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WO2025082777A1 - Double ciblage de cellules tumorales co-exprimant muc16 et mésothéline par complémentation fonctionnelle de molécules de demi-corps cycat® - Google Patents

Double ciblage de cellules tumorales co-exprimant muc16 et mésothéline par complémentation fonctionnelle de molécules de demi-corps cycat® Download PDF

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Publication number
WO2025082777A1
WO2025082777A1 PCT/EP2024/078140 EP2024078140W WO2025082777A1 WO 2025082777 A1 WO2025082777 A1 WO 2025082777A1 EP 2024078140 W EP2024078140 W EP 2024078140W WO 2025082777 A1 WO2025082777 A1 WO 2025082777A1
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seq
amino acid
acid sequence
present disclosure
muc16
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Inventor
Christoph ERKEL
Felicitas STEIN
Simon Schuster
Steffen DICKOPF
Markus Moosmeier
Sebastian Jäger
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • 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
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention provides novel human antibodies that specifically bind to Mucin-16 (MUC16) and novel human antibodies that specifically bind to Mesothelin (MSLN).
  • MUC16 Mucin-16
  • MSLN Mesothelin
  • the invention further relates to complementary pairs of CyCAT ® halfbody molecules comprising the MUC16 or MSLN specific antibodies of the present disclosure and their use in redirecting T-cell mediated killing of MUC16 and MSLN co-expressing tumor cells via on-cell formation of T-cell engaging trispecific antibodies.
  • MUC16 is a well-characterized target for Ovarian Cancer (OVCA) as it is over- expressed on the majority of epithelial OVCA tumor cells (see, e.g., Giamougiannis et al., Carcinogenesis 42(3): 327–343 (Mar 2021); Aithal et al., PLoS ONE 13(4): e0193907 (Feb 2018)).
  • OVCA Ovarian Cancer
  • cancer antigen 125 CA-125
  • OVCA cancer antigen 125
  • Felder et al. Mol Cancer 29(13):129. doi: 10.1186/1476-4598-13-129 (May 2014); Sölétormos et al., Int J Gynecol Cancer 26: 43-51 (Jan 2016)).
  • MUC16 targeting antibody in the form of a bispecific T-cell engager is in clinical development (see, e.g., Crawford et al., Sci Trans Med 11(497): eaau7534 (Jun 2019); ClinicalTrials.gov Identifier: NCT03564340).
  • Mesothelin (“MSLN”) is a well-characterized tumor antigen over-expressed in a variety of solid tumors including OVCA (see, e.g., Frierson et al., Hum Pathol 34: 605–609 (Jun 2003); Weidemann et al., Biomedicines 9(397) (Apr 2021)).
  • MSLN ⁇ HSA ⁇ CD3 trispecific T-cell engager
  • CD3 is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor complex (TCR) and is required for T cell activation.
  • Functional CD3 is formed from the dimeric association of two of four different chains: epsilon, zeta, delta and gamma.
  • the CD3 dimeric arrangements include gamma/epsilon, delta/epsilon and zeta/zeta.
  • Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby causing T cell activation in a manner similar to the engagement of the TCR by peptide-loaded MHC molecules. Thus, anti-CD3 antibodies have been proposed for therapeutic purposes involving the activation of T cells.
  • bispecific antibodies that are capable of binding CD3 and a target antigen have been proposed for therapeutic uses involving targeting T cell immune responses to tissues and cells expressing the target antigen.
  • Co-expression as well as co-localization of MUC16 and MSLN can be found on the majority of OVCA patient tumor cells and furthermore, both antigens are known to interact with each other (see, e.g., Kakimoto et al., Pathol Oncol Res 26(4): 2299– 2306 (Oct 2020); Gubbels et al., Mol Cancer 5(1): (Oct 2006)).
  • the presence of two cell surface tumor antigens provides an opportunity for the development of highly selective targeted therapies.
  • TCR2 Therapeutics Simultaneous targeting of MUC16 and MSLN in form of a CAR-T cell-like approach has been described in the art (TCR2 Therapeutics). Based on this dual targeting approach (TRuC-T technology), engineered T cells are directed to both, MUC16 and MSLN double positive cells but also to cells expressing either one or the other antigen. While this simultaneous targeting approach appears to be therapeutically attractive to overcome tumor escape mechanisms by downregulation of one or the other antigen, it may also increase the risk for unwanted side effects associated with targeting of single target-positive healthy cells and tissues. Hence a need for improved therapeutic approaches remains, especially with regard to improved safety.
  • two complementary CyCAT halfbody molecules are designed, each composed of a Fab targeting moiety with specificity for either MUC16 (“ ⁇ MUC16-Fab”) or Mesothelin (“ ⁇ MSLN-Fab”), respectively, and either carrying an antibody variable light chain domain (“ ⁇ CD3-VL”) or an antibody variable heavy chain domain (“ ⁇ CD3-VH”) of a functional ⁇ CD3-Fv binding fragment (“ ⁇ CD3-Fv”).
  • ⁇ CD3- VH alone nor the ⁇ CD3-VL alone and neither one of the corresponding CyCAT halfbody molecules alone is able to bind to CD3 expressed on effector T-cells and to redirect T-cells for tumor cell destruction.
  • the two halfbody molecules may bind to a malignant cell expressing both MUC16 and Mesothelin on its cell surface.
  • the ⁇ CD3-VL and ⁇ CD3-VH domain present on the two halfbody molecules 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 are disclosed in WO2022/063819.
  • the present disclosure pertains to a trispecific antibody with specificity for MUC16, MSLN and CD3.
  • the present disclosure pertains to a trispecific antibody comprising a) a first Fab specific for MUC16 ( ⁇ MUC16-Fab), b) a second Fab specific for MSLN ( ⁇ MSLN-Fab), and c) an antibody Fv fragment (Fv) specific for CD3 ( ⁇ CD3-Fv).
  • the present disclosure provides a trispecific antibody, wherein a) the ⁇ MUC16-Fab binds to human MUC16 and cynomolgus monkey MUC16, b) the ⁇ MSLN-Fab binds to human MSLN and cynomolgus monkey MSLN, and c) the ⁇ CD3-Fv to human CD3 and cynomolgus monkey CD3.
  • said ⁇ CD3-Fv comprises the VH of an antibody specific for CD3 ( ⁇ CD3-VH) and the VL of an antibody specific for CD3 ( ⁇ CD3-VL) according to the present disclosure.
  • said ⁇ CD3-Fv consists of the VH of an antibody specific for CD3 ( ⁇ CD3-VH) and the VL of an antibody specific for CD3 ( ⁇ CD3-VL) according to the present disclosure
  • the trispecific antibody according to the present disclosure is composed of a first halfbody molecule (HB-1) and a second halfbody molecule (HB-2), wherein a) HB-1 comprises the ⁇ MUC16-Fab and either the VH ( ⁇ CD3-VH1) or the VL ( ⁇ CD3-VL1) of the ⁇ CD3-Fv, and b) HB-2 comprises the ⁇ MSLN-Fab and either the complementary VL ( ⁇ CD3-VL2) or the complementary VH ( ⁇ CD3-VH2) of the ⁇ CD3-Fv.
  • the trispecific antibody according to the present disclosure is composed of a first halfbody molecule (HB-1) and a second halfbody molecule (HB-2), wherein a) HB-1 comprises the ⁇ MUC16-Fab and either the VH ( ⁇ CD3-VH1) or the VL ( ⁇ CD3-VL1) of the ⁇ CD3-Fv, and b) HB-2 comprises the ⁇ MSLN-Fab and either the complementary VL ( ⁇ CD3-VL2) or the complementary VH ( ⁇ CD3-VH2) of the ⁇ CD3-Fv, and wherein HB-1 and HB-2 are not linked to each other by a covalent bond.
  • HB-1 comprises the ⁇ MUC16-Fab and either the VH ( ⁇ CD3-VH1) or the VL ( ⁇ CD3-VL1) of the ⁇ CD3-Fv
  • HB-2 comprises the ⁇ MSLN-Fab and either the complementary VL ( ⁇ CD3-VL2) or the complementary VH ( ⁇
  • the present disclosure pertains to a pair of a first halfbody molecule (HB-1) and a second halfbody molecule (HB-2), wherein a) HB-1 comprises a first Fab specific for MUC16 ⁇ ⁇ ⁇ MUC16-Fab) and either the VH ( ⁇ CD3- VH1) or the VL ( ⁇ CD3-VL1) of the Fv specific for CD3 ( ⁇ CD3-Fv), and b) HB-2 comprises a second Fab specific for Mesothelin ( ⁇ MSLN-Fab) and either the complementary VL ( ⁇ CD3-VL2) or the complementary VH ( ⁇ CD3-VH2) of the Fv specific for CD3 ( ⁇ CD3-Fv).
  • HB-1 comprises a first Fab specific for MUC16 ⁇ ⁇ ⁇ MUC16-Fab) and either the VH ( ⁇ CD3- VH1) or the VL ( ⁇ CD3-VL1) of the Fv specific for CD3 ( ⁇ CD3-Fv
  • the present disclosure pertains to a complementary pair of a first halfbody molecule (HB-1) and a second halfbody molecule (HB-2), wherein a) HB-1 comprises a first Fab specific for MUC16 ⁇ ⁇ ⁇ MUC16-Fab) and either the VH ( ⁇ CD3- VH1) or the VL ( ⁇ CD3-VL1) of the Fv specific for CD3 ( ⁇ CD3-Fv), and b) HB-2 comprises a second Fab specific for Mesothelin ( ⁇ MSLN-Fab) and either the complementary VL ( ⁇ CD3-VL2) or the complementary VH ( ⁇ CD3-VH2) of the Fv specific for CD3 ( ⁇ CD3-Fv).
  • HB-1 comprises a first Fab specific for MUC16 ⁇ ⁇ ⁇ MUC16-Fab) and either the VH ( ⁇ CD3- VH1) or the VL ( ⁇ CD3-VL1) of the Fv specific for CD3 ( ⁇ CD3-F
  • the present disclosure pertains to a complementary pair of a first halfbody molecule (HB-1) and a second halfbody molecule (HB-2), wherein a) HB-1 comprises a first Fab specific for MUC16 ⁇ ⁇ ⁇ MUC16-Fab) and either the VH ( ⁇ CD3- VH1) or the VL ( ⁇ CD3-VL1) of the Fv specific for CD3 ( ⁇ CD3-Fv), b) HB-2 comprises a second Fab specific for Mesothelin ( ⁇ MSLN-Fab) and either the complementary VL ( ⁇ CD3-VL2) or the complementary VH ( ⁇ CD3-VH2) of the Fv specific for CD3 ( ⁇ CD3-Fv), and wherein HB-1 and HB-2 are not linked to each other by a covalent bond.
  • HB-1 comprises a first Fab specific for MUC16 ⁇ ⁇ ⁇ MUC16-Fab) and either the VH ( ⁇ CD3- VH1) or the VL (
  • said HB-1 comprises a first Fab specific for MUC16 ( ⁇ MUC16-Fab) and either the VH ( ⁇ CD3-VH1) or the VL ( ⁇ CD3-VL1) of an Fv specific for CD3 ( ⁇ CD3-Fv).
  • said HB-1 comprises an ⁇ MUC16-Fab and an ⁇ CD3- VH1.
  • said HB-1 comprises an ⁇ MUC16-Fab and an ⁇ CD3-VL1.
  • said HB-1 comprises an ⁇ MUC16-Fab and an ⁇ CD3-VH1 but not an ⁇ CD3-VL1.
  • said HB-1 comprises an ⁇ MUC16-Fab and an ⁇ CD3-VL1 but not an ⁇ CD3-VH1.
  • said HB-2 comprises a second Fab specific for Mesothelin ( ⁇ MSLN-Fab) and either the complementary VH ( ⁇ CD3-VH2) or the complementary VL ( ⁇ CD3-VL2) of the Fv specific for CD3 ( ⁇ CD3-Fv).
  • said HB-2 comprises an ⁇ MSLN-Fab and an ⁇ CD3-VH2.
  • said HB-2 comprises an ⁇ MSLN-Fab and an ⁇ CD3-VL2.
  • said HB-2 comprises an ⁇ MSLN- Fab and an ⁇ CD3-VH2 but not an ⁇ CD3-VL2. In an aspect, said HB-2 comprises an ⁇ MSLN- Fab and an ⁇ CD3-VL2 but not an ⁇ CD3-VH2. In an aspect of the present disclosure, said HB-1 and said HB-2 according to the present disclosure are a pair of halfbody molecules. In an aspect, said HB-1 and HB-2 are a set of halfbody molecules. In an aspect, said HB-1 and HB-2 are a complementary pair of halfbody molecules. In an aspect of the present disclosure, said HB-1 and HB-2 are not linked by a covalent bond.
  • said HB-1 comprises the ⁇ MUC16-Fab and the ⁇ CD3- VH1 and said HB-2 comprises the ⁇ MSLN-Fab and the ⁇ CD3-VL2.
  • said HB-1 comprises the ⁇ MUC16-Fab and the ⁇ CD3-VL1 and said HB-2 comprises the ⁇ MSLN-Fab and the ⁇ CD3-VH2.
  • neither ⁇ CD3-VH1 and ⁇ CD3-VH2 by themselves nor ⁇ CD3-VL1 and ⁇ CD3-VL2 by themselves are capable of binding to CD3.
  • neither HB-1 by itself nor HB-2 by itself is capable of binding to CD3.
  • neither HB-1 by itself nor HB-2 by itself is capable of mediating T-cell redirected killing of cells expressing MUC16 and/or Mesothelin on their cell surface.
  • neither HB-1 by itself nor HB-2 by itself is capable of inducing T-cell mediated killing of cells expressing MUC16 and/or Mesothelin on their cell surface.
  • the ⁇ CD3-VH1 or the ⁇ CD3-VH2 comprises an amino acid substitution at position 37, 44 or 45 (utilizing the number scheme according Kabat).
  • the ⁇ CD3-VH1 or the ⁇ CD3-VH2 comprises an amino acid substitution at position 37, 44 or 45 (utilizing the number scheme according Kabat), wherein the amino acid substitution inhibits the trispecific antibody or the pair of HB-1 and HB-2 induced T-cell mediated killing of cells expressing either MUC16 or Mesothelin on their cell surface as compared to the trispecific antibody or pair of HB-1 and HB-2 induced T-cell mediated killing of such cells before the amino acid substitution.
  • the amino acid substitution at position 37, 44 or 45 does not substantially inhibit the trispecific antibody or the pair of HB-1 and HB-2 induced T-cell mediated killing of cells expressing MUC16 and Mesothelin on their cell surface as compared to the trispecific antibody or the pair of HB-1 and HB-2 induced T-cell mediated killing of such cells before the amino acid substitution.
  • the amino acid at position 37, 44 or 45 (utilizing the number scheme according Kabat) in the ⁇ CD3-VH1 or the ⁇ CD3-VH2 is substituted for M or L, the amino acid at position 44 is substituted for A, and/or the amino acid at position 45 is substituted for I or F.
  • the amino acid substitution in the ⁇ CD3-VH1 or the ⁇ CD3-VH2 is a V37M amino acid substitution.
  • the ⁇ CD3-VH1 or the ⁇ CD3-VH2 comprises a V or a M at position 37 (utilizing the number scheme according Kabat).
  • the ⁇ MUC16-Fab comprises an antibody variable heavy chain region (VH) comprising an heavy chain complementary determining region (HCDR)1 region comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 11 or SEQ ID NO: 21, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 12 or SEQ ID NO: 22 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23; 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, SEQ ID NO: 14, or SEQ ID NO: 24, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 15 or SEQ ID NO: 25, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 16, or SEQ ID NO: ii.
  • VH antibody variable heavy chain region
  • the ⁇ MSLN-Fab comprises a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 42, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 43; and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 34, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 35, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 46, and iii.
  • the ⁇ CD3-VH1 or ⁇ CD3-VH2 comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 52 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 53; and the ⁇ CD3- VL1 or ⁇ CD3-VL2 comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO : 54, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 56.
  • the present disclosure pertains to a pair of a first halfbody molecule (HB-1) and a second halfbody molecule (HB-2), wherein a) HB-1 comprises a first Fab specific for MUC16 ⁇ ⁇ ⁇ MUC16-Fab) and either the VH ( ⁇ CD3- VH1) or the VL ( ⁇ CD3-VL1) of an Fv specific for CD3 ( ⁇ CD3-Fv), and b) HB-2 comprises a second Fab specific for Mesothelin ( ⁇ MSLN-Fab) and either the complementary VL ( ⁇ CD3-VL2) or the complementary VH ( ⁇ CD3-VH2) of the Fv specific for CD3 ( ⁇ CD3-Fv), wherein i.
  • HB-1 comprises a first Fab specific for MUC16 ⁇ ⁇ ⁇ MUC16-Fab) and either the VH ( ⁇ CD3- VH1) or the VL ( ⁇ CD3-VL1) of an Fv specific for CD3 ( ⁇
  • the ⁇ MUC16-Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 17 or SEQ ID NO: 27 and a VL comprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 18 or SEQ ID NO: 28, and ii.
  • the ⁇ MSLN-Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 47 and a VL comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 48, and iii.
