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WO2025087681A1 - Anticorps bispécifiques contre cd3 et cd20 - Google Patents

Anticorps bispécifiques contre cd3 et cd20 Download PDF

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Publication number
WO2025087681A1
WO2025087681A1 PCT/EP2024/078149 EP2024078149W WO2025087681A1 WO 2025087681 A1 WO2025087681 A1 WO 2025087681A1 EP 2024078149 W EP2024078149 W EP 2024078149W WO 2025087681 A1 WO2025087681 A1 WO 2025087681A1
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amino acid
domain
seq
acid sequence
present disclosure
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Christina HEITMÜLLER
Eveline Schaadt
Karin FELDERER
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Morphosys AG
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Morphosys AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency

Definitions

  • the present disclosure relates to bispecific antibodies, which interact with CD20 and CD3.
  • the present disclosure also relates to nucleic acid compositions, vector composition and host cells capable of expressing said bispecific antibody, pharmaceutical compositions comprising said bispecific antibody and uses of said bispecific antibody for the treatment of specific diseases, in particular B-cell related disorders.
  • 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.
  • 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.
  • CD20 is a non-glycosylated phosphoprotein expressed on the cell membranes of mature B cells.
  • CD20 is considered a B cell tumor-associated antigen because it is expressed by more than 95% of B-cell non-Hodgkin lymphomas (NHLs) and other B-cell malignancies, but it is absent on precursor B-cells, dendritic cells and plasma cells.
  • NHLs B-cell non-Hodgkin lymphomas
  • rituximab has been used or suggested for use in treating cancers such as NHL, chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL).
  • Rituximab is believed to kill CD20-expressing tumor cells by complement dependent cytotoxicity (CDC), antibody-dependent cell mediated cytotoxicity (ADCC) and/or induction of apoptosis and sensitization to chemotherapy.
  • CDC complement dependent cytotoxicity
  • ADCC antibody-dependent cell mediated cytotoxicity
  • anti-CD20 tumor targeting strategies have shown great promise in clinical settings, not all patients respond to anti-CD20 therapy, and some patients have been shown to develop resistance to or exhibit incomplete responses to anti-CD20 therapy (e.g., resistance to rituximab).
  • Bispecific antibodies that bind to both, CD3 and CD20 have been demonstrated to be useful in therapeutic settings. Recently, three bispecific anti-CD3xanti-CD20 antibodies (Lunsumio® (mosunetuzumab-axgb), Columvi® (glofitamab-gxbm) and Epkinly® (Epcoritamab) have been approved for the treatment of B-cell malignancies.
  • bispecific CD3xCD20 antibodies that effectively treat a subject suffering from a B cell-related disorder or cancer, in particular, a subject who has developed resistance to traditional anti-CD20, e.g., rituximab, therapy.
  • the present invention primarily takes a new approach of providing optimized bispecific antibodies targeting CD20 and CD3 by employing a fully human anti-CD3 Fv fragment characterized by the removal of potential T cell epitopes present in the CDR regions of their parental antibody counterpart.
  • the antibody format realized in the CD20xCD3 antibody of the present disclosure combines high affinity and avidity binding for CD20 but low affinity and monovalent binding to CD3 which further reduces the risk for side effects associated with CD3 directed therapies.
  • the present disclosure pertains to a bispecific CD3xCD20 antibody comprising one binding domain specific for CD3 and two binding domains specific for CD20.
  • the two CD20 binding domains are identical.
  • the CD3xCD20 antibody disclosed herein further comprises an Fc region.
  • the CD3xCD20 antibody according to the present disclosure has a structure has shown in FIG. 1.
  • the present disclosure pertains to a bispecific antibody comprising a first and a second binding domain which specifically bind to CD20 and a third binding domain which specifically binds to CD3 (CD3xCD20 antibody);
  • the first and the second binding domain of the CD3xCD20 antibody that bind to CD20 each comprises: (i) a VH domain comprising a HCDR1 , HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and
  • VL domain comprising a LCDR1, LCDR2, and LCDR3 having an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively;
  • VH domain comprising a HCDR1 , HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively;
  • VL domain comprising a LCDR1, LCDR2, and LCDR3 having an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure wherein:
  • the first and the second binding domain of the CD3xCD20 antibody that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16; and
  • the third binding domain of the CD3xCD20 antibody that binds to CD3 comprises a VH domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8;
  • the CD3xCD20 antibody according to the present disclosure is composed of
  • the present disclosure pertains to the CD3xCD20 according to the present disclosure, wherein:
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure, wherein the Fc region is a human IgGl Fc region.
