 Morphosys Ag Nouveaux procedes permettant la presentation de (poly)peptides/proteines sur des particules bacteriophages au moyen de liaisons disulfure
US7527791B2 (en) 2004-03-31 2009-05-05 Genentech, Inc. Humanized anti-TGF-beta antibodies
US8728981B2 (en) 2010-11-19 2014-05-20 Morphosys Ag Collection and methods for its use
WO2013104804A2 (fr) 2012-01-13 2013-07-18 Julius-Maximilians-Universität Würzburg Complémentation fonctionnelle bipartite induite par un antigène double
WO2016201065A1 (fr) * 2015-06-12 2016-12-15 Seattle Genetics, Inc. Anticorps anti-cd123 et conjugués de ceux-ci
WO2017021362A1 (fr) * 2015-07-31 2017-02-09 Amgen Research (Munich) Gmbh Constructions d'anticorps pour flt3 et cd3
WO2017210443A1 (fr) 2016-06-01 2017-12-07 Xencor, Inc. Anticorps bispécifiques qui lient cd123 et cd3
WO2021089748A2 (fr) 2019-11-06 2021-05-14 Amgen Research (Munich) Gmbh Molécules de liaison à des antigènes à cibles multiples destinées à être utilisées dans des maladies prolifératives
WO2022063819A1 (fr) 2020-09-24 2022-03-31 Morphosys Ag Nouveaux anticorps humains se liant au cd3 epsilon humain

Non-Patent Citations (40)

* Cited by examiner, † Cited by third party
Title
"Short Protocols in Molecular Biology", 2002, JOHN WILEY AND SONS
"Uniprot", Database accession no. P36888-1
ABU-DUHIER ET AL., BR J HAEMATOL., vol. 111, no. 1, October 2000 (2000-10-01), pages 190 - 5
AL-LAZIKANI ET AL., J. MOL. BIOL., vol. 273, 1997, pages 927 - 948
ALMAGROFRANSSON, FRONT BIOSCI, vol. 13, 2008, pages 1619 - 1633
BIRG ET AL., BLOOD, vol. 80, no. 10, November 1992 (1992-11-01)
CAROW ET AL., BLOOD, vol. 87, no. 3, February 1996 (1996-02-01)
CHOTHIA ET AL., J. MOL. BIOL., vol. 186, 1985, pages 651 - 663
CHOTHIA ET AL., J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
CHOTHIA ET AL., NATURE, vol. 341, 1989, pages 544 - 546
DALL'ACQUA ET AL., METHODS, vol. 36, 2005, pages 61 - 68
FRIPPIAT ET AL., HUM. MOL. GENET., vol. 4, 1995, pages 983 - 991
GOLDSTEIN ET AL., CANCER RES, vol. 82, 2022, pages 6313
HOLLINGERHUDSON, NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1126 - 1136
HUSTON ET AL., PROC. NATL. ACAD. SCI., vol. 85, 1988, pages 5879 - 5883
JONES ET AL., NATURE, vol. 321, 1986, pages 522 - 525
KABAT, E.A. ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES, article "Sequences of Proteins of Immunological Interest"
KAWASAKI ET AL., GENOME RESEARCH, vol. 7, no. 3, 1997, pages 250 - 61
KLIMKA ET AL., BR J CANCER, vol. 83, 2000, pages 252 - 260
KNAPPIK ET AL., J MOL BIOL, vol. 296, 2000, pages 57 - 86
KOHLER, NATURE, vol. 256, 1975, pages 495
LAZIKANI ET AL., J. MOL. BIO., vol. 273, 1997, pages 927 - 948
LEFRANC MP, EXP CLIN IMMUNOGENET, vol. 18, no. 2, 2001, pages 100 - 16
LEFRANC MP, EXP CLIN IMMUNOGENET., vol. 18, 2001, pages 242 - 254
MATSUDA ET AL., J EXP MED, vol. 188, no. 11, 1998, pages 2151 - 62
MORDENTI ET AL., PHARMACEUT. RES., vol. 8, 1991, pages 1351
MORRISON ET AL., PROC NATL ACAD SCI, vol. 81, 1984, pages 6851 - 6855
MORRISONOI, ADV IMMUNOL, vol. 44, 1988, pages 65 - 92
NOVOTNYHABER, PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 4592 - 4596
PADLAN, MOL IMMUNOL, vol. 28, 1991, pages 489 - 498
PADLAN, MOLEC IMMUN, vol. 31, no. 3, 1994, pages 169 - 217
QUEEN ET AL., PROC NATL ACAD SCI USA, vol. 86, 1989, pages 10029 - 10033
RIECHMANN ET AL., NATURE, vol. 332, 1988, pages 323 - 329
SAMBROOK ET AL.: "A laboratory manual", 1989, COLD SPRING HARBOR LABORATORY PRESS, article "Molecular cloning"
SUN ET AL., ONCOTARGETS AND THERAPY, vol. 12, 2019, pages 4907 - 4925
TILLER ET AL., MABS, vol. 5, no. 3, May 2013 (2013-05-01), pages 1 - 26
TOMLINSON ET AL., J. MOL. BIOL., vol. 227, 1992, pages 776 - 798
VERHOEYEN ET AL., SCIENCE, vol. 239, 1988, pages 1534 - 1536
YAMAMOTO ET AL., BLOOD, vol. 97, no. 8, April 2001 (2001-04-01)
ZAPATA ET AL., PROTEIN ENG, vol. 8, 1995, pages 1057 - 1062

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