  • the present disclosure pertains to a trispecific antibody according to the present disclosure, wherein said trispecific antibody comprises a) an ⁇ MUC16-Fab comprising i.
  • VH variable heavy chain region
  • HCDR heavy chain complementary determining region
  • VL variable light chain region
  • LCDR light chain complementary determining region
  • LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 15 or SEQ ID NO: 25
  • LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 16, or SEQ ID NO: 26 and b) an ⁇ MSLN-Fab comprising i.
  • VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 42, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 43, and ii.
  • a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 34, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 35, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 46, and c) an ⁇ CD3-Fv comprising a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 52 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 53 and a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 54, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 56.
  • the present disclosure pertains to a trispecific antibody according to the present disclosure, wherein said trispecific antibody comprises i. an ⁇ MUC16-Fab comprising a VH comprising the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 17 or SEQ ID NO: 27 and a VL comprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 18 or SEQ ID NO: 28, and ii. an ⁇ MSLN-Fab comprising a VH comprising the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 47 and a VL comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 48, and iii.
  • an ⁇ CD3-Fv comprises a VH comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 159 and a VL comprising the amino acid sequence of SEQ ID NO: 58.
  • the present disclosure pertains to a trispecific antibody according to the present disclosure, wherein said trispecific antibody comprises i.
  • an ⁇ MUC16-Fab comprising 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 of SEQ ID NO: 7, SEQ ID NO: 17 or SEQ ID NO: 27 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 of SEQ ID NO: 8, SEQ ID NO: 18 or SEQ ID NO: 28, and ii.
  • an ⁇ MSLN-Fab comprising a VH 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 of SEQ ID NO: 37 or SEQ ID NO: 47 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 of SEQ ID NO: 38 or SEQ ID NO: 48, and iii.
  • an ⁇ CD3-Fv 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 of SEQ ID NO: 57 or SEQ ID NO: 159 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 of SEQ ID NO: 58.
  • said ⁇ CD3-VH1 or ⁇ CD3-VH2 comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 51, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 52 and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 53.
  • said ⁇ CD3-VL1 or ⁇ CD3-VL2 comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO: 54, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 56.
  • said ⁇ CD3-VH1 or ⁇ CD3-VH2 comprises the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 159 and said ⁇ CD3-VL1 or ⁇ CD3-VL2 comprises the amino acid sequence of SEQ ID NO: 58.
  • the trispecific antibody according to the present disclosure further comprises a first Fc region and/or a second Fc region.
  • said HB-1 further comprises a first Fc region.
  • said HB-2 further comprises a second Fc region.
  • said first Fc region is positioned between the ⁇ MUC16-Fab and the either ⁇ CD3- VH1 or ⁇ CD3-VL1. In an aspect, the said first Fc region is positioned at the C-terminus of the either ⁇ CD3-VH1 or ⁇ CD3-VL1. In an aspect, said second Fc region is positioned between the ⁇ MSLN-Fab and the ⁇ CD3-VH2 or ⁇ CD3-VL2. In an aspect, said second Fc region is positioned at the C-terminus of the ⁇ CD3-VH2 or ⁇ CD3-VL2. In an aspect, each of said first and said second Fc region is composed of a first and second Fc region subunit.
  • each of said Fc region subunit is composed of a CH2 and CH3 immunoglobulin constant domain.
  • the Fc region of the trispecific antibody according to the present disclosure or of HB-1 and/or HB-2 according to the present disclosure comprises one or more amino acid mutations promoting the association of the first and the second Fc region subunit.
  • the Fc region of HB-1 and/or HB-2 according to the present disclosure comprises one or more amino acid mutations that reduces the binding affinity of the Fc region to an Fc receptor and/or to C1q and/or reduces its effector function.
  • the ⁇ MUC16-Fab, the ⁇ CD3-VH1 or ⁇ CD3-VL1 and the first Fc region of HB-1 are fused to each other via peptide linkers.
  • said peptide linkers each have a length of between 1 – 50 amino acid residues.
  • the C-terminus of the heavy chain of the ⁇ MUC16-Fab is fused to the N-terminus of the either ⁇ CD3-VH1 or ⁇ CD3-VL1 via a first peptide linker.
  • the C-terminus of the either ⁇ CD3-VH1 or ⁇ CD3-VL1 is fused to the N-terminus of the first Fc region subunit of the first Fc region via a second peptide linker.
  • the C-terminus of the heavy chain of the ⁇ MUC16-Fab is fused to the N-terminus of the first Fc region subunit of the first Fc region.
  • the C-terminus of the first Fc region subunit of the first Fc region is fused to the N- terminus of the either ⁇ CD3-VH1 or ⁇ CD3-VL1.
  • the ⁇ MSLN-Fab, the ⁇ CD3-VH2 or ⁇ CD3-VL2, and the second Fc region of HB-2 are fused to each other via peptide linkers.
  • the C-terminus of the heavy chain of the ⁇ MSLN-Fab is fused to the N-terminus of the either ⁇ CD3-VH2 or ⁇ CD3-VL2 via a first peptide linker.
  • the C-terminus of the ⁇ CD3-VH2 or ⁇ CD3-VL2 is fused to the N-terminus of the first Fc region subunit of the second Fc region via a second peptide linker.
  • the C-terminus of the heavy chain of the ⁇ MSLN-Fab is fused to the N- terminus of the first Fc region subunit of the second Fc region via a first peptide linker.
  • the C-terminus of the first Fc region subunit of the second Fc region is fused to the N-terminus of the ⁇ CD3-VH2 or aCD3-VL2 via a second peptide linker.
  • HB-1 is composed of a first and second polypeptide, wherein a) the first polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the ⁇ MUC16-Fab, a first peptide linker and either the ⁇ CD3-VH or ⁇ CD3-VL and b) the second polypeptide comprises the light chain of the ⁇ MUC16-Fab.
  • HB-1 is composed of a first, second and third polypeptide, wherein a) the first polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the ⁇ MUC16-Fab, a first peptide linker, either the ⁇ CD3-VH1 or ⁇ CD3-VL1, a second peptide linker, and a first Fc region subunit, b) the second polypeptide comprises the light chain of the ⁇ MUC16-Fab, c) the third polypeptide comprises from its N-terminus to its C-terminus, a third peptide linker and a second Fc region subunit.
  • HB-1 is composed of a first, second and third polypeptide, wherein a) the first polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the ⁇ MUC16-Fab, a first peptide linker, a first Fc region subunit, a second peptide linker, either the ⁇ CD3-VH1 or ⁇ CD3-VL1, b) the second polypeptide comprises the light chain of the ⁇ MUC16-Fab, and c) the third polypeptide comprises from its N-terminus to its C-terminus, a third peptide linker and the second Fc region subunit.
  • said first, second and/or third peptide linker comprises 1 – 50 amino acid residues. In an aspect, said first, second and/or third peptide linker has a length of 1 – 50 amino acid residues. In an aspect, said first, second and/or third peptide linker comprises an amino acid sequence selected from the group of: SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142; SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 128, S
  • HB-1 is composed of a first, second and third polypeptide, wherein said first polypeptide comprises any one of the amino acid sequences selected from the group of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 and SEQ ID NO: 169.
  • said HB-1 is composed of a first, second and third polypeptide, wherein said second polypeptide of HB-1 comprises any one of the amino acid sequences selected from the group of SEQ ID NO: 10, SEQ ID NO: 20, and SEQ ID NO: 30.
  • said HB-1 is composed of a first, second and third polypeptide, wherein said third polypeptide of HB-1 comprises the amino acid sequence of SEQ ID NO: 87.
  • HB-1 is composed of a first, second and third polypeptide, wherein a) said first polypeptide comprises any one of the amino acid sequences selected from the group of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 and SEQ ID NO: 169, b) said second polypeptide comprises any one of the amino acid sequences selected from the group of SEQ ID NO: 10, SEQ ID NO: 20, and SEQ ID NO: 30, and c) said third polypeptide comprises the amino acid sequence of SEQ ID NO: 87.
  • HB-1 is composed of a first, second and third polypeptide, wherein a) said first polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 and SEQ ID NO: 169, b) said second polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%,
  • HB-2 is composed of a first and second polypeptide, wherein a) the first polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the ⁇ MSLN-Fab, a first peptide linker and either the ⁇ CD3-VL2 or ⁇ CD3-VH2 and b) the second polypeptide comprises the light chain of the ⁇ MSLN-Fab.
  • HB-2 is composed of a first, second and third polypeptide, wherein a) the first polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the ⁇ MSLN-Fab, a first peptide linker, either the ⁇ CD3-VL2 or ⁇ CD3-VH2, a second peptide linker, and a first Fc region subunit. b) the second polypeptide comprises the light chain of the ⁇ MSLN-Fab, and c) the third polypeptide comprises from its N-terminus to its C-terminus, a third peptide linker and the second Fc region subunit.
  • HB-2 is composed of a first, second and third polypeptide, wherein a) the first polypeptide comprises from its N-terminus to its C-terminus, the heavy chain of the ⁇ MSLN-Fab, a first peptide linker, a first Fc region subunit, a second peptide linker, and either the ⁇ CD3-VL2 or ⁇ CD3-VH2, and b) the second polypeptide comprises the light chain of the ⁇ MSLN-Fab, and c) the third polypeptide comprises from its N-terminus to its C-terminus, a third peptide linker and the second Fc region subunit.
  • said first, second and/or third peptide linker comprises 1 – 50 amino acid residues. In an aspect, said first, second and/or third peptide linker has a length of 1 – 50 amino acid residues. In an aspect, said first, second and/or third peptide linker comprises an amino acid sequence selected from the group of: SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142; SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 128, S
  • said HB-2 is composed of a first, second and third polypeptide, wherein the first polypeptide of HB-2 comprises any one of the amino acid sequence selected from the group of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163.
  • said HB-2 according to the present disclosure is composed of a first, second and third polypeptide, wherein said second polypeptide of HB-2 comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 50.
  • said HB-2 according to the present disclosure is composed of a first, second and third polypeptide, wherein said third polypeptide of HB-2 comprises the amino acid sequence of SEQ ID NO: 87.
  • said HB-2 is composed of a first, second and third polypeptide, wherein a) the first polypeptide of HB-2 comprises any one of the amino acid sequence selected from the group of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, b) wherein said second polypeptide of HB-2 comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 50 and c) wherein said third polypeptide of HB-2 comprises the amino acid sequence of SEQ ID NO: 87.
  • said HB-2 is composed of a first, second and third polypeptide, wherein d) the first polypeptide of HB-2 comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, e) wherein said second polypeptide of HB-2 comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%
  • said HB-1 and HB-2 according to the present disclosure are not linked by a covalent bond. In an aspect, said HB-1 and HB-2 are not linked to each other by a covalent bond. In an aspect, said HB-1 and HB-2 are capable of non-covalently associating with each other. In an aspect, said HB-1 and HB-2 are capable of non-covalently associating which each other thereby forming a trispecific antibody according to the present disclosure. In an aspect, said HB-1 by itself is capable of binding to MUC16. In an aspect, said HB-2 by itself is capable of binding to MSLN. In an aspect, neither said HB-1 by itself nor said HB-2 by itself is capable of binding to CD3.
  • said HB-1 by itself is not capable of binding to CD3.
  • said HB-2 by itself is not capable of binding to CD3.
  • neither said ⁇ CD3-VH by itself nor said ⁇ CD3-VL by itself is capable of binding to CD3.
  • the ⁇ CD3-VH and the ⁇ CD3-VL according to the present disclosure are capable of non-covalently associating with each other thereby forming the ⁇ CD3-Fv.
  • the ⁇ CD3-VH1 present on HB-1 and the complementary ⁇ CD3-VL2 present on HB-2 are capable of non-covalently associating with each other thereby forming the ⁇ CD3-Fv.
  • said ⁇ CD3-VL1 present on HB-1 and said complementary ⁇ CD3-VH2 present on HB-2 are capable of non-covalently associating with each other thereby forming the ⁇ CD3-Fv.
  • said non-covalent association of the ⁇ CD3-VH1 with the ⁇ CD3-VL2 or of the ⁇ CD3-VL1 with the ⁇ CD3-VH2 occurs upon binding of HB-1 to MUC16 and upon binding of HB-2 to MSLN.
  • said non-covalent association of HB-1 and HB-2 occurs in solution.
  • said non-covalent association occurs when HB-1 and HB-2 are mixed in solution.
  • said non-covalent association occurs when HB-1 and HB-2 are present in the same solution. In an aspect, said non-covalent association occurs on the surface of a cell. In some aspects, said non-covalent association preferably occurs on the surface of a cell. In some aspects, said non-covalent association occurs in the presence of MUC16 and MSLN. In some aspects, said non-covalent association occurs upon binding of HB-1 to MUC16. In some aspects, said non-covalent association occurs upon binding of HB-2 to MSLN. In an aspect, said MUC16 and MSLN are expressed or are present on the surface of the same cell. In some aspects, said non-covalent association occurs upon binding of HB-1 to MUC16 expressed on a cell.
  • said non-covalent association occurs upon binding of HB-1 to MUC16 present on the surface a cell. In some aspects, said non-covalent association occurs upon binding of HB-2 to MSLN expressed on a cell. In some aspects, said non-covalent association occurs upon binding of HB-2 to MSLN present on the surface of a cell. In a preferred aspect, said non-covalent association occurs upon binding of HB-1 to MUC16 and upon binding of HB-2 to MSLN. More preferably, said non-covalent association occurs upon binding of HB-1 to MUC16 expressed or present on the surface of a cell and upon binding of HB-2 to MSLN expressed or present on the surface of the same cell.
  • MUC16 and MSLN are expressed or present on the same cell.
  • the presence of a cell expressing or having both, MUC16 and MSLN on its cell surface induces the non-covalent association of HB-1 and HB-2.
  • said non-covalent association of HB-1 with HB-2 occurs via dimerization of the either VH or VL of the Fv fragment specific for CD3 present on HB-1 with the complementary VL or VH of the Fv fragment specific for CD3 present on HB-2.
  • the either VH or VL of the ⁇ CD3-Fv present on HB-1 and the complementary VL or VH of the Fv ⁇ CD3-Fv fragment present on HB-2 are capable of non-covalently associating with each other thereby forming the ⁇ CD3-Fv.
  • the presence of a cell expressing or having MUC16 and MSLN on its surface induces dimerization of the either ⁇ CD3-VH1 or ⁇ CD3-VL1 present of HB-1 with the complementary ⁇ CD3-VL2 or ⁇ CD3-VH2, respectively, present of HB-2.
  • the presence of a cell expressing or having MUC16 and MSLN on its surface induces the non-covalent association of the either ⁇ CD3-VH1 or ⁇ CD3-VL1 present of HB-1 with the ⁇ CD3-VL2 or ⁇ CD3-VH2, respectively present of HB-2.
  • the amount of non-covalently associated HB-1 and HB-2 formed under conditions where MUC16 and MSLN are present is higher than under conditions where MUC16 and MSLN are absent.
  • the amount of non-covalently associated HB-1 and HB-2 formed under conditions where MUC16 and MSLN are present is higher than under conditions where MUC16 is present but MSLN is absent or where MSLN is present but MUC16 is absent. In some aspects, the amount of non-covalently associated HB-1 and HB-2 formed under conditions where cells expressing MUC16 and MSLN on their cell surface are present, is higher than under conditions where cells expressing MUC16 but not MSLN on their cell surface are present or where cells expressing MSLN but not MUC16 on their cell surface are present.
  • the amount of non-covalently associated HB-1 and HB-2 formed under conditions where MUC16 and MSLN 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 amounts formed under conditions where cells expressing MUC16 but not MSLN on their cell surface are present or where cells expressing MSLN but not MUC16 on their cell surface are present.
  • the amount of the ⁇ CD3-Fv formed under conditions where MUC16 and MSLN are present is higher than under conditions where MUC16 and MSLN are absent. In some aspects, the amount of the ⁇ CD3-Fv formed under conditions where MUC16 and MSLN are present is higher than under conditions where MUC16 is present but MSLN is absent or where MSLN is present but MUC16 is absent. In some aspects, the amount of the ⁇ CD3-Fv formed under conditions where cells expressing MUC16 and MSLN on their cell surface are present, is higher than under conditions where cells expressing MUC16 and MSLN on their cell surface are absent.
  • the amount of the ⁇ CD3-Fv formed under conditions where cells expressing MUC16 and MSLN on their cell surface are present is higher than under conditions where cells expressing MUC16 but not MSLN on their cell surface are present or where cells expressing MSLN but not MUC16 on their cell surface are present.
  • the amount of the ⁇ CD3-Fv formed under conditions where MUC16 and MSLN 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 amounts formed under conditions where cells expressing MUC16 but not MSLN on their cell surface are present or where cells expressing MSLN but not MUC16 on their cell surface are present.
  • HB-1 by itself is not capable of inducing or does not induce T-cell mediated killing of a cell expressing MUC16 on its cell surface. In some aspects, HB-1 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. In some aspects, HB-2 by itself is not capable of inducing or does not induce T-cell mediated killing of a cell expressing MUC16 on its cell surface. In some aspects, HB-2 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 trispecific antibody according to the present disclosure is formed upon the non- covalent association of the either VH or VL of the Fv fragment specific for CD3 present on HB-1 with the complementary VL or VH of the Fv fragment specific for CD3 present on HB-2.