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure, wherein the first and the second Fc region subunit of the human IgGl Fc region comprises one or more amino acid substitutions promoting the association of the first and second Fc region subunit.
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure, wherein:
  • 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
  • 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.
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure, wherein in the first and the second Fc region subunit at least 5 amino acid residues in the positions corresponding to positions L234, L235, G237, A330, P331 with numbering according EU index are mutated to A, E, A, S, and S, respectively.
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure, wherein the CD3xCD20 antibody is composed of 4 polypeptides, wherein:
  • the first polypeptide comprises the light chain of the first Fab
  • the second polypeptide comprises from N-terminus to C-terminus: i. the heavy chain of the first Fab, ii. a first peptide linker, iii. the VL domain of the third binding domain, iv. a second peptide linker, and v. the first Fc region subunit of the Fc region;
  • the second polypeptide comprises from N-terminus to C-terminus: i. the heavy chain of the second Fab, ii. a third peptide linker, iii. the VH domain of the third binding domain, iv. a fourth peptide linker, and v. the second Fc region subunit of the Fc region; and
  • the fourth polypeptide comprises the light chain of the second Fab.
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure, wherein each fusion occurs via a peptide linker.
  • the peptide linker is selected of having a length of 1 - 50 amino acid residues.
  • the first peptide linker and the third peptide linker are identical and/or the second and fourth peptide linker are identical.
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure, wherein the first peptide linker and the third peptide linker each comprises the ammo acid sequence of: AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 20) or EPKSCAQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 21) and/or wherein the second and fourth peptide linker each comprises the amino acid sequence of DQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 22) or DQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 23).
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure, wherein the first Fab comprising the first binding domain which binds to CD20 and the second Fab comprising the second binding domain which binds to CD20 are identical.
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure, wherein:
  • the first and the fourth polypeptide each comprises the amino acid sequence of SEQ ID NO: 17,
  • the second polypeptide comprises the amino acid sequence of SEQ ID NO: 18, and
  • the third polypeptide comprises the amino acid sequence of SEQ ID NO: 19.
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure, wherein the CD3xCD20 antibody provides bivalent binding to CD20 and monovalent binding to CD 3.
  • the present disclosure pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising the CD3xCD20 antibody according to the present disclosure and a pharmaceutically acceptable carrier or excipient.
  • the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure or the pharmaceutical composition according to the present disclosure for use in medicine. In one embodiment, the present disclosure pertains to the CD3xCD20 antibody according to the present disclosure or the pharmaceutical composition according to the present disclosure in the treatment of cancer. In one embodiment, said cancer is a hematological cancer. In one embodiment, said cancer is a B-cell malignancy. BRIEF description of the drawings
  • FIG. 1 depicts the structure of the bispecific CD3xCD20 antibody described herein.
  • the bispecific antibody has a 2+1 (Fab)2-Fv-Fc antibody format.
  • This antibody format is built from a human IgGl backbone incorporating one additional Fv fragment between the Fc region and the two Fab arms of the IgG. Both Fabs arms are identical and bind to CD20 while the additional Fv fragment comprises the variable regions of an fully human deimmunized anti-CD3 antibody.
  • the Fc region of the source IgGl backbone also includes substitutions that result in an immunocompromised Fc region that is unable to interact with IgG Fc receptors (Fc gamma receptors) and complement.
  • amino acid mutations were incorporated according to the knobs-into-holes technology (KiH) to promote heterodimerization of the two non-identical heavy or main chains of this bispecific antibody format.
  • FIG. 2 FIG. 2A & B: Tumor growth curves of subcutaneously engrafted Raji tumor cells with co-engrafted human T-cells in NOD-SCID mice treated with the CD20xCD3 antibody according to the present disclosure or vehicle control (FIG. 2B) or Isotype Control (FIG. 2A).
  • FIG. 2C & D Progression-free survival curves using a tumor cut-off value of TV>1.5 cm A 3, of subcutaneously engrafted Raji tumor cells with co-engrafted T-cells in NOD-SCID mice treated with CD20xCD3 antibody according to the present disclosure or vehicle control (FIG. 2D) or Isotype Control (FIG. 2C).
  • FIG. 3 Tumor growth curves of a subcutaneously implanted Raji tumor cell model in CD34 hematopoietic stem cell humanized NCG mice, boosted with human IL-15 plasmid DNA, and treated with CD20xCD3 antibody according to the present disclosure or vehicle control.