  • said trispecific antibody according to the present disclosure is formed upon binding of HB-1 to MUC16 and upon binding of HB-2 to MSLN.
  • said trispecific antibody according to the present disclosure is formed upon binding of HB-1 to MUC16 and upon binding of HB-2 to MSLN, wherein MUC16 and MSLN are both expressed on the surface of the same cell.
  • the amount of trispecific antibody formed under conditions where MUC16 and MSLN are present is higher than under conditions where MUC16 and MSLN are absent. In some aspects, the amount of trispecific antibody formed under conditions where MUC16 and MSLN are present is higher than under conditions where MUC16 is present but MSLN is absent or where MSLN is present but MUC16 is absent. In some aspects, the amount of trispecific antibody formed under conditions where cells expressing MUC16 and MSLN on their cell surface are present is higher than under conditions where cells expressing MUC16 and MSLN on their cell surface are absent.
  • the amount of trispecific antibody formed under conditions, where cells expressing MUC16 and MSLN on their cell surface are present is higher than under conditions where cells expressing MUC16 but not MSLN on their cell surface are present or where cells expressing MSLN but not MUC16 on their cell surface are present.
  • the amount of trispecific antibody formed under conditions where MUC16 and MSLN 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 amounts formed under conditions where cells expressing MUC16 but not MSLN on their cell surface are present or where cells expressing MSLN but not MUC16 on their cell surface are present.
  • the trispecific antibody according to the present disclosure is capable of inducing or induces T cell-mediated cell killing of cells expressing MUC16 and MSLN on their cell surface.
  • the trispecific antibody according to the present disclosure induces T cell mediated killing of cells expressing MUC16 and MSLN on their cell surface with an IC50 concentration of less than 500 pM, less than 450 pM, less than 400 pM, less than 350 pM,300 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than 100 pM, less than 90 pM, less than 80 pM, less than 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, or less than about 20 pM.
  • the complementary pair of HB-1 and HB-2 according to the present disclosure is capable of inducing or induces T cell mediated cell killing of cells expressing MUC16 and MSLN on their cell surface.
  • the complementary pair of HB-1 and HB-2 according to the present disclosure induces T cell mediated cell killing of cells expressing MUC16 and MSLN on their cell surface with an IC50 concentration of less than 500 pM, less than 450 pM, less than 400 pM, less than 350 pM,300 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than 100 pM, less than 90 pM, less than 80 pM, less than 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, or less than about 20 pM.
  • the trispecific antibody according to the present disclosure induces T cell-mediated cell killing of cells expressing MUC16 and MSLN on their cell surface with an IC50 concentration of between 10 pM and 500 pM.
  • the complementary pair of HB-1 and HB-2 according to the present disclosure induces T cell-mediated cell killing of cells expressing MUC16 and MSLN on their cell surface with an IC 50 concentration of between 10 pM and 500 pM.
  • the IC50 concentration for the induced T cell-mediated cell killing of cells expressing MUC16 and MSLN 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 lower compared to the IC 50 concentration for the induced T cell-mediated cell killing of cells expressing MUC16 or MSLN or neither of MUC16 and MSLN on their cell surface.
  • said T cell-mediated cell 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. In an aspect, said T cell-mediated cell killing is measured in presence PBMCs. In an aspect, said T cell-mediated cell killing is measured in presence of CD8+ and CD4+ T cells. In an aspect, said T cell-mediated cell killing is measured in presence of CD8+ T cells. In an aspect, said T cell-mediated cell killing is measured in presence of pan T cells. In an aspect, said T cells or PBMCs are human cells. In an aspect, said T cell-mediated cell killing is measured in an in vitro T cell-mediated tumor cell killing assay as described in Example 7. In an aspect, said cells are cancer cells. In an aspect, said cells are human cancer cells. In an aspect, said cells are solid cancer cells.
  • said cells are ovarian cancer cells. In an aspect, said cells are OVAR-3 cells (ATCC, HTB-161). In an aspect of the present disclosure, neither said HB-1 by itself nor said HB-2 by itself is capable of mediating T-cell redirected killing of cells expressing MUC16 and/or Mesothelin on their cell surface. In an aspect of the present disclosure, neither said HB-1 by itself nor said HB- 2 by itself is capable of inducing T-cell mediated killing of cells expressing MUC16 and/or Mesothelin on their cell surface. In an aspect of the present disclosure, HB-1 by itself is not capable of mediating T-cell redirected killing of cells expressing MUC16 on their cell surface.
  • HB-1 by itself is not capable of inducing T-cell mediated killing of cells expressing MUC16 on their cell surface.
  • HB-2 by itself is not capable of mediating T-cell redirected killing of cells expressing Mesothelin on their cell surface.
  • HB-2 by itself is not capable of inducing T-cell mediated killing of cells expressing Mesothelin on their cell surface.
  • the present disclosure provides a trispecific antibody as disclosed herein, wherein said trispecific antibody is composed of 6 polypeptides, wherein the first polypeptide comprises an amino acid sequence selected from the group of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 and SEQ ID NO: 169, the second polypeptide comprises an amino acid sequence selected from the group of SEQ ID NO: 10, SEQ ID NO: 20, and SEQ ID NO: 30, the third and sixth polypeptide each comprises the amino acid sequence of SEQ ID NO: 87, the fourth polypeptide comprises an amino acid sequence of
  • said first and second polypeptide are associated to form the Fab fragment specific for MUC16.
  • the fourth and the fifth polypeptide are associated to form the Fab fragment specific for MSLN.
  • said first and third polypeptide are associated to form the first Fc region.
  • said fourth and sixth polypeptide are associated to form the second Fc region.
  • the first and second and that first and third polypeptide are linked to each other via a covalent bond.
  • the fourth and fifth polypeptide and that fourth and sixth polypeptide are linked to each other via a covalent bond.
  • neither said first, second and third polypeptide is covalently linked to the fourth, fifth and sixth polypeptide.
  • the first polypeptide and the fourth polypeptide are capable of non-covalently associating with each other thus forming a Fv fragment specific for CD3.
  • the VH of the Fv fragment specific for CD3 is present on the first polypeptide and the VL of the Fv fragment specific for CD3 is present on the fourth polypeptide.
  • the VL of the Fv fragment specific for CD3 is present on the first polypeptide and the VH of the Fv fragment specific for CD3 is present on the fourth polypeptide.
  • MUC16 specific CyCAT halfbody molecules In an aspect, the present disclosure provides a first halfbody molecule (HB-1), wherein said HB- 1 is part of the trispecific antibody as disclosed herein.
  • the present disclosure provides a first halfbody molecule (HB-1), wherein said HB- 1 comprises a first Fab specific for MUC16 and either the VH or VL of an Fv specific for CD3.
  • said first Fab specific for MUC16 is any of the MUC16-Fabs of the present disclosure
  • said first Fab specific for MUC16 comprises the 6 CDRs of any one of the antibodies listed in Table 5, Table 6 or Table 7 of the present disclosure.
  • said first Fab specific for MUC16 comprises the VH and the VL of any one of the antibodies listed in Table 5, Table 6 or Table 7 of the present disclosure.
  • the VH of the Fv specific for CD3 comprises the HCDRs of any one of the CD3 specific antibodies of the present disclosure.
  • the VL of the Fv specific for CD3 comprises the LCDRs of any one of the CD3 specific antibodies of the present disclosure.
  • the VH of the Fv specific for CD3 comprises the 3 HCDRs of any one of the antibodies listed in Table 10 or Table 17 of the present disclosure.
  • the VL of the Fv specific for CD3 comprises the 3 LCDRs of any one of the antibodies listed in Table 10 or Table 17 of the present disclosure.
  • said HB-1 has a molecular weight more than 70 kilodalton (kDa), more than 75 kDa, more than 80 kDa, or more than 90 kDa.
  • said HB-1 comprise a Fc region.
  • said Fc region is composed of two Fc region subunits, each composed of a CH2 and CH3 immunoglobulin constant domain.
  • said HB-1 comprises a Fab fragment specific for MUC-16 and the VH but not the VL of a Fv specific for CD3 or the VL but not the VH of a Fv specific for CD3.
  • said HB-1 by itself is capable of binding to MUC-16.
  • said HB-1 by itself is not capable of binding to CD3. In some aspects, said HB-1 by itself is not capable of inducing T-cell mediated killing of a cell expressing MUC-16 on its cell surface. In some aspects, said HB-1 by itself is not capable of inducing proliferation and/or activation of a T-cell expressing CD3 on its cell surface.
  • the present disclosure provides a first halfbody molecule (HB-1), wherein said HB- 1 comprises from its N-terminus to its C-terminus, a first Fab specific for MUC-16, a first peptide linker, and either the VH or the VL of an antibody Fv fragment (Fv) specific for CD3.
  • the present disclosure provides a first halfbody molecule (HB-1), wherein said HB- 1 comprises from its N-terminus to its C-terminus, a first Fab fragment specific for MUC-16, a first peptide linker, either the VH or the VL of an antibody Fv fragment (Fv) specific for CD3, a second peptide linker, and a Fc region.
  • HB-1 first halfbody molecule
  • said HB- 1 comprises from its N-terminus to its C-terminus, a first Fab specific for MUC-16, a Fc region, a first peptide linker, and either the VH or the VL of an antibody Fv fragment (Fv) specific for CD3.
  • the present disclosure provides a first halfbody molecule (HB-1), wherein said HB- 1 comprises from its N-terminus to its C-terminus, a first Fab specific for MUC-16, a first peptide linker, a Fc region, a second peptide linker, and either the VH or the VL of an antibody Fv fragment (Fv) specific for CD3.
  • the peptide linker comprises any one of the peptide linkers according to the present disclosure.
  • said peptide linker comprises 1 – 50 amino acid residues.
  • said peptide linker has a length of 1 – 50 amino acid residues.
  • the Fc region comprises any one of the Fc regions according to the present disclosure.
  • the present disclosure provides a first halfbody molecule (HB-1), wherein the first polypeptide of HB-1 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 of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 and SEQ ID NO: 169, and wherein the second polypeptide of HB-1
  • the present disclosure provides a first halfbody molecule (HB-1), wherein the first polypeptide of HB-1 comprises an amino acid sequence selected from the group of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 and SEQ ID NO: 169, and wherein the second polypeptide of HB-1 comprises an amino acid sequence selected from the group of: SEQ ID NO: 10, SEQ ID NO: 20, and SEQ ID NO: 30, and wherein the third polypeptide of HB-1 comprises an amino acid sequence of SEQ ID NO: 87.
  • the present disclosure provides a first halfbody molecule (HB-1), wherein (a) the first polypeptide of HB-1 comprises an amino acid sequence selected from the group of SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 164, and SEQ ID NO: 165, wherein the second polypeptide of HB-1 comprises the amino acid sequence of SEQ ID NO: 10, and wherein the third polypeptide of HB-1 comprises the amino acid sequence of SEQ ID NO: 87; or (b) wherein the first polypeptide of HB-1 comprises an amino acid sequence selected from the group of SEQ ID NO: 79; SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 166 and SEQ ID NO: 167, wherein the second polypeptide of HB-1 comprises the amino acid sequence of SEQ ID NO: 20 and wherein the third polypeptide of HB-1 comprises the amino acid sequence of SEQ
  • the present disclosure pertains to an antibody or antibody fragment specific for MUC16.
  • said antibody fragment is the ⁇ MUC16-Fab according to the present disclosure.
  • said antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab comprises the 6 CDRs of any one of the antibodies listed in Tables 5 – 7.
  • said antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab comprises the 6 CDRs defined by Kabat of any one of the antibodies listed in Tables 5 – 7.
  • the present disclosure pertains to an antibody or antibody fragment specific for MUC16 or of ⁇ MUC16-Fab, comprising a VH and a VL of any one of the antibodies listed in Tables 5 – 7. In an aspect, the present disclosure pertains to an antibody or antibody fragment specific for MUC16 or of ⁇ MUC16-Fab, comprising a HC and a LC of any one of the antibodies listed in in Tables 5 – 7.
  • said ⁇ antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab comprises a) an antibody variable heavy chain region (VH) comprising an heavy chain complementary determining region (HCDR)1 region comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 11, or SEQ ID NO: 21; an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 12, or SEQ ID NO: 22, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 13, or SEQ ID NO: 23; and b) 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, SEQ ID NO: 14, or SEQ ID NO: 24, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 15, or SEQ ID NO: 25, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:
  • VH antibody variable heavy chain
  • said ⁇ antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab comprises a) a VH comprising an HCDR1 comprising the amino acid sequence of SHTAAWT (SEQ ID NO: 1), an HCDR2 comprising the amino acid sequence of RTYYRSKWYYDYAQSVKS (SEQ ID NO: 2) and an HCDR3 comprising the amino acid sequence of ISYGYLKAYSMDA (SEQ ID NO: 3); and a VL comprising a LCDR1 comprising the amino acid sequence of RASQGISSYLA (SEQ ID NO: 4), a LCDR2 comprising the amino acid sequence of AASTLQS (SEQ ID NO: 5), and a LCDR3 comprising the amino acid sequence of QQHIQIPLT (SEQ ID NO: 6); or b) a VH comprising an HCDR1 comprising the amino acid sequence of SYYIH (SEQ ID NO: 11), an HCDR2 comprising the amino acid
  • said ⁇ antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 17 or SEQ ID NO: 27 and a VL comprising the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 18, or SEQ ID NO: 28.
  • said ⁇ antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab 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 the amino acid sequence of QVQLQQSGPGLVKPSQTLSLTCAISGVSVSSHTAAWTWIRQSPSRGLEWLGRTYYRSKWYYD YAQSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARISYGYLKAYSMDAWGQGTLVTVSS (SEQ ID NO: 7) 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 of DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTL
  • said ⁇ antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab comprises a) a VH comprising the amino acid sequence of QVQLQQSGPGLVKPSQTLSLTCAISGVSVSSHTAAWTWIRQSPSRGLEWLGRTYYRSKWYYD YAQSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARISYGYLKAYSMDAWGQGTLVTVSS (SEQ ID NO: 7) and a VL comprising the amino acid sequence of DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGS GSGTEFTLTISSLQPEDFATYYCQQHIQIPLTFGQGTKVEIKRT (SEQ ID NO: 8); or b) a VH comprising the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIH
  • said ⁇ antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab comprises: a) a HC comprising the amino acid sequence of: QVQLQQSGPGLVKPSQTLSLTCAISGVSVSSHTAAWTWIRQSPSRGLEWLGRTYYRSKWYYD YAQSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARISYGYLKAYSMDAWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC (SEQ ID NO: 9) and a LC comprising the amino acid sequence of: DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGS GSGTEFTLTISSLQPEDFATYY
  • said antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab is an isolated antibody or antibody fragment.
  • the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab is a human, humanized or chimeric antibody or antibody fragment.
  • said antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab is a recombinant or a synthetic antibody or antibody fragment.
  • said antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab is a monoclonal antibody or antibody fragment.
  • said antibody fragment is selected from the group 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 antibody fragment is a Fab.
  • the antibody or antibody fragment specific for MUC16 according to the present disclosure is of the IgG isotype. In an aspect, the antibody or antibody fragment specific for MUC16 is an IgG1.
  • the present disclosure pertains to an multispecific antibody comprising a first binding domain of an antibody or antibody fragment specific for MUC16 or of said ⁇ MUC16-Fab according to the present disclosure and a second binding domain specific for a different antigen.
  • the present disclosure pertains to an multispecific antibody comprising a first binding domain of an antibody or antibody fragment specific for MUC16 or of said ⁇ MUC16-Fab according to the present disclosure and a second binding domain of an antibody or antibody fragment specific for CD3.
  • the present disclosure provides a pharmaceutical composition comprising the antibody or antibody fragment specific for MUC16 or the ⁇ MUC16-Fab according to the present disclosure and a pharmaceutically acceptable carrier or excipient.
  • the present disclosure provides an antibody or antibody fragment specific for MUC16 or the ⁇ MUC16-Fab according to the present disclosure for use as a medicament.
  • the present disclosure provides an antibody or antibody fragment specific for MUC16 or the ⁇ MUC16-Fab according to the present disclosure for use in the treatment of a subject in need thereof.
  • said antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab according to the present disclosure is specific for human MUC16.
  • said antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab specifically binds to the extracellular region of human MUC16.
  • said antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab is specific for human MUC16 having the amino acid sequence of SEQ ID NO: 59. In an aspect, said antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab is specific for a polypeptide comprising the amino acid sequence of SEQ ID NO: 59. In an aspect, the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab specifically binds to or is specific for a human MUC16 polypeptide having an amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 97.
  • the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab is specific for human MUC16 and cynomolgus monkey MUC16. In an aspect, said antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab is specific for human MUC16 and is cross-reactive to cynomolgus monkey MUC16. In an aspect, the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab is specific for human MUC16 and cross-reactively binds to cynomolgus monkey MUC16.
  • the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab specifically binds to human MUC16 and cross-reactively binds to cynomolgus MUC16.
  • said cynomolgus MUC16 comprises the amino acid sequence of SEQ ID NO: 60.
  • the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab specifically binds to the extracellular region of cynomolgus MUC16.
  • the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab specifically binds to a cynomolgus MUC16 polypeptide having an amino acid sequence of SEQ ID NO: 60 or SEQ ID NO: 98.
  • HB-1 and/or the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab binds to an human MUC16 polypeptide comprising the amino acid sequence of SEQ ID NO: 97 with a K D of about 1 nM or less, such as 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, such as about 0.45 nM or less, about 0.4 nM or less, about 0.35 nM or less, about 0.3 nM or less, about 0.25 nM or less, about 0.2 nM or less, about 0.15 nM or less, about 0.1 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.
  • HB-1 and/or the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab according to the present disclosure bind to a cynomolgus monkey MUC16 polypeptide comprising the amino acid sequence of SEQ ID NO: 98, with a KD of about 1 nM or less, such as 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, such as about 0.45 nM or less, about 0.4 nM or less, about 0.35 nM or less, about 0.3 nM or less, about 0.25 nM or less, about 0.2 nM or less, about 0.15 nM or less, about 0.1 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.
  • HB-1 and/or the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab bind to a human MUC16 polypeptide comprising the amino acid sequence of SEQ ID NO: 97 and to a cynomolgus monkey MUC16 polypeptide comprising the amino acid sequence of SEQ ID NO: 98 with a K D of about 1 nM or less, such as 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, such as about 0.45 nM or less, about 0.4 nM or less, about 0.35 nM or less, about 0.3 nM or less, about 0.25 nM or less, about 0.2 nM or less, about 0.15 nM or less, about 0.1 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. 0. 0.
  • said 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.
  • HB-1 and/or the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab according to the present disclosure bind to MUC16 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 or less, 0.07 nM or less, 0.06 nM or less, 0.05 nM or less, 0.04 nM or less, 0.03 nM or less, or 0.02
  • HB-1 and/or the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab according to the present disclosure bind to MUC16 expressed on cells with an EC 50 concentration of between 1 nM and 10 nM. In certain aspects of the present disclosure, HB-1 and/or the antibody or antibody fragment specific for MUC16 or said ⁇ MUC16-Fab according to the present disclosure bind to MUC16 expressed on cells with an EC50 concentration of about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM or 10 nM. In certain aspects, said MUC16 is human MUC16.
  • said cells are human cells. In certain aspects, said cells are cancerous cells. In certain aspects, said cells are malignant cells. In certain aspects, said cells are OVCAR-3 cells. In an aspect, said cell is a cell of a solid cancer. In an aspect, said cell is a cell of an ovarian cancer. In some aspects, said EC50 concentration is determined by an FACS assay as described herein in Example 6. MSLN specific CyCAT halfbody molecules In an aspect, the present disclosure provides a second halfbody molecule (HB-2), wherein said HB-2 is part of the trispecific antibody as disclosed herein.
  • HB-2 second halfbody molecule
  • the present disclosure provides a second halfbody molecule (HB-2), wherein said HB-2 comprises a second Fab specific for MSLN and either the VH or VL of an Fv specific for CD3.
  • said second Fab specific for MSLN is any of the ⁇ MSLN-Fabs of to the present disclosure.
  • said second Fab specific for MSLN comprises the 6 CDRs of any one of the antibodies listed in Table 8 or Table 9 of the present disclosure.
  • said second Fab specific for MSLN comprises the VH and the VL of any one of the antibodies listed in Table 8 or Table 9 of the present disclosure.
  • the VH of the Fv specific for CD3 comprises the HCDRs of any one of the CD3 specific antibodies of the present disclosure.
  • the VL of the Fv specific for CD3 comprises the LCDRs of any one of the CD3 specific antibodies of the present disclosure.
  • the VH of the Fv specific for CD3 comprises the 3 HCDRs of any one of the antibodies listed in Table 10 or Table 17 of the present disclosure.
  • the VL of the Fv specific for CD3 comprises the 3 LCDRs of any one of the antibodies listed in Table 10 or Table 17 of the present disclosure.
  • said HB-2 has a molecular weight more than 70 kilodalton (kDa), more than 75 kDa, more than 80 kDa, or more than 90 kDa.
  • said HB-2 comprise a Fc region.
  • said Fc region is composed of two Fc region subunits, each composed of a CH2 and CH3 immunoglobulin constant domain.
  • said HB-2 comprises a Fab specific for MSLN and the VH but not the VL of a Fv specific for CD3 or the VL but not the VH of a Fv specific for CD3.
  • said HB-2 by itself is capable of binding to MSLN.
  • said HB-2 by itself is not capable of binding to CD3. In some aspects, said HB-2 by itself is not capable of inducing T-cell mediated killing of a cell expressing MSLN on its cell surface. In some aspects, said HB-2 by itself is not capable of inducing proliferation and/or activation of a T-cell expressing CD3 on its cell surface.
  • the present disclosure provides a second halfbody molecule (HB-2), wherein said HB-2 comprises from its N-terminus to its C-terminus, a second Fab specific for MSLN, a third peptide linker, and either the VH or the VL of an antibody Fv fragment (Fv) specific for CD3.
  • the present disclosure provides a second halfbody molecule (HB-2), wherein said HB-2 comprises from its N-terminus to its C-terminus, a second Fab fragment specific for MSLN, a third peptide linker, either the VH or the VL of an antibody Fv fragment (Fv) specific for CD3, a fourth peptide linker, and a Fc region.
  • HB-2 second halfbody molecule
  • said HB-2 comprises from its N-terminus to its C-terminus, a second Fab specific for MSLN, a Fc region, a third peptide linker, and either the VH or the VL of an antibody Fv fragment (Fv) specific for CD3.
  • the present disclosure provides a second halfbody molecule (HB- 2), wherein said HB-2 comprises from its N-terminus to its C-terminus, a second Fab specific for MSLN, a third peptide linker, a Fc region, a fourth peptide linker, and either the VH or the VL of an antibody Fv fragment (Fv) specific for CD3.
  • the peptide linker comprises any one of the peptide linkers according to the present disclosure.
  • said peptide linker comprises 1 – 50 amino acid residues.
  • said peptide linker has a length of 1 – 50 amino acid residues.
  • the Fc region comprises any one of the Fc regions according to the present disclosure.
  • the present disclosure provides a second halfbody molecule (HB-2), wherein the first polypeptide of HB-2 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 of : SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, and wherein the second polypeptide of HB-2 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
  • the present disclosure provides a second halfbody molecule (HB-2), wherein the first polypeptide of HB-2 comprises an amino acid sequence selected from the group of: SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, and SEQ ID NO: 163, and wherein the second polypeptide of HB-2 comprises an amino acid sequence selected from the group of: SEQ ID NO: 40 or SEQ ID NO: 50, and wherein the third polypeptide of HB-2 comprises an amino acid sequence of SEQ ID NO: 87.
  • the first polypeptide of HB-2 comprises an amino acid sequence selected from the group of: SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO
  • the present disclosure provides a second halfbody molecule (HB-2), wherein (a) the first polypeptide of HB-2 comprises an amino acid sequence selected from the group of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 160, and SEQ ID NO: 161, wherein the second polypeptide of HB-2 comprises the amino acid sequence of SEQ ID NO: 40, and wherein the third polypeptide of HB-2 comprises the amino acid sequence of SEQ ID NO: 87; or (b) wherein the first polypeptide of HB-2 comprises an amino acid sequence selected from the group of SEQ ID NO: 67; SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 162 and SEQ ID NO: 163, wherein the second polypeptide of HB-2 comprises the amino acid sequence of SEQ ID NO: 50 and wherein the third polypeptide of HB-2 comprises the amino acid sequence of SEQ ID NO:
  • the present disclosure pertains to an antibody or antibody fragment specific for MSLN.
  • said antibody fragment is the ⁇ MSLN-Fab according to the present disclosure.
  • said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab comprise the 6 CDRs of any one of the antibodies listed in Tables 8 – 9.
  • said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab comprises the 6 CDRs defined by Kabat of any one of the antibodies listed in Tables 8 – 9.
  • the present disclosure pertains to an antibody or antibody fragment specific for MSLN or of ⁇ MSLN-Fab comprising a VH and a VL of any one of the antibodies listed in in Tables 8 – 9.
  • said ⁇ antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab according to the present disclosure comprises i. a VH comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 42, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 33 or SEQ ID NO: 43; and ii.
  • a VL comprising a LCDR1 comprising the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 24, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 25, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 46.
  • said ⁇ antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab comprises a) a VH comprising an HCDR1 comprising the amino acid sequence of SHWIH (SEQ ID NO: 31), an HCDR2 comprising the amino acid sequence of LISSGGSGVEYAESVKG (SEQ ID NO: 32), and an HCDR3 comprising the amino acid sequence GSWRYHGFDI (SEQ ID NO: 33) and a VL comprising a LCDR1 comprising the amino acid sequence of TGTSSDVGSYNLVS (SEQ ID NO: 34), a LCDR2 comprising the amino acid sequence of EGSKRPS (SEQ ID NO: 35), and a LCDR3 comprising the amino acid sequence of SSWVLRSVKV (SEQ ID NO: 36); or b) a VH comprising an HCDR1 comprising the amino acid sequence of RYALN (SEQ ID NO: 41), an HCDR2 comprising the amino acid sequence of HI
  • said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab comprises a VH comprising the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 47 and a VL comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 48.
  • said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab 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 the amino acid sequence of QVQLVESGGGLVKPGGSLRLSCAASGFTFGSHWIHWIRQAPGKGLEWVSLISSGGSGVEYAE SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGSWRYHGFDIWGQGTLVTVSS (SEQ ID NO: 37) 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 of QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNR FSGSKSGNT
  • said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab comprises a) a VH comprising the amino acid sequence of QVQLVESGGGLVKPGGSLRLSCAASGFTFGSHWIHWIRQAPGKGLEWVSLISSGGSGVEYAE SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGSWRYHGFDIWGQGTLVTVSS (SEQ ID NO: 37) and a VL comprising the amino acid sequence of QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNR FSGSKSGNTASLTISGLQAEDEADYYCSSWVLRSVKVFGGGTKLTVLGQ (SEQ ID NO: 38); or b) VH comprising the amino acid sequence of EVQLVESGGGLVKPGGSLRLSCAASGFTFSRYALNWVRQAPGKGLEWVGHIKTQAAGGTTEY
  • said ⁇ antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab comprises: a) a HC comprising an amino acid sequence of: QVQLVESGGGLVKPGGSLRLSCAASGFTFGSHWIHWIRQAPGKGLEWVSLISSGGSGVEYAE SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGSWRYHGFDIWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC (SEQ ID NO: 39) and a LC comprising an amino acid sequence of: QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQQHPGKAPKLMIYEGSKRPSGVSNR FSGSKSGNTASLTISGLQAEDEADYYCSSWVLRSVK
  • the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab is a human, humanized or chimeric antibody or antibody fragment.
  • said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab is a recombinant or a synthetic antibody or antibody fragment.
  • said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab is a monoclonal antibody or antibody fragment.
  • said antibody fragment is selected from the group 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 present disclosure provides a pharmaceutical composition comprising the antibody or antibody fragment specific for MSLN or the ⁇ MSLN-Fab according to the present disclosure and a pharmaceutically acceptable carrier or excipient.
  • the present disclosure provides an antibody or antibody fragment specific for MSLN or the ⁇ MSLN-Fab according to the present disclosure for use as a medicament.
  • the present disclosure provides an antibody or antibody fragment specific for MSLN or the ⁇ MSLN-Fab according to the present disclosure for use in the treatment of a subject in need thereof.
  • said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab according to the present disclosure is specific for human MSLN.
  • said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab is specific for human MSLN having the amino acid sequence of SEQ ID NO: 61. In an aspect, said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab is specific for a polypeptide comprising the amino acid sequence of SEQ ID NO: 61. In an aspect, said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab specifically binds to the extracellular region of human MSLN. In an aspect, said extracellular region of human MSLN comprises the amino acid sequence of SEQ ID NO: 62.
  • the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab specifically binds to or is specific for a human MSLN polypeptide having an amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 62, or SEQ ID NO: 99.
  • the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab is specific for human MSLN and cynomolgus monkey MSLN.
  • said antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab is specific for human MSLN and is cross- reactive to cynomolgus monkey MSLN.
  • the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab is specific for human MSLN and cross-reactively binds to cynomolgus monkey MSLN.
  • the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab specifically binds to human MSLN and cross-reactively binds to cynomolgus MSLN.
  • said cynomolgus MSLN comprises the amino acid sequence of SEQ ID NO: 91.
  • the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab specifically binds to the extracellular region of cynomolgus MSLN.
  • said extracellular region of cynomolgus MSLN comprises the amino acid sequence of SEQ ID NO: 92.
  • the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab specifically binds to a cynomolgus MSLN polypeptide having an amino acid sequence of SEQ ID NO: 91, SEQ ID NO: 92 or SEQ ID NO: 100.
  • HB-2 and/or the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab according to the present disclosure bind to a human MSLN polypeptide comprising the amino acid sequence of SEQ ID NO: 99 with a KD of about 1 nM or less, such as 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, such as about 0.45 nM or less, about 0.4 nM or less, about 0.35 nM or less, about 0.3 nM or less, about 0.25 nM or less, about 0.2 nM or less, about 0.15 nM or less, about 0.1 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.
  • HB-2 and/or the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab according to the present disclosure bind to a cynomolgus monkey MSLN polypeptide comprising the amino acid sequence of SEQ ID NO: 100, with a K D of about 1 nM or less, such as 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, such as about 0.45 nM or less, about 0.4 nM or less, about 0.35 nM or less, about 0.3 nM or less, about 0.25 nM or less, about 0.2 nM or less, about 0.15 nM or less, about 0.1 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.
  • HB-2 and/or the antibody or antibody fragment specific for MSLN or said ⁇ MSLN- Fab bind to a human MSLN polypeptide comprising the amino acid sequence of SEQ ID NO: 99 and to a cynomolgus monkey MSLN polypeptide comprising the amino acid sequence of SEQ ID NO: 100 with a K D of about 1 nM or less, such as 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, such as about 0.45 nM or less, about 0.4 nM or less, about 0.35 nM or less, about 0.3 nM or less, about 0.25 nM or less, about 0.2 nM or less, about 0.15 nM or less, about 0.1 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
  • said 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.
  • HB-2 and/or the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab according to the present disclosure bind to MSLN 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 or less, 0.07 nM or less, 0.06 nM or less, 0.05 nM or less, 0.04 nM or less, 0.03 nM or less, or 0.02
  • HB-2 and/or the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab according to the present disclosure bind to MSLN expressed on cells with an EC 50 concentration of between 1 nM and 10 nM.
  • HB-1 and/or the antibody or antibody fragment specific for MSLN or said ⁇ MSLN-Fab according to the present disclosure bind to MSLN expressed on cells with an EC 50 concentration of about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM or 10 nM.
  • said MSLN is human MSLN.
  • said cells are human cells. In certain aspects, said cells are cancerous cells. In certain aspects, said cells are malignant cells. In certain aspects, said cells are OVCAR-3 cells. In an aspect, said cell is a cell of a solid cancer. In an aspect, said cell is a cell of an ovarian cancer. In some aspects, said EC50 concentration is determined by an FACS assay as described herein in Example 6.
  • ⁇ CD3 Antibodies Preferably, the VH and VL domain that form the ⁇ CD3-Fv according to the present disclosure are those described in published International Application Number PCT/EP2021/076052, which is incorporated herein by reference in its entirety.
  • the VH and VL of the Fv specific for CD3 according to the present disclosure originates from or is a derivative or variant of any one of the CD3 specific antibodies disclosed in International Application No. PCT/EP2021/076052.
  • the VH of the Fv specific for CD3 is the ⁇ CD3-VH, the ⁇ CD3-VH1 or the ⁇ CD3-VH2 of the present disclosure.
  • the VL of the Fv specific for CD3 is the ⁇ CD3-VL, the ⁇ CD3-VL1 or the ⁇ CD3-VL2 of the present disclosure.
  • the ⁇ CD3-VH is the ⁇ CD3-VH1 or the ⁇ CD3-VH2 of the present disclosure.
  • the ⁇ CD3-VL is the ⁇ CD3-VL1 or the ⁇ CD3-VL2 of the present disclosure.
  • 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 Fv 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 ⁇ CD3-VL according to the present disclosure is of the V ⁇ 1 human germline gene family.
  • the framework regions of the ⁇ CD3-VL are encoded by the V ⁇ 1-47 human germline gene.
  • the ⁇ CD3-VH according to the present disclosure comprises VH3 human germline protein sequences.
  • the framework regions of the ⁇ CD3-VH comprises VH3-07 human germline protein sequences.
  • the ⁇ CD3-VL comprises V ⁇ ⁇ ⁇ human germline protein sequences. In some aspects, the framework regions of the ⁇ CD3-VL comprises V ⁇ 1-47 human germline protein sequences. In some aspects, the ⁇ CD3-VH according to the present disclosure comprises a V or M at position 37 (utilizing the nomenclature according Kabat). In some aspects, the ⁇ CD3-VH according to the present disclosure comprises a M at position 37 (utilizing the nomenclature according Kabat). In some aspects, the ⁇ CD3-VH according to the present disclosure comprises a V at position 37 (utilizing the nomenclature according Kabat).