  • FIG 3B Progression-free survival curves, using a tumor cut-off value of TV>1500 mm
  • EMBODIMENTS EMBODIMENTS
  • any numerical values are to be understood as being modified in all instances by the term “about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
  • a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v).
  • an antigen refers to any molecule of interest that can be bound by one of the binding sites present in an antibody.
  • an antigen is a peptide, a protein or any other proteinaceous molecule.
  • an antigen may be any other organic or inorganic molecule, such as carbohydrate, fatty acid, lipid, dye or flourophor.
  • the terms “comprises”, “comprising”, “includes”, “including”, “has”, “having”, “contains”, “containing,” or “composed” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended.
  • a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus.
  • the term “comprise” “comprises” or “comprising” as used herein, may be replaced with “consists of’, “consisting of’, “consists essentially of’, or “consisting essentially of’.
  • CD3 also known as “cluster of differentiation 3,” refers to a T-cell co-receptor that helps in activation of both cytotoxic T-cell (e.g., CD8+ naive T cells) and T helper cells (e.g., CD4+ naive T cells) and is composed of four distinct chains: one CD3y chain (e.g., Genbank Accession Numbers NM_000073 and MP_000064 (human)), one CD38 chain (e.g., Genbank Accession Numbers NM_000732, NM_001040651, NP_00732 and NP_001035741 (human)), and two CD3s (CD3 epsilon) chains (e.g., Genbank Accession Numbers NM_000733 and NP_00724 (human)).
  • CD3y chain e.g., Genbank Accession Numbers NM_000073 and MP_000064 (human)
  • one CD38 chain e.g., Genbank Accession
  • the chains of CD3 are highly related cell-surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain.
  • the CD3 molecule associates with the T-cell receptor (TCR) and ⁇ -chain to form the T-cell receptor (TCR) complex, which functions in generating activation signals in T lymphocytes.
  • TCR T-cell receptor
  • TCR T-cell receptor
  • TCR T-cell receptor
  • TCR TCR
  • the CD 3 is human CD3.
  • the CD3 is human CD3 epsilon.
  • Human CD3epsilon (or human CD3e) has the amino acid sequence of UniProt P07766:
  • the mature extracellular domain of human CD3epsilon without signal sequence comprises amino acid residues 22-126 and has the amino acid sequence of:
  • Cynomolgus CD3epsilon (or cyno CD3e) has the amino acid sequence of UniProt Q95LI5
  • the mature extracellular region of cynomolgus monkey CD3 epsilon without the signal sequence comprises amino acid residues 22-198 and has the amino acid sequence of:
  • CD20 also known as “B-lymphocyte antigen CD20”, “CD20 antigen”, “CD20 Receptor”, “Membrane Spanning 4-Domains Al”, “Membrane- Spanning 4-Domains, Subfamily A, Member 1”, “Leukocyte Surface Antigen Leu- 16”, “Bp35”, “B-Lymphocyte Cell-Surface Antigen 1”, “LEU-16”, “CVID5”, “MS4A”, “Bl”, and “S7”, refers to an activated-glycosylated phosphoprotein expressed on the surface of B-cells and is encoded by the MS4A1 gene in humans (e.g, Genbank Accession Numbers NM_J 52866, NM_021950, NP_068769 and NP_690605 (human)).
  • CD20 plays a role in the development and differentiation of B-cells into plasma cells.
  • the CD20 is human CD20.
  • administering 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.
  • 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.
  • antibody refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds which interacts with an antigen.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions 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 amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with 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.
  • antibody includes for example, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies and chimeric antibodies.
  • the antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.
  • immunoglobulin variable domains e.g., CDRs
  • 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. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NTH Publication no. 91-3242 U.S.
  • antibody as used herein is intended to include monospecific specific antibodies as well as bispecific and multispecific antibodies.
  • antibody fragment or “antigen-binding 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 or antigen-binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI 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 CHI domains; a Fv fragment 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).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • 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 CHI domains
  • 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” or “antigen-binding 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 or antigenbinding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) 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).
  • antigen binding domain or just “binding domain” as used herein refer to a set of six CDRs that, when present as part of a polypeptide sequence, specifically binds a target antigen as discussed herein.
  • these CDRs are generally present as a first set of variable heavy CDRs (HCDRs) and a second set of variable light CDRs (LCDRs), each comprising three CDRs: HCDR1, HCDR2, HCDR3 for the heavy chain and LCDR1, LCDR2 and LCDR3 for the light chain.