  • the ⁇ CD3-VH according to the present disclosure comprises an amino acid substitution in framework regions 1, 2, 3 or 4. In some aspects, the ⁇ CD3-VH according to the present disclosure comprises not more than one amino acid substitution. In an aspect, said amino acid substitution is a V37M amino acid substitution (utilizing the nomenclature according Kabat). In some aspects, in the ⁇ CD3-VH of an halfbody molecule according to the present disclosure, V at position 37 is replaced for a M (V37M). In some aspects of the present disclosure, the M at position 37 (utilizing the nomenclature according Kabat) inhibits the association of the ⁇ MUC16-Fab light chain with the ⁇ CD3-VH present on HB-1 during recombinant production of HB-1.
  • the M at position 37 inhibits the association of the MSLN-Fab light chain with the ⁇ CD3-VH present of HB-2 during recombinant production of HB-2. In some aspects, the M at position 37 (utilizing the nomenclature according Kabat) reduces the amount of ⁇ MUC16-Fab light chain associated with the ⁇ CD3-VH during recombinant production of HB-1. In some aspects, the M at position 37 (utilizing the nomenclature according Kabat) reduces the amount of ⁇ MSLN-Fab light chain associated with the ⁇ CD3-VH during recombinant production of HB-2.
  • said M at position 37 reduces the amount of detectable ⁇ MUC16-Fab light chain after production of HB-1. In some aspects, the M at position (utilizing the nomenclature according Kabat) reduces the amount of detectable ⁇ MSLN-Fab light chain after production of HB-2. In an aspect, said reduction is determined under non-reducing conditions. In an aspect, said reduction is determined by SDS chromatography under non-reducing conditions. In an aspect, said SDS chromatography is CE-SDS.
  • CE-SDS as used herein refers to capillary electrophoresis sodium dodecyl sulfate.
  • the amount of detectable ⁇ MUC16-Fab light chain or ⁇ MSLN-Fab light chain is below 10%, such as 9%, 8%, 7%, %, 5%, 4%, 3%, 2%, 1% or 0%. In some aspects, the amount of detectable ⁇ MUC16-Fab light chain or ⁇ MSLN-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 ⁇ MUC16-Fab light chain or ⁇ MSLN-Fab light chain, respectively, 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 method for reducing the amount of ⁇ MUC16- or ⁇ MSLN-Fab light chain associated with the ⁇ CD3-VH present on a halfbody according to the present disclosure 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 ⁇ MUC16- or ⁇ MSLN-Fab light chain detectable after recombinant production of the halfbody according to the present disclosure, 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 trispecific antibody or a complementary pair of HB-1 and HB-2, wherein in presence of cells expressing either MUC16 or MSLN or expressing neither of MUC16 nor MSLN 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 HB-1 with ⁇ CD3-VL present on HB-2 or of ⁇ CD3-VH present on HB-2 with ⁇ CD3-VL present on HB-1, and/or b) reduces the dimerization of HB-1 and HB-2; and/or c) reduces the formation of the aCD3-Fv; and/or d) reduces the binding of the ⁇ CD3-Fv to the CD3 in presence of CD3; and/or e) reduces the binding of the ⁇ CD3-VH to the ⁇ CD3-VL and/or f) reduces
  • said reduction is compared to the trispecific antibody or the complementary pair of HB-1 and HB-2 before said amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB-1 and HB-2 comprising a M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH, wherein the IC50 concentration determined for the antibody or complementary pair of HB-1 and HB-2 induced T cell-mediated killing of cells expressing either MUC16 or MSLN 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 concentration determined for the tri
  • the present disclosure provides a trispecific antibody or a complementary pair of HB-1 and HB-2 according to the present disclosure comprising a M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH, where in presence of either MUC16 or MSLN, the KD determined for the binding of the ⁇ CD3-VH to the ⁇ CD3-VL, or vice versa, is greater than 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 K D determined for CD3 binding of the ⁇ CD3-VH to the ⁇ CD3-VL, or vice versa, before the amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB-1 and HB-2 according to the present disclosure, wherein the M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH does not inhibit the dimerization of ⁇ CD3-VH present on HB-1 with ⁇ CD3-VL present on HB-2 or of ⁇ CD3-VH present on HB-2 with ⁇ CD3-VL present on HB-1 in presence of a cells expressing MUC16 and MSLN on their cell surface.
  • the M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3- VH does not inhibit the formation of the ⁇ CD3-Fv in presence of a cells expressing MUC16 and MSLN on their cell surface. In some aspects, the M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3- VH according to the present does not inhibit the binding of the ⁇ CD3-Fv to CD3 in presence of cells expressing MUC16 and MSLN 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 HB-1 with HB-2 in presence of a cells expressing MUC16 and MSLN on their cell surface. In some aspects, 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 MUC16 and MSLN 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 HB-1 and HB-2 according to the present disclosure to induce T cell mediated killing of cells expressing MUC16 and MSLN on their cell surface.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB-1 and HB-2 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 or complementary pair of HB-1 and HB-2 induced T cell mediated killing of cells expressing MUC16 and MSLN 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 HB-1 and HB-2 before the amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB-1 and HB-2 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 or complementary pair of HB-1 and HB-2 induced T cell mediated killing of cells expressing MUC16 and MSLN on their cell surface is substantially the same as the IC 50 concentration determined for the trispecific antibody or complementary pair of HB-1 and HB-2 before the amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB-1 and HB-2 according to the present disclosure comprising a M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH, where in presence of MUC16 and MSLN, 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 HB-1 and HB-2 before the amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the present disclosure provides a trispecific antibody or a complementary pair of HB-1 and HB-2 according to the present disclosure comprising a M at position 37 (utilizing the nomenclature according Kabat) in the ⁇ CD3-VH, wherein in presence of MUC16 and MSLN, the KD determined for CD3 binding is substantially the same as the KD determined for CD3 binding of the trispecific antibody or of the complementary pair of HB-1 and HB-2 before the amino acid substitution at position 37 in the ⁇ CD3-VH.
  • the ⁇ CD3-Fv according to the present disclosure competes with an antibody specific for CD3 for binding to an epitope on CD3, in particular CD3 epsilon.
  • the ⁇ CD3-Fv according to the present disclosure competes with any one of the antibodies specific for CD3 disclosed in International Application No. PCT/EP2021/076052.
  • the ⁇ CD3-Fv comprises any of the VH and/or VL domains disclosed in PCT/EP2021/076052.
  • the ⁇ CD3-Fv competes with the antibody specific for CD3 disclosed in Table 10 or Table 17 of the present specification for binding to an epitope on CD3.
  • the ⁇ CD3- Fv according to the present disclosure competes with an antibody comprising an ⁇ CD3-VH having an amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 159 and an ⁇ CD3-VL having an amino acid sequence of SEQ ID NO: 58 for binding to an epitope on CD3.
  • the VH and VL domain and/or their corresponding CDR regions incorporated in the CyCAT halfbody molecule according to the present disclosure that are capable of forming the ⁇ CD3-Fv is shown in Tables 10 or Table 17 of the present specification.
  • the present disclosure pertains to an antibody or antibody fragment specific for CD3.
  • said antibody fragment is the ⁇ CD3-Fv according to the present disclosure.
  • the ⁇ CD3-VH and ⁇ CD3-VL form the ⁇ CD3- Fv.
  • said ⁇ CD3-Fv is composed of consists of an ⁇ CD3-VL and ⁇ CD3-VH, such as an ⁇ CD3-VL1, ⁇ CD3-VH1, ⁇ CD3-VL2 or ⁇ CD3-VH2 as disclosed herein.
  • said ⁇ CD3-VH and ⁇ CD3-VL are complementary to each other.
  • said ⁇ CD3-VH and ⁇ CD3-VL are capable of non-covalently dimerization with each other.
  • said ⁇ CD3-VH and ⁇ CD3-VL are capable of non-covalently associating with each other.
  • said non-covalent association results in the formation of the ⁇ CD3-Fv.
  • said dimerization or non-covalent association occurs in solution.
  • said dimerization or non-covalent association occurs in solution in the presence of a target cell.
  • the ⁇ CD3-VH and said ⁇ CD3-VL are not linked by a covalent bond.
  • said ⁇ CD3-VH by itself is not capable of binding to CD3.
  • said ⁇ CD3-VL by itself is not capable of binding to CD3.
  • neither the ⁇ CD3-VH by itself nor the ⁇ CD3-VL by itself of the ⁇ CD3-Fv is capable of binding to CD3.
  • said ⁇ CD3-Fv comprise the 6 CDRs of the antibody listed in Table 10 or Table 17.
  • said ⁇ CD3-Fv comprises the 6 CDRs defined by Kabat of the antibodies listed in Table 10 or Table 17.
  • the ⁇ CD3-Fv according to the present disclosure comprises the VH and the VL of the antibody listed in in Table 10.
  • the ⁇ CD3-Fv according to the present disclosure comprises the VH and the VL of the antibody listed in in Table 17.
  • said ⁇ ⁇ CD3-Fv according to the present disclosure comprises a VH comprising an HCDR1 comprising the amino acid sequence of SHYMT (SEQ ID NO: 51), an HCDR2 comprising the amino acid sequence of NIDYEGTRTYYAESVKG (SEQ ID NO: 52), and an HCDR3 comprising the amino acid sequence of GYSAEFAHRSGLDV (SEQ ID NO: 53) and a VL comprising a LCDR1 comprising the amino acid sequence of SGSSSNIGANYVY (SEQ ID NO: 54), a LCDR2 comprising the amino acid sequence of NNQRPS (SEQ ID NO: 55) and a LCDR3 comprising the amino acid sequence of AAWDRHSHGAV (SEQ ID NO: 56)
  • said ⁇ ⁇ CD3-Fv according to the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 58.
  • said ⁇ ⁇ CD3-Fv according to the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 159 and a VL comprising the amino acid sequence of SEQ ID NO: 58.
  • said ⁇ ⁇ CD3-Fv according to the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 159 and a VL comprising the amino acid sequence of SEQ ID NO: 58.
  • said ⁇ ⁇ CD3-Fv 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 of EVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAE SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 57) 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 QSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRF SGSKSGTSASLAISGLRSED
  • said ⁇ ⁇ CD3-Fv comprises a VH comprising an amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWMRQAPGKGLEWVANIDYEGTRTYYAE SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 159) and a VL comprising an amino acid sequence of QSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRF SGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 58).
  • said ⁇ ⁇ CD3-Fv 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 of SEQ ID NO: 57 or SEQ ID NO: 159 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: 58.
  • said ⁇ ⁇ CD3-Fv comprises a VH comprising an amino acid sequence of: EVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEWVANIDYEGTRTYYAE SVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 57) and a VL comprising an amino acid sequence of: QSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRF SGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 58)
  • said ⁇ CD3-Fv is an isolated antibody fragment.
  • said ⁇ CD3-Fv is a human, humanized or chimeric antibody fragment. In some aspects, said ⁇ CD3-Fv is a recombinant or a synthetic antibody fragment. In an aspect, said ⁇ CD3-Fv is a monoclonal antibody fragment. In an aspect, the ⁇ CD3-Fv according to the present disclosure specifically is specific for human CD3. In an aspect, the ⁇ CD3-Fv according to the present disclosure specifically is specific for human CD3 and cynomolgus monkey CD3. In an aspect, the ⁇ CD3-Fv according to the present disclosure is specific to human CD3 and cross-reactively binds to cynomolgus CD3.
  • said ⁇ CD3-Fv binds to immune cells expressing CD3.
  • the ⁇ CD3-Fv binds to T-cells expressing CD3.
  • the ⁇ CD3-Fv binds to cytotoxic T-cells expressing CD3.
  • the ⁇ CD3-Fv binds to human cells expressing human CD3.
  • the ⁇ CD3-Fv binds to cynomolgus monkey cells expressing cynomolgus monkey CD3.
  • the ⁇ CD3-Fv binds to human cells expressing human CD3 and to cynomolgus monkey cells expressing cynomolgus monkey CD3.
  • said CD3 is CD3 epsilon. In an aspect, said CD3 is human CD3 epsilon. In an aspect, said human CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 93. In an aspect, said CD3 is cynomolgus CD3 epsilon. In an aspect, the cynomolgus CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 95. In an aspect, the ⁇ CD3-Fv according to the present disclosure binds to the extracellular region of CD3 epsilon. In an aspect, said ⁇ CD3- Fv binds to the extracellular region human CD3 epsilon.
  • said ⁇ CD3-Fv binds to the extracellular region cynomolgus CD3 epsilon. In an aspect, said ⁇ CD3-Fv binds to the extracellular region human and cynomolgus CD3 epsilon. In an aspect, said extracellular region of human CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 94. In an aspect, the extracellular region of cynomolgus CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 96.
  • said ⁇ CD3-Fv binds to a human CD3 epsilon polypeptide comprising an amino acid sequence of SEQ ID NO: 93 or SEQ ID NO: 94. In an aspect, said ⁇ CD3-Fv binds to a cynomolgus CD3 epsilon polypeptide comprising an amino acid sequence of SEQ ID NO: 95 or SEQ ID NO: 96. In an aspect, said ⁇ CD3-Fv according to the present disclosure specifically binds to a polypeptide comprising the amino acid sequence selected from the group of: SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 101.
  • said ⁇ CD3-Fv specifically binds to a polypeptide comprising the amino acid sequence selected from the group of: SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106.
  • said CD3 is a complex of CD3epsilon/CD3delta.
  • said CD3 is human CD3epsilon/CD3delta.
  • said human 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: 101.
  • said ⁇ CD3-Fv binds to a human CD3 polypeptide comprising the amino acid sequence of SEQ ID NO: 101 with a K D of about 10 nM or less, such as 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or about 1 nM or less.
  • said ⁇ CD3-Fv binds to a human CD3 polypeptide comprising the amino acid sequence of SEQ ID NO: 101 with a KD of between 1 nM and 10 nM.
  • said ⁇ CD3-Fv binds to a human CD3 polypeptide comprising the amino acid sequence of SEQ ID NO: 101 with a KD of about 1 nM, about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6, about 7 nM, about 8 nM, about 9nM or about 10 nM.
  • said ⁇ CD3-Fv binds to a CD3 with a KD of about 10 nM or less, such as 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or about 1 nM or less.
  • said ⁇ CD3-Fv binds to a CD3 with a K D of between 1 nM and 10 nM.
  • said KD is determined by an in vitro biolayer interferometry binding assay (BLI) at 25°C.
  • the monovalent affinity or K D is determined by an in vitro bio-layer interferometry binding assay (BLI) as described herein in Example 5.
  • Peptide Linkers A CyCAT halfbody molecule according to the present disclosure is designed such that its individual components (such as the targeting Fab fragment and the ⁇ CD3-VH or ⁇ CD3-VH) are fused to each other either directly or indirectly through a linker. Such fusion can be achieved by a number of strategies, which include, but are not limited to peptide or polypeptide fusions between the N- and C-terminus of peptides or polypeptides, fusion via disulfide bonds, and fusion via chemical cross-linking reagents.
  • the linker is a peptide linker.
  • the linker is a peptide linker comprising one or more amino acid residues, joined by peptide bonds that are known in the art.
  • the composition and length of a peptide linker may be determined in accordance with methods well known in the art and may be tested for efficacy.
  • the peptide linker should have a length that is adequate to fuse two components in such a way that they assume the correct conformation relative to one another so that they retain or obtain the desired activity or functionality.
  • a peptide linker according to the present disclosure is composed of only naturally occurring amino acid residues.
  • the peptide linker is non-immunogenic.
  • the peptide linker is an unstructured peptide linker. In an aspect, said peptide linker is a flexible peptide linker. In an aspect, said peptide linker does not comprise a protease cleavage site.
  • the peptide linker may comprise G-A polymers, A-S polymers, P-A polymers or P- A-S polymers, such as (GS)n (SEQ ID NO: 114), (G4S)n (SEQ ID NO: 115), (SG4)n (SEQ ID NO: 116), (GSGGS)n (SEQ ID NO: 117), (GGGS)n (SEQ ID NO: 118), G4(SG4)n (SEQ ID NO: 119), (GGSG)n (SEQ ID NO: 120), (GGSGG)n (SEQ ID NO: 121), (GSGSG)n (SEQ ID NO: 122),(GSGGG)n (SEQ ID NO: 123), (GGGSG)n (SEQ ID NO: 124), and (GSSSG)n (SEQ ID NO: 125), wherein n is an integer between 1 and 10, typically between 2 and 4.
  • Suitable peptide linkers can be also derived from immunoglobulin light or heavy chain constant domains, such as CL ⁇ or CL ⁇ domains or the CH1 domain, but not all residues of such a constant domain, for example only the first 5 – 12 amino acid residues.
  • the peptide linker according to the present disclosure comprises an amino acid sequence of: QPKAAP (SEQ ID NO: 126) or ASTKGP (SEQ ID NO: 127).
  • a peptide linker may also comprise an immunoglobulin hinge (e.g. a human IgG1 hinge or part thereof) or any peptide derived from such hinge.
  • the truncated hinge may still include one or more of its interchain cysteines.
  • the presence of the interchain cysteines allows for the formation of a dimeric peptide linker (or hinge region) by disulphide bridges, in situations where two of such hinge peptide linkers are used in two neighbouring polypeptides.
  • the presence of a dimeric peptide linker or hinge region additionally promotes and stabilizes the dimerization of the two Fc region subunits which may be present in an CyCAT halfbody molecule according to the present disclosure.
  • a peptide linker according to the present disclosure is composed of the amino acid residues A, Q, D, P, H, G, S, E, T, K, and C.
  • the peptide linker according to the present disclosure is composed of amino acid residues selected from the group of: A, Q, D, P, H, and G.
  • a peptide linker comprises an amino acid sequence selected from the group of: KTHT (SEQ ID NO: 128), KTHTCPPCP (SEQ ID NO: 129), DKTHTCPPCP (SEQ ID NO: 130), EPKSCDKTHTCPPCP (SEQ ID NO: 131), EPKSC (SEQ ID NO: 132), GQPSG (SEQ ID NO: 133), EPKSCGQPSG (SEQ ID NO: 134) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 135), AQPAAPDAHEAPAPAQGS (SEQ ID NO: 136), AQPAAPAPDAHEAPAPAQGSKTHT (SEQ ID NO: 137), AQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 138), AQPAAPAPAE (SEQ ID NO: 139), AQPAAPAPAEKTHTCPPCP (SEQ ID NO: 140), AQPAAPAPDAHEAPAPAQGA
  • the peptide linker according to the present disclosure has a length of about 4 to 100, 4 to 50, or 4 to 40 amino acids residues.
  • the peptide linker comprises 1 to 200, 1 to 100, 1 to 70, 1 to 65, 1 to 50, 1 to 40, 1 to 25 or 1 to 20 amino acid residues.
  • the peptide linker has a length of at least 4, of at least 5, of at least 10, at least 15, or at least 20 amino acids residues.
  • the peptide linker has a length of not more than 50, not more than 60, not more than 70, not more than 80, not more than 90, or not more than 100 amino acids residues.
  • the peptide linker according to the present disclosure has a length of between 4 and 50 amino acid residues, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues.
  • the peptide linker has a length of 5 to 50 amino acids residues.
  • a peptide linker according to the present disclosure has a length of 4 to 50 amino acids residues, 4 to 45 amino acid residues, 4 to 40 amino acid residues, 4 to 35 amino acid residues, 4 to 30 amino acid residues, 4 to 25 amino acid residues, 4 to 20 amino acid residues, 4 to 15 amino acid residues, or 4 to 10 amino acid residues.
  • the peptide linker has a length of 4 to 50 amino acid residues.
  • the peptide linker has a length of 9 to 29 amino acid residues.
  • the peptide linker has a length of 5 to 29 amino acid residues.
  • the peptide linker has a length of 5 amino acids residues, 8 amino acids residues, 9 amino acid residues, 10 amino acids residues, 15 amino acids residues, 20 amino acids residues, 29 amino acids residues, 40 amino acids residues, or 49 amino acid residues. In an aspect, the peptide linker has a length of 5 amino acids residues. In an aspect, the peptide linker has a length of 8 amino acids residues. In an aspect, the peptide linker has a length of 9 amino acids residues. In an aspect, the peptide linker has a length of 10 amino acids residues. In an aspect, the peptide linker has a length of 15 amino acids residues.
  • the peptide linker has a length of 19 amino acids residues. In an aspect, the peptide linker has a length of 20 amino acids residues. In an aspect, the peptide linker has a length of 25 amino acids residues. In an aspect, the peptide linker has a length of 29 amino acids residues. In an aspect, the peptide linker has a length of 40 amino acids residues. In an aspect, the peptide linker has a length of 45 amino acids residues. In an aspect, the peptide linker has a length of 49 amino acids residues.
  • the different components of a halfbody molecule according to the present disclosure are fused in a “non-cleavable” fashion.
  • non-cleavable is meant the inability to be cleaved and released from a CyCAT halfbody molecule through the activity of an enzyme expressed by a cancer cell to which such CyCAT halfbody molecules binds to.
  • an ⁇ CD3-VH or ⁇ CD-VL domain of the present disclosure may be fused to halfbody molecule through a peptide linker, in particular, by a non-cleavable peptide linker.
  • non cleavable peptide linker herein is meant an amino acid sequence that cannot be cleaved by a human protease under normal physiological conditions. Accordingly, a peptide linker according to the present disclosure does not comprise a protease cleavage site.
  • a peptide linker is not cleavable by a protease.
  • said protease comprises a tumor specific protease.
  • said protease comprises a matrix metalloprotease (MMP) or a serine protease.
  • said matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14.
  • said serine protease comprises matriptase, urokinase, or hepsin.
  • a “protease cleavage site“ as meant herein, is an amino acid sequence that can be cleaved by a protease, such as, for example, a matrix metalloproteinase or a furin.
  • a protease such as, for example, a matrix metalloproteinase or a furin.
  • sites include: Gly-Pro-Leu-Gly-Ile-Ala-Gly- Gln (SEQ ID NO: 111) or Ala-Val-Arg-Trp-Leu-Leu-Thr-Ala (SEQ ID NO: 112), which can be cleaved by metalloproteinases, or Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 113), which is cleaved by a furin.
  • the protease cleavage site could be cleaved by a protease that is produced by target cells, for example cancer cells or infected cells, or pathogens.
  • target cells for example cancer cells or infected cells, or pathogens.
  • Cancer cells are known to express proteases such as matrix-metalloproteinases and proteases associated with cancer as well as corresponding protease cleavage sites (i.e. an particular amino acid sequence recognized by such protease) are well known in the art.
  • Protease databases include ExPASy Peptide Cutter and PMAP.Cut DB. Further databases which helps to identify particular protease cleavage sites are Oncomine or the European Bioinformatic Institute.
  • a peptide linker does not comprise a protease cleavage site, in particular a cancer associated protease cleavage site, in particular a protease cleavage site cleaved or cleavable by a cancer associated protease.
  • a peptide linker as used herein is not limited to only one of the aforementioned and exemplified peptide linkers but may comprise any combination of two or more such linker which are fused to each other.
  • a peptide linker as used herein may be built from a G-S polymer and an immunoglobulin hinge derived sequence.
  • a peptide linker as used herein does not comprise an IgG Fc region. In an aspect, a peptide linker does not comprise a monomer of an IgG Fc region subunit.
  • the peptide linkers present in a CyCAT halfbody molecules according to the present disclosure are identical. In an aspect, such peptide linkers are different. In an aspect, such peptide linkers are of identical length. In an aspect, such peptide linkers are of different length.
  • Fc region The Fc region of a CyCAT halfbody molecule according to the present disclosure may comprise or consists of a pair of polypeptides comprising heavy chain constant domains of a regular immunoglobulin.
  • the Fc region of a regular IgG exists as a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains.
  • the two Fc region subunits are capable of stable association with each other.
  • the two Fc region subunits of HB-1 and/or HB-2 according to the present disclosure are capable of stable association with each other.
  • the Fc region of HB-1 and/or HB-2 according to the present disclosure is an IgG Fc region.
  • said Fc region is a human Fc region.
  • the Fc region is a human IgG1 Fc region.
  • the heterodimeric Fc region The two Fc region subunits of HB-1 and/or HB-2 according to the present disclosure may be comprised in two non-identical polypeptide chains. To improve the yield and purity of the halfbody molecule in recombinant production, it is advantageous to introduce in the Fc region one or more modifications promoting the association of the two non-identical polypeptides forming the Fc region subunits.
  • the present disclosure provides HB-1 and/or HB-2 according to the present disclosure that each rely on the use of two different variant Fc region subunits that will self- assemble to form a heterodimeric molecule.
  • the Fc region of HB-1 and/or HB-2 comprises one or more modifications promoting the association of the first and the second Fc region subunit.
  • the first and second Fc region subunit of HB-1 and/or HB-2 may comprise one or more modification promoting the association of the first and the second Fc region subunit.
  • the first Fc region subunit and second Fc region subunit comprise one or more modifications that reduce homodimerization or reduce homodimer formation between two identical polypeptide chains comprising the same Fc region subunit.
  • the first and second Fc region subunit comprises different amino acid modifications, such that the heterodimeric Fc region is more stable than the homodimeric Fc region.
  • the first and second Fc region subunit comprise different amino acid modification, such that the association of the first and second Fc region subunit is promoted.
  • a modification may be present in the first Fc region subunit and/or the second Fc region subunit. In an aspect, such modification is present in the first and second Fc region subunit. In an aspect, such modification occurs in the CH3 domain of each Fc region subunit.
  • a modification can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
  • the CH3 domain of one polypeptide chain e.g.
  • immunoglobulin heavy chain and the CH3 domain of the other polypeptide chain are both engineered in a complementary manner so that the polypeptide comprising one engineered CH3 domain can no longer homodimerize with another polypeptide chain of the same structure. Thereby the polypeptide comprising one engineered CH3 domain is forced to heterodimerize with the other polypeptide comprising the CH3 domain, which is engineered in a complementary manner.
  • WO 96/027011 Ridgway, J.B., et al, Protein Eng. 9 (1996) 617-621; Merchant, A.M., et al, Nat. Biotechnol.16 (1998) 677-681; US 5,731,168; US 7,695,936; WO 98/ 050431, Carter, J Immunol Meth 248, 7- 15 (2001) which are incorporated herein by reference.
  • the "knobs-into-holes” technology broadly involves: (1) mutating the CH3 domains in each Fc region subunit to promote heterodimerization; and (2) combining the mutated Fc region subunits under conditions that promote heterodimerization.
  • “Knobs” or “protuberances” are typically created by replacing a small amino acid in a parental antibody with a larger amino acid (e.g., T366Y or T366W); "Holes” or “cavities” are created by replacing a larger residue in a parental antibody with a smaller amino acid (e.g., Y407T, T366S, L368A and/or Y407V) with numbering according EU index.
  • the modification present in the Fc region of a HB-1 and/or HB-2 according to the present disclosure is a "knobs-into-holes" modification, comprising "knob mutations” in one of the two Fc region subunits and "hole mutations” in the other complementary Fc region subunit.
  • the knob modifications and hole modifications can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.
  • the CH3 domain of each Fc region subunit is modified according to the knobs-into-holes technology.
  • the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and in the CH3 domain of the second Fc region subunit the tyrosine residue at position 407 is replaced with a valine residue (Y407V) with numbering according EU index.
  • the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) with numbering according EU index.
  • the serine residue at position 354 is replaced with a cysteine residue (S354C)
  • the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) with numbering according EU index based.
  • Introduction of these two cysteine residues results in formation of a disulfide bridge between the two Fc region subunits, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
  • the present disclosure provides HB-1 and/or HB-2 as described herein, wherein in the CH3 domain of first Fc region subunit, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue (S354C) and in the CH3 domain of the second Fc region subunit the tyrosine residue at position 407 is replaced with a valine residue (Y407V), the threonine residue at position 366 is replaced with a serine residue (T366S), the leucine residue at position 368 is replaced with an alanine residue (L368A) and the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) with numbering according EU index.
  • T366W tryptophan residue
  • S354C cysteine residue
  • the tyrosine residue at position 407 is replaced with a valine residue (Y407V)
  • the present disclosure provides HB-1 and/or HB-2, wherein the ⁇ CD3-VH or ⁇ CD3-VL domain and the Fc region subunit comprising the knob-mutations are present on the same polypeptide chain.
  • the present disclosure provides HB-1 and/or HB- 2, wherein the ⁇ CD3-VH or ⁇ CD3-VL domain and the Fc region subunit comprising the hole- mutations are present on the same polypeptide chain.
  • the Fab heavy chain and the ⁇ CD3-VH or ⁇ CD3-VL domain and the Fc region subunit comprising the knob-mutations are present on the same polypeptide chain.
  • the Fab heavy chain and the Fc region subunit comprising the knob-mutations are present on the same polypeptide chain and the Fc region subunit comprising the hole-mutations and the ⁇ CD3-VH or ⁇ CD3-VL domain are present on a second different polypeptide chain.
  • Fc receptor binding and effector function For certain therapeutic situations, it may be desirable to reduce or inhibit the normal or wildtype binding of an IgG Fc region to one or more or all of the Fc ⁇ receptors and/or binding to a complement component, such as C1q. For instance, it may be desirable to reduce or prevent the binding of an Fc region to one or more or all of the Fc ⁇ receptors (e.g.
  • Fc ⁇ Rl, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIIa when a complementary pair of HB-1 and HB-2 according to the present disclosure co-engages CD3, it is advisable to prevent Fc ⁇ RIIIa binding to abolish or significantly reduce ADCC activity and/or to prevent C1q binding to eliminate or significantly reduce CDC activity.
  • the reduced or abolished effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced or abolished antibody-dependent cell-mediated cytotoxicity (ADCC), reduced or abolished antibody- dependent cellular phagocytosis (ADCP).
  • the reduced or abolished effector function is one or more selected from the group consisting of CDC, ADCC and ADCP.
  • the reduced or abolished effector function is ADCC.
  • the reduced or abolished effector function is CDC.
  • the reduced or abolished effector function is ADCP.
  • the reduced or abolished effector function is CDC, ADCC and ADCP.
  • the Fc region of HB-1 and/or HB-2 according to the present disclosure is engineered to have a reduced binding affinity to an Fc receptor and/or to C1q and/or to have reduced effector function when compared to a non-engineered Fc region.
  • the Fc region of HB-1 and/or HB-2 according to the present disclosure is engineered to have reduced effector function when compared to a non-engineered Fc region.
  • the Fc region of HB-1 and/or HB-2 according to the present disclosure comprises one or more amino acid mutations that reduces the binding affinity of the Fc region to an Fc receptor and/or to C1q and/or reduces its effector function.
  • the same one or more amino acid mutation(s) is present in each one of the two Fc region subunits forming the Fc region.
  • the one or more amino acid mutations reduces the binding affinity of the Fc region to an Fc receptor.
  • the engineered Fc region does not bind substantially to an Fc receptor and/or C1q and/or induce effector function.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an activating Fc receptor.
  • the Fc receptor is an Fc ⁇ receptor.
  • the Fc receptor is an activating human Fc ⁇ receptor, more specifically human Fc ⁇ RIIIa, Fc ⁇ RI or Fc ⁇ RIIa, most specifically human Fc ⁇ RIIIa.
  • the binding affinity of the Fc region to a complement component in particular the binding affinity to C1q, is reduced or abolished.
  • the reduced or abolished effector function is one or more selected from the group of reduced or abolished CDC, reduced or abolished ADCC and reduced or abolished ADCP.
  • the reduced or abolished effector function is reduced ADCC, CDC, and ADCP.
  • the Fc region of HB-1 and/or HB-2 according to the present disclosure comprises one or more amino acid mutation(s) that reduce(s) the binding affinity of the Fc region to an Fc receptor and/or to C1q and/or reduces the effector function.
  • the amino acid mutation is an amino acid substitution.
  • the Fc region of HB-1 and/or HB-2 according to the present disclosure comprises one or more amino acid mutations that reduces the binding affinity of the Fc region to an Fc receptor and/or to C1q and/or reduces its effector function, wherein each Fc region subunit comprises an amino acid substitution at a position selected from the group of 234, 235, 237, 330 and 331 with numbering according EU index.
  • each Fc region subunit of HB-1 and/or HB-2 according to the present disclosure comprises an amino acid substitution at a position selected from the group of L234, L235 and G237 (numbering according EU index).
  • each Fc subunit comprises the amino acid substitutions L234A and L235E with numbering according EU index.
  • each Fc region subunit comprises the amino acid substitutions L234A, L235E and G237A with numbering according EU index. In an aspect, each Fc region subunit comprises an amino acid substitution at a position selected from the group of 330 and 331 with numbering according EU index. In an aspect, each Fc region subunit comprises an amino acid substitution at the positions 330 and 331 with numbering according EU index. In an aspect, the amino acid substitution is A330S or P331S.
  • the Fc region of HB-1 and/or HB-2 comprises one or more amino acid mutations in each Fc region subunit that reduces the binding affinity of the Fc region to an Fc receptor and/or to C1q and/or reduces the effector function, wherein said one or more amino acid mutations are L234A, L235E, G237A, A330S and P331S.
  • the Fc region of HB-1 and/or HB-2 consists of one or more amino acid mutation in each Fc region subunit that reduces the binding affinity of the Fc region to an Fc receptor and/or to C1q and/or reduces the effector function, wherein the one or more amino acid mutations are L234A, L235E, G237A, A330S and P331S.
  • the Fc region is an IgG1 Fc region, particularly a human IgG1 Fc region.
  • Mutant Fc regions or Fc region subunits can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art.
  • the Fc region subunit according to the present disclosure comprises the amino acid sequence of (Fc-AEASS-Knob): PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 108)
  • the Fc region subunit according to the present disclosure comprises the amino acid sequence of (Fc-AEASS-hole): PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIE
  • the present disclosure provides a method for inducing lysis of a cancer cell expressing MUC16 and MSLN, comprising contacting said cancer cell in the presence of a cytotoxic T cell with a complementary pair of HB-1 and HB-2 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 MUC16 and MSLN, comprising contacting said cancer cell in the presence of a cytotoxic T cell with a complementary pair of HB-1 and HB-2 or with the trispecific antibody, according to the present disclosure.