  • the CDRs are present in the variable heavy and variable light domains, respectively, and together form an Fv region or Fv fragment.
  • the six CDRs of the antigen binding domain are contributed by a variable heavy and a variable light domain.
  • the set of 6 CDRs are contributed by two different polypeptide sequences, the variable heavy domain (VH; containing the HCDR1, HCDR2 and HCDR3) and the variable light domain (VL; containing the LCDR1, LCDR2 and LCDR3), with the C-terminus of the VH domain being attached to the N-terminus of the CHI domain of the heavy chain and the C-terminus of the VL domain being attached to the N-terminus of the constant light domain (and thus forming the light chain).
  • VH variable heavy domain
  • VL variable light domain
  • an antibody “binds specifically to”, “specifically binds to”, is “specific to/for” or “specifically recognizes” an antigen if such antibody is able to discriminate between such antigen and one or more reference antigen(s), since binding specificity is not an absolute, but a relative property.
  • determination of binding specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like.
  • bispecific antibody means any non-native or alternate antibody format that engages two or more different antigens (e.g., CD3xCD20 bispecific antibodies).
  • cell proliferative disease or “proliferative disease” as used herein refer to a disease that is associated with some degree of abnormal cell proliferation.
  • the cell proliferative disease is cancer.
  • the cell proliferative disease is a tumor.
  • cancer and “cancerous” as used herein refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • cell surface means one or more protein(s) that is/are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of the protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion or fragment of an antibody.
  • Cell surface expressed CD3 includes CD3 proteins contained within the context of a functional T cell receptor in the membrane of a cell.
  • the expression "cell surface expressed CD3” includes CD3 protein expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., gamma/epsilon, delta/epsilon, and zeta/zeta CD3 dimers).
  • cell surface expressed CD3 also includes a CD3 chain (e.g., CD3epsilon) that is expressed by itself, without other CD3 chain types, on the surface of a cell.
  • a “cell surface expressed CD3” can comprise or consist of a CD3 protein expressed on the surface of a cell which normally expresses CD3 protein.
  • “cell surface expressed CD3” can comprise or consist of CD3 protein expressed on the surface of a cell that normally does not express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.
  • chimeric antibody or “chimeric antibody fragment” are 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.
  • compositions of the present disclosure may be used for therapeutic or prophylactic applications.
  • the present disclosure therefore, includes a pharmaceutical composition containing an antibody (or antigen-binding fragment thereof) as disclosed herein and a pharmaceutically acceptable carrier or excipient therefor.
  • the present disclosure provides a method for treating cancer. Such method contains the steps of administering to a subject in need thereof an effective amount of the pharmaceutical composition that contains an antibody (or antigen-binding fragment thereof) according to the present disclosure.
  • the present disclosure provides therapeutic methods comprising the administration of a therapeutically effective amount of a CD20xCD3 antibody as disclosed herein to a subject in need of such treatment.
  • cross-reactively binds or the term “is cross-reactive” are used herein interchangeably and refers to an antibody or antigen-binding fragment which has the ability to specifically bind to more than one antigen.
  • the antibody according to the present disclosure antibody cross-reactively binds to cynomolgus CD3, such as cynomolgus CD3 epsilon.
  • Deimmunization refers to a method, wherein amino acids within an given antibody sequence that are predicted to bind effectively to HLA molecules are changed such that they no longer bind HLA and thus can no longer stimulate a T cell response. Accordingly, deimmunization renders a given protein or polypeptide non-immunogenic or less immunogenic to a given species.
  • T cell epitopes from proteins has been previously disclosed (see WO 98/52976).
  • One suitable technique for deimmunizing antibodies is described, for example, WO 00/34317 or WO 2003/105058.
  • For a therapeutic antibody preferably all of the potential T cell epitopes are removed whilst retaining the functional activity of the unmodified parental antibody.
  • a “deimmunized” antibody or antibody fragment or binding domain as used herein refers to an antibody, antibody fragment or binding domains which was subject to deimmunization.
  • a deimmunized” antibody may be less or non-immunogenic in a given species compared to the unmodified parental antibody when used in vivo.
  • Fab refers to the polypeptide that comprises the VH, CHI, VL, and CL immunoglobulin domains, generally on two different polypeptide chains (e.g.,VH-CHl on one chain and VL-CL on the other).
  • Fab may refer to this region in isolation, or this region in the context of a bispecific antibody provided herein.
  • the Fab comprises