  • the present disclosure provides a method for killing a cancer cell expressing MUC16 and MSLN, comprising contacting said cancer cell, in the presence of a cytotoxic T cell, with a complementary pair of HB-1 and HB-2 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 MUC16 and MSLN, comprising contacting said cancer cell in the presence of a T-cell with a complementary pair of HB-1 and HB-2 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 MUC16 and MSLN, comprising contacting said cancer cells in the presence of said T cell with a complementary pair of HB-1 and HB-2 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 MUC16 and MSLN, comprising contacting said cancer cell, in the presence of said T cell, with a complementary pair of HB-1 and HB-2 or with the trispecific antibody, according to the present disclosure.
  • the present disclosure provides a method for the treatment of cancer, preferably a non-hematological cancer, more preferably ovarian cancer.
  • the trispecific antibody or the complementary pair of HB-1 and HB-2, according to the present disclosure may be used in therapeutic methods.
  • the present disclosure provides a complementary pair of HB-1 and HB-2 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 HB-1 and HB-2 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 HB-1 and HB-2 according or the trispecific antibody, according to the present disclosure, for use in the treatment of a disease in a subject in need thereof.
  • the present disclosure provides the use of a complementary pair of HB-1 and HB-2 or of the trispecific antibody, according to the present disclosure, for the manufacture of a medicament.
  • the present disclosure provides a complementary pair of HB-1 and HB-2 or the trispecific antibody, according to the present disclosure, for use as a medicament.
  • the present disclosure provides a complementary pair of HB-1 and HB-2 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 HB-1 and HB-2 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 HB-1 and HB-2 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 MUC16 and MSLN are involved.
  • the present disclosure provides the use of a complementary pair of HB-1 and HB-2 or the trispecific antibody according to the present disclosure for the treatment of cancer, comprising cancer cells expressing MUC16 and MSLN 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 MUC16 and MSLN expressing cancer cells in the one or more samples; and (d) administering to the subject an effective amount of a complementary pair of HB-1 and HB-2 according to the present disclosure or the trispecific antibody according to the present disclosure.
  • said disease is associated with the undesired presence of MUC16.
  • said disease is associated with the undesired presence of MSLN. In an aspect, said disease is associated with the undesired presence of MUC16 and MSLN.
  • the present disclosure provides a complementary pair of HB-1 and HB-2 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 HB-1 and HB-2 according to the present disclosure. In an aspect, 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.
  • HB-1 and HB-2 or the trispecific antibody according to the present disclosure would be formulated, dosed, and administered in a way consistent with good medical practice.
  • the complementary pair of HB-1 and HB-2 according to the present disclosure are administered separately.
  • said HB-1 and HB-2 are administered consecutively.
  • HB-1 and HB-2 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 HB-1 and HB-2 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 HB-1 and HB-2 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 HB-1 and HB-2 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 HB-1 and HB-2 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 MUC16.
  • said cancer is a cancer expressing MSLN.
  • said cancer is a cancer expressing MUC16 and MSNL.
  • said cancer is a carcinoma.
  • said cancer is a ovarian cancer.
  • the present disclosure provides a method for the treatment of ovarian cancer with the trispecific antibody or with the complementary pair of HB-1 and HB-2 according to the present disclosure.
  • the present disclosure provides the trispecific antibody or the complementary pair of HB-1 and HB-2 according to the present disclosure for use in the treatment of ovarian cancer.
  • Compositions and Treatments In an aspect, the present disclosure provides a pharmaceutical composition comprising the trispecific antibody according to the preset disclosure and a pharmaceutical acceptable carrier. In an aspect, the present disclosure provides the pharmaceutical composition comprising the trispecific antibody of the present disclosure for use in the treatment of a cancer expressing MUC16 and MSLN. In an aspect, the present disclosure provides a kit comprising the pharmaceutical composition comprising the trispecific antibody of the present disclosure. In an aspect, the present disclosure provides a pharmaceutical composition comprising HB-1 according to the present disclosure and a pharmaceutical acceptable carrier.
  • the present disclosure provides a pharmaceutical composition comprising HB-2 according to the present disclosure and a pharmaceutical acceptable carrier.
  • the present disclosure provides a kit comprising the pharmaceutical composition comprising HB-1 and the pharmaceutical composition comprising HB-2 of the present disclosure.
  • the present disclosure provides the pharmaceutical composition comprising HB-1 and the pharmaceutical composition comprising HB-2 according to the present disclosure for use in the treatment of a cancer expressing MUC16 and MSLN.
  • said cancer is a solid cancer.
  • said cancer is ovarian cancer.
  • 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 HB-1 and HB-2, or (c) the pharmaceutical composition comprising HB-1 and the pharmaceutical composition comprising HB-2, according to the present disclosure, to a subject in need thereof.
  • the present disclosure provides the use as described herein, wherein (a) HB-1 and HB-2 are administered separately or (b) wherein the pharmaceutical composition comprising HB-1 and the pharmaceutical composition comprising HB-2 are administered separately.
  • 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 HB-1 and HB-2 according to the present disclosure, said pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • HB-1 and HB-2 may be formulated or comprised in a separate pharmaceutical composition.
  • the present disclosure provides a first pharmaceutical composition comprising HB-1 according to the present disclosure and at least one pharmaceutically acceptable carrier.
  • the present disclosure provides a second pharmaceutical composition comprising HB-2 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 MUC16 and/or MSLN.
  • the present disclosure provides (a) a first pharmaceutical composition comprising HB-1 and a second pharmaceutical composition comprising HB-2, 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 HB-1 and a second pharmaceutical composition comprising HB-2, 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 MUC16 and MSLN.
  • the present disclosure provides (a) a first pharmaceutical composition comprising HB-1 and a second pharmaceutical composition comprising HB-2, 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 HB-1 and a second pharmaceutical composition comprising HB-2, 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 HB-1 and a pharmaceutical composition comprising HB-2 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 MUC16.
  • said cancer is a cancer expressing MSLN.
  • said cancer is a cancer expressing MUC16 and MSLN.
  • said cancer is a carcinoma.
  • said cancer is a ovarian cancer.
  • compositions according to the present disclosure may comprise a therapeutically effective amount of (a) HB-1 and HB-2 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 HB-1 and HB-2 or the trispecific antibody according to the present disclosure.
  • the present disclosure provides a kit comprising a first pharmaceutical composition comprising HB-1 and a second pharmaceutical composition comprising HB-2, 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 HB-1 and HB-2 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 HB-1 and HB-2 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 or trispecific antibody, and the discretion of the attending physician.
  • Effective dosages and schedules for administering pharmaceutical compositions comprising HB- 1 and HB-2 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 HB-1 and HB-2 or the trispecific antibody according to the present disclosure, wherein said HB-1 and HB-2 or said trispecific antibody are administered at a dose sufficient to achieve a therapeutically effective serum level.
  • the administration of HB-1 and HB-2 according to the present disclosure encompass separate administration, in which case, administration of HB-1 occurs prior to, simultaneously, and/or following, administration of the HB-2 or vice versa.
  • HB-1 and HB-2 or the trispecific antibody according to the present disclosure are administered intravenously.
  • HB-1 and HB-2 are administered subcutaneously.
  • the trispecific antibody is administered subcutaneously.
  • Combination Therapies HB-1 and HB-2 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 HB-1 and HB-2 or of the trispecific antibody according to the present disclosure can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent.
  • HB-1 and HB-2 or the trispecific antibody according to the present disclosure can also be used in combination with radiation therapy.
  • Nucleic acids In an aspect, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the antibody or antibody fragment specific for MUC16 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 MUC16 as disclosed in Tables 5 – 7 of the present disclosure.
  • 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 MSLN 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 MSLN as disclosed in Tables 8 – 9 of the present disclosure.
  • the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding HB-1 according to the present disclosure. In an aspect, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding HB-2 according to the present disclosure. In an aspect, the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the trispecific antibody according to the present disclosure.
  • 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 MUC16 according to the present disclosure, or an antibody or antibody fragment specific for MSLN according to the present disclosure, or an HB-1 according to the present disclosure, or an HB-2 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 MUC16 according to the present disclosure, an antibody or antibody fragment specific for MSLN according to the present disclosure, an HB-1 according to the present disclosure, an HB-2 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 MUC16 according to the present disclosure, an antibody or antibody fragment specific for MSLN according to the present disclosure, an HB-1 according to the present disclosure, an HB-2 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 MUC16 according to the present disclosure, or an antibody or antibody fragment specific for MSLN according to the present disclosure, or an HB-1 according to the present disclosure, or an HB-2 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 MUC16 according to the present disclosure, or an antibody or antibody fragment specific for MSLN according to the present disclosure, or an HB-1 or an HB-2 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 MUC16, an antibody or antibody fragment specific for MSLN, or an HB-1, or an HB-2, or an trispecific antibody according to the present disclosure, under conditions suitable for expression of the antibody or antibody fragment specific for MUC16 , or the antibody or antibody fragment specific for MSLN, or HB-1 or HB-2, or the trispecific antibody according to the present disclosure, and isolating the same from the host cell or host cell culture medium.
  • 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.
  • Dual-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.
  • 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.
  • 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) inhibits the binding of the VL of the Fab light chain with the ⁇ CD3-VH present on HB during recombinant production of said HB, and/or iii) reduces the amount of Fab light chain associated with the ⁇ CD3-VH during recombinant production of said HB, and/or iv) reduces the amount of detectable
  • 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 the ⁇ CD3-VH of the present disclosure.
  • the present disclosure provides a method for reducing the amount of Fab light chain associated with the ⁇ CD3-VH present of a HB during recombinant production of said HB, 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 HB, 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 HB 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 HB with a natural occurring amino acid except cysteine and producing said HB 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 HB 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 HB is produced as a pharmaceutical composition.
  • said HB 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 of a trispecific antibody composed of said complementary pair of halfbody molecules, wherein a) the first halfbody molecule (HB-1) comprises i. a first antibody fragment (Fab1) specific for a first antigen (AG1) ii.
  • Fab2 second antibody fragment specific for a second antigen
  • ⁇ 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, ii.
  • said inhibition is compared to the trispecific antibody or the complementary pair of HB-1 and HB-2 according to the present disclosure before said amino acid substitution.
  • said binding of ⁇ CD3-VH1 to ⁇ CD3-VL2 or of ⁇ CD3-VL1 to ⁇ CD3-VH2, respectively is reduced by at least 1.5-fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or at least 10 fold when compared to the respective binding before said amino acid substitution.
  • said binding is provided as a KD value or EC50 concentration.
  • said binding is determined in presence of AG1 and CD3 and absence of AG2.
  • said binding is determined in presence of AG2 and CD3 and absence of AG1.
  • said K D value is determined by surface plasmon resonance (SPR) or Bio-layer interferometry (BLI).
  • 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 HB-1 with HB-2, iii.
  • said inhibition is compared to trispecific antibody or the complementary pair of HB-1 and HB-2 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 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 IC50 concentration determined for the complementary pair of HB-1 and HB-2 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 IC50 (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 IC
  • 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 IC 50 (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 10 or Table 17 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 aspects, said amino acid substitution is located in framework region 1 of the ⁇ CD3-VH. In some aspects, said amino acid substitution is located in framework region 2 of the ⁇ CD3-VH. In some aspects, said amino acid substitution is located in framework region 3 of the ⁇ CD3-VH. In some aspects, said amino acid substitution is located in framework region 4 of the ⁇ CD3-VH. Accordingly, in some aspects, 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).
  • said amino acid substitution is a V37M, V37L, G44A, L45I or L45F amino acid substitution (utilizing the nomenclature according Kabat).
  • V at position 37 is replaced with a M (V37M) or L (V37L)
  • G at position 44 is replaced with A (G44A)
  • 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 aspects, the ⁇ CD3-VH according to the present disclosure comprises a G or A at position 44 (utilizing the nomenclature according Kabat). In some aspects, the ⁇ CD3-VH according to the present disclosure comprises a L, F, or I at position 45 (utilizing the nomenclature according Kabat). In some aspects, the ⁇ CD3-VH according to the present disclosure comprises the amino acid sequence of SEQ ID NO: 57 or 159. In some aspects, the ⁇ CD3-VL according to the present disclosure comprises the amino acid sequence of SEQ ID NO: 58.
  • HB-1 and HB-2 are capable of forming a heterodimer with each other, wherein said formation of a heterodimer of HB-1 and HB-2 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 HB-1 and HB-2 results in the formation of the T-cell engaging trispecific antibody.
  • HB-1, HB-2, ⁇ 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.
  • said Fab or Fab1 is specific for MUC16.
  • said Fab or Fab2 is specific for MSLN.
  • said Fab or Fab1 specific for MUC16 is any of the ⁇ MUC16-Fabs disclosed in Table 5, 6 or 7 of the present disclosure.
  • said Fab light chain is any of the Fab light chains disclosed in Table 5, 6 or 7 of the present disclosure.
  • said Fab or Fab2 specific for MSLN is any of the ⁇ MSLN-Fabs disclosed in Table 8 or 9 of the present disclosure.
  • said Fab light chain is any of the Fab light chains disclosed in Table 8 or 9 of the present disclosure.
  • Fig.1A Schematic representation of a complementary pair of CyCAT halfbody molecules according to the present invention, each carrying a Fab with specificity for MUC16 or MSLN, respectively, one half of an ⁇ CD3-Fv binding fragment (aCD3-VH or aCD3-VL) and an effector compromised IgG Fc region including Knob-into-Holes (KiH) mutations.
  • the individual components of the halfbody molecules are fused to each other via peptide linkers.
  • Fig 1B Illustration of on-cell complex formation of a pair of two complementary CyCAT halfbody molecules resulting in the re-constitution of a functional Fv binding fragment being able to bind to CD3 on the surface of a T-cell.
  • Figure 2 Cell binding of MUC16 specific halfbody molecules and of Mesothelin specific halfbody molecules according to the present invention. Shown is cell binding as a function of halfbody concentration determined on the MUC16 / Mesothelin double positive ovarian cancer cell lines OVCAR-3.
  • Figure 3 Exemplary results of cytotoxicity assays according to Example 7 for Dual-Targeting pairs of halfbody molecules according to the present invention on the MUC16 / MSLN double-positive ovarian cancer cell line OVCAR-3 in presence of human pan T effector cells.
  • Figure 4 Exemplary results of cytotoxicity assays according to Example 7 for Dual-Targeting halfbody pairs according to the present invention with specificity for MUC16 and MSLN in comparison to Mono-Targeting halfbody pairs with specificity for MUC16 and Lysozyme or MSLN and Lysozyme on the MUC16 / MSLN double-positive ovarian cancer cell line OVCAR-3 in presence of human pan T effector cells.
  • Figure 5 Exemplary results of cytotoxicity assays according to Example 8 for Dual-Targeting halfbody pairs with specificity for MUC16 and MSLN in comparison to a conventional bispecific antibody molecule with specificity for MSLN and CD3 on the MSLN positive / MUC16 negative epithelial cell line MeT-5A in presence of human pan T effector cells.
  • Figure 6 Exemplary results of cytotoxicity assays according to Example 11 for complementary pairs of CyCAT halfbody molecules with specificity for MUC16 and MSLN, respectively, utilizing the mutated ⁇ CD3-VHV37M domain on the MUC16 / Mesothelin double positive ovarian cancer cell line OVCAR-3.
  • FIG.2 A – C: Exemplary results of cytotoxicity assays according to Example 12 for complementary pairs of CyCAT halfbody molecules with specificity for MUC16 and MSLN, respectively, utilizing the unmutated ⁇ CD3-VH domain in comparison to corresponding halfbody pairs utilizing the mutated aCD3-VHV37M domain, on the MUC16 / Mesothelin double positive ovarian cancer cell line OVCAR-3.
  • MUC16 refers to a protein also known as “Mucin-16” or “CA-125”.
  • Human MUC16 (Uniprot: Q8WXI7) has the amino acid sequence of: MLKPSGLPGSSSPTRSLMTGSRSTKATPEMDSGLTGATLSPKTSTGAIVVTEHTLPFTSPDKTL ASPTSSVVGRTTQSLGVMSSALPESTSRGMTHSEQRTSPSLSPQVNGTPSRNYPATSMVSGL SSPRTRTSSTEGNFTKEASTYTLTVETTSGPVTEKYTVPTETSTTEGDSTETPWDTRYIPVKITS PMKTFADSTASKENAPVSMTPAETTVTDSHTPGRTNPSFGTLYSSFLDLSPKGTPNSRGETSLE LILSTTGYPFSSPEPGSAGHSRISTSAPLSSSASVLDNKISETSIFSGQSLTSPLSPGVPEARAST MPNSAIPFSMTLSNAETSAERVRSTISSLGTPSISTKQTAETILTFHAFAETMDIPSTHIAKTLASE WLGSPGTLGGTSTSALTTTSPSTTLVSEETNTHHSTSGKETEGTLNTSMTPLETSAPGEESE
  • Human Mesothelin (Uniprot: Q13421-3) has the amino acid sequence of: MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPR QLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPD AFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDL PGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPV LGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYK KWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDI RKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFV
  • the mature extracellular domain of cynomolgus MSLN without signal sequence has the amino acid sequence of: LAGETRQEAAPLDGILTNAPDIASLSPRQLLGFTCVEVSGLSTELVQELAVALGQKNVKLSAEQL RCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTHFFSRVAKANVDLLPRGAPERQRLLPA ALTCWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVVLPRLVRCLGPLDQDQQEAARAAL QRGGPPYGPPSTWSISTLDDLQSLLPVLGQPVIHSIPQGILAAWRQRSSRDPSWQQPEQTVLR PRFRRDVERTTCPPEKEVHEIDESLIFYKKRELEACVDAALLAAQMDRVDAIPFTYEQLDVLKHK LDELYPQGYPESVIRHLGHLFLKMSPEDIRKWNVTSLETLKALLKVSKGHEMSAQVATLIDRVVVG GRGQLDKDTADTLTAFCPGCL
  • 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: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHND KNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD VMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYE PIRKGQRDLYSGLNQRRI (SEQ ID NO: 93) (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: QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLK EFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 94) Cynomolgus monkey CD3epsilon (or cynomolgus CD3e or cyno CD3e) has the amino acid sequence of UniProt Q95LI5: MQSGTRWRVLGLCLLSIGVWGQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHN GKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATI VIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKG
  • the mature extracellular region of cynomolgus monkey CD3 epsilon without the signal sequence comprises has the amino acid sequence of: QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQS GYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKA KAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI (SEQ ID NO: 96).
  • the term “antigen” or “target antigen” as used herein 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 fluorophore.
  • 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. Unless otherwise indicated, a particular amino acid sequence of a polypeptide also implicitly encompasses conservatively modified variants thereof (e.g. by replacing an amino acid residue with another amino acid residue having similar structural and/or chemical properties).
  • a 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. A polypeptide may also comprise one or more disulfide bonds.
  • 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.
  • “Complementary” halfbody molecule(s) as used herein refers to any 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 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 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).
  • a “Fc region subunit” as used herein refers to one of the two polypeptides forming the dimeric Fc region of an immunoglobulin, i.e. a polypeptide comprising the C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. Accordingly, the two Fc region subunits (e.g.
  • IgG Fc region subunit comprises an IgG CH2 and an IgG CH3 constant domain.
  • the term includes native sequence Fc region subunits and variant Fc region subunits.
  • the boundaries of the Fc region subunits of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region subunit is usually defined to extend from Cys226, or from Pro230, to the C-terminus of the heavy chain.
  • the C- terminal lysine (Lys447) of the Fc region subunit may or may not be present.
  • EU numbering system also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • EU index 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%.
  • amino acid residues or “amino acid” will be indicated either by their full name or according to the standard three-letter or one-letter amino acid code. “Natural occurring amino acids” means the following amino acids listed in Table 1.
  • 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 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 therefore represents the concentration that reduces a given response by 50%.
  • 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, halfbody molecule or CyCAT molecule is able to specifically bind to two or more different antigens.
  • a multispecific antibody, halfbody molecule or CyCAT 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, halfbody molecule or CyCAT molecule is able to specifically bind to two different antigens.
  • a bispecific antibody or halfbody molecule or CyCAT molecule comprises two antigen binding sites, each of which is specific for a different antigen or epitope.
  • the term “trispecific” means that an antibody or halfbody molecule or CyCAT molecule is able to specifically bind to three different antigens.
  • a trispecific antibody, halfbody molecule or CyCAT 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 are well known in the art and 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”, “sixth” “seventh”, “eights” and so on, with respect to a 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 a 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 aspects, 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 K D 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).
  • Antigen Sequences Table 2: Amino acid sequences of recombinant human and cynomolgus MUC16 extracellular domain antigens.
  • Table 3 Amino acid sequences of recombinant human and cynomolgus MSLN extracellular domain antigens.
  • Table 4 Amino acid sequences of recombinant human and cynomolgus CD3 epsilon extracellular domain antigens.
  • Antibody Sequences Table 5 Antibody sequences of the MUC16 specific antibody ⁇ MUC16-Fab1 Table 6: Antibody sequences of the MUC16 specific antibody ⁇ MUC16-Fab2 Table 7: Antibody sequences of the MUC16 specific antibody ⁇ MUC16-Fab3 Table 8: Antibody sequences of the MSLN specific antibody ⁇ MSLN-Fab1 Table 9: Antibody sequences of the MSLN specific antibody ⁇ MSLN-Fab2 Table 10: Antibody sequences of the human CD3 specific antibody ⁇ CD3-Fv used in the CyCAT halfbody molecules in the present examples. CyCAT Halfbody Sequences Each CyCAT halfbody molecule is composed of three polypeptide chains.
  • a main chain comprising the Fab heavy chain, either the ⁇ CD3-VH or ⁇ CD3-VL domain, and one Fc region subunit; a second chain comprising the second Fc region subunit; and a third chain comprising the light chain of the Fab.
  • the MorphoSys Ylanthia® library (Tiller et al. mAbs 5:3, 1–26; May/June (2013) and U.S. Patent No. 8,728,981) is a commercially available phagemid library and employs the CysDisplay® technology for displaying the Fab on the phage surface (Lohning et al., WO2001/05950).
  • the LCDR3 and HCDR1/HCDR2 regions of isolated MUC16 or MSLN specific antibodies were optimized in parallel using diversified Ylanthia ® maturation modules that were generated previously with the Slonomics® technology (van den Brulle et al.2008).
  • ⁇ MUC16 antibodies namely ⁇ MUC16-Fab1, ⁇ MUC16-Fab2 and ⁇ MUC16-Fab3 as well as two optimized MSLN specific human antibodies, namely ⁇ MSLN-Fab1 and ⁇ MSLN- Fab2 were used as Fab fragments in the CyCAT halfbody molecules as described herein.
  • Amino acid sequences of the optimized antibodies are shown in Tables 5 – 9.
  • nucleotide sequences encoding the VH and VL domains of the antibody " ⁇ CD3-Fv” as described in Table 10 and Table 17 were used.
  • Generation of ⁇ CD3-Fv is described in International Publication Number: WO2022/063819, which is incorporated herein in its entirety.
  • Example 2 Cloning and production of CyCAT halfbody molecules comprising a Fab fragment with specificity for MUC16 or MSLN, respectively, and a single ⁇ CD3 antibody variable domain.
  • the following are examples of molecules and methods according to the present disclosure. It is understood that various other aspects may be practiced, given the general description provided herein. Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A.
  • each CyCAT halfbody molecule is composed of three polypeptide chains providing the Fab fragment with specificity for MUC16 or MSLN and either the VL or VH of an ⁇ CD3 Fv antibody fragment and an IgG Fc region. Fc regions were further modified by introducing mutations into the CH3 domain of each Fc region subunit according to the knob-into-holes technology.
  • the Fc region subunit comprising one mutated CH3 domain is forced to heterodimerize with the other Fc region subunit comprising the other CH3 domain, which is engineered in a complementary manner.
  • the Fc region was additionally modified by introducing mutations into the CH2 domain of each Fc region subunit in order to abolish its activity to mediate effector function, such as ADCC, CDC, and ADCP.
  • Exemplary peptide linkers which may be used to link the different components of an halfbody molecule may be selected of having an amino acid sequence selected from the group of SEQ ID NOs: 114 – 158 and SEQ ID NO: 170.
  • CyCAT halfbody molecules For expression of CyCAT halfbody molecules, exponentially growing eukaryotic HEK293-6E cells were transfected with mammalian expression vectors encoding all components of a halfbody molecule according to the present disclosure, resulting in an 1:1:1 heterotrimer of three polypeptides. Cell culture supernatants were harvested on day 6 post transfection and subjected to Protein A or anti-CH1 affinity chromatography (Capture Select IgG-CH1 or CH1-XL ⁇ ThermoFisher Scientific). Buffer exchange was performed to 1x Dulbcecco ⁇ s PBS (pH 7.2 ⁇ Invitrogen) and samples were sterile filtered (0.2 ⁇ m pore size).
  • Protein concentrations were determined by UV-spectrophotometry and purities of the constructs were analyzed under denaturing, reducing and non-reducing conditions using CE-SDS (LabChip GX Touch ⁇ Perkin Elmer). UHP-SEC was performed to analyze CyCAT halfbody molecule preparations in native state.
  • Example 4 K D determination on human and cynomolgus monkey MUC16 and Mesothelin by Biolayer Interferometry (BLI) For KD determinations, ⁇ MUC16 or ⁇ MSLN CyCAT halfbody molecules of the present disclosure were used. Affinity determination by determining kinetic rate constants was performed on an Octet RED (FortéBIO, Sartorius AG) instrument as described below.
  • Streptavidin-coated biosensors (FortéBIO, Sartorius AG) were immobilized with biotinylated human MUC16 (SEQ ID NO: 97), cynomolgus monkey MUC16 (SEQ ID NO: 98), human Mesothelin (SEQ ID NO: 99) or cynomolgus monkey Mesothelin (SEQ ID NO: 100) antigen, respectively, in immobilization buffer (DPBS, 0.05% (v/v) Tween 20) resulting in immobilization levels of approx.0.2 nm.
  • immobilization buffer DPBS, 0.05% (v/v) Tween 20
  • the different halfbody molecules were diluted with assay buffer in 2 n serial dilution with seven concentrations in total starting with a concentration between 20 nM and 100 nM.
  • a blank sample with assay buffer was included for referencing, i.e. correcting for dissociation of loaded halfbody molecules.
  • the association phase was recorded for 360 s, followed by a dissociation phase of 3600 s.
  • the biosensors were regenerated two times for 30 s with 10 mM Glycine HCl pH 1.5 to remove bound halfbody, while maintaining the integrity of the capture surface. Between regeneration steps, biosensors were washed with assay buffer for 20 s.
  • the sensorgrams were fitted using Octet Data Analysis Software 12.2 (FortéBio, Sartorius AG) to determine kon and koff rate constants (using a 1:1 binding model), which were used to calculate K D .
  • Table 12 Mesothelin binding kinetics (BLI) *the ⁇ CD3-VH domain present in the tested halfbody molecules comprised a single point amino acid substitution (VHmut) in its VH framework region.
  • Example 5 KD determination on human and cynomolgus monkey CD3 antigens by Biolayer Interferometry (BLI)
  • KD determinations on CD3 antigens complementary pairs of MUC16- and MSLN specific CyCAT halfbody molecules according to the present disclosure were used. Affinity determination by determining kinetic rate constants was performed on an Octet RED (FortéBIO, Sartorius AG) instrument at 27°C as described below.
  • 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 (DPBS, 0.05% (v/v) Tween 20) resulting in a high-density anti-human Fab kappa/lambda sensor surface.
  • unpaired CyCAT halfbody molecules 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.
  • Example 6 Binding of ⁇ MUC16- and ⁇ MSLN CyCAT halfbody molecules to endogenous MUC16 and MSLN expressed on OVCAR-3 cells
  • flow cytometry analyses using the MUC16- and MSLN-positive human ovarian carcinoma cell line OVCAR-3 (ATCC, HTB-161) were performed. Briefly, a single cell suspension of OVCAR-3 cells using 0.05% Trypsin-EDTA (GibcoTM Trypsin-EDTA (0.05%), phenol red (Fisher Scientific)) was prepared.
  • Trypsin-EDTA GibcoTM Trypsin-EDTA (0.05%), phenol red (Fisher Scientific)
  • Example 7 T cell mediated killing of MSLN and MUC16 double positive human ovarian cancer cells induced by functional complementation of ⁇ MUC16 and ⁇ MSLN CyCAT halfbody molecules
  • Complementary pairs of CyCAT halfbody molecules with specificity for MSLN and MUC16 were allowed for on-target functional complementation of trispecific antibodies with specificity for MSLN, MUC16 and CD3.
  • the potency of functionally complemented pairs of ⁇ MSLN and ⁇ MUC16 CyCAT halfbody molecules according to the present invention in redirecting effector pan T cells against the MSLN and MUC16 double positive human ovarian cancer cell line OVCAR-3 was measured in a 48 hour cytotoxicity assay using different readouts.
  • Method 1E+06 cells/mL OVCAR-3 cells (ATCC, HTB-161) were treated with Mitomycin C (10 ⁇ g/ml, Sigma-Aldrich #M4287-2MG) for 1 hour at 37°C. Cells were washed twice with PBS. 5E+03 cells/well were seeded out into black 384 well assay plate (clear bottom, Perkin Elmer, #6007460) and incubated overnight in a humidified, 5% CO2 incubator at 37°C.
  • pan T cells were extracted from frozen PBMC (prior isolation from whole blood using Pancoll human, cat# P04-60500 according to the manufacturerer’s manual) using the EasySepTM human T-Cell isolation kit (STEMCELL Technologies #17951) and the RoboSepTM-S instrument (STEMCELL Technologies, # 21000) according to the manufacturer’s instructions. Respective amount of Pan T cells were added to reach an effector to target ratio(E:T) of 1:1.
  • Granzyme B (GrB) release was quantified in the supernatants using the Human Granzyme B Duo Set ELISA (R&D Systems, #DY2906-05) according to the manufacturer’s recommended protocol and results were visualized using the GraphPad PRISM software.
  • the absolute cytotoxicity was quantified using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega # G7571) according to the manufacturer’s manual with the exception that before applying the reagent, Pan T cells were carefully removed by manual washes (80 ⁇ L/well) with pre-warmed assay media. Values were plotted using the GraphPad PRISM software.
  • Table 15 Results of the cytotoxicity assays for Dual-Targeting OVCAR-3 cells with complementary pairs of CyCAT halfbody molecules with specificity for MSLN and MUC16.
  • HPN536 is a single chain antibody composed of a single domain antibody with specificity for Mesothelin, a single domain antibody with specificity for albumin and a scFv fragment with specificity for CD3.
  • HPN536 has the amino acid sequence of : QVQLVESGGGVVQAGGSLTLSCAASGSTFSIRAMRWYRQAPGTERDLVAVIYGSSTYYADAVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNADTIGTARDYWGQGTLVTVSSGGGGSGGGS EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAD SVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSE VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYAD QVKDRFTISRDDSKNT
  • Example 9 MUC16 and MSLN specific CyCAT halfbodies with improved production and heterodimerization properties
  • MUC16 specific halfbody molecules as well as MSLN specific halfbody molecules utilizing an ⁇ CD3-VH domain 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: 57); mutated ⁇ CD3- VH V37M (SEQ ID NO: 159)).
  • the variant halfbody molecules were mammalian produced as described before, in parallel to their counterpart halfbodies utilizing the unmutated ⁇ CD3-VH domain.
  • a summary of the amino acid sequences of the produced halfbodies according to Example 9 are provided below.
  • Example 10 KD determination on human CD3 antigen by Biolayer Interferometry (BLI) after functional complementation of halfbody molecules utilizing the mutated ⁇ CD3-VHV37M domain Affinity determination by determining kinetic rate constants was performed on an Octet HTX (FortéBIO, Sartorius AG) instrument as described in Example 5. The results of the KD determination are summarized in Table 19. Result Functionally complemented halfbody pairs utilizing the mutated ⁇ CD3-VH V37M domain revealed similar binding affinities to human CD3 when compared to the binding affinity of corresponding halfbody pairs utilizing the unmutated ⁇ CD3-VH domain (see Table 19 last column vs. Table 13 last column).
  • Table 20 Results of the cytotoxicity assays on OVCAR-3 cells with complementary pairs of halfbody molecules with specificity for MUC16 and MSLN utilizing the mutated ⁇ CD3- VH V37M domain.
  • Example 12 T cell mediated killing of MSLN and MUC16 double positive human ovarian cancer cells induced by functional complementation of ⁇ MUC16 and ⁇ MSLN CyCAT halfbody molecules utilizing the unmutated ⁇ CD3-VH or mutated ⁇ CD3-VHV37M domain. Cytotoxicity assays were conducted as described in Example 7. Cytotoxicity activity was assessed by measuring incorporated CellToxGreen (Promega) fluorescence.
  • results of the cytotoxicity assays for selected pairs of halfbody molecules are summarized in Table 21 and shown in Figure 7.
  • Table 21 Results of the cytotoxicity assays on OVCAR-3 cells with complementary pairs of halfbody molecules with specificity for MUC16 and MSLN, respectively, either utilizing the unmutated ⁇ CD3-VH or mutated ⁇ CD3-VHV37M domain

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Abstract

La présente invention concerne de nouveaux anticorps humains qui se lient spécifiquement à MUC16 ainsi que de nouveaux anticorps humains qui se lient spécifiquement à la mésothéline. La présente invention concerne en outre des paires complémentaires de molécules de demi-corps CyCAT englobant les nouveaux anticorps spécifiques de MUC16 et de mésothéline de la présente invention et leur utilisation dans la redirection de la destruction médiée par les lymphocytes T de cellules cancéreuses co-exprimant MUC16/mésothéline, par l'intermédiaire d'une formation sur cellule, sur cible, d'anticorps trispécifiques de recrutement de lymphocytes T.
PCT/EP2024/078140 2023-10-17 2024-10-07 Double ciblage de cellules tumorales co-exprimant muc16 et mésothéline par complémentation fonctionnelle de molécules de demi-corps cycat® Pending WO2025082777A1 (fr)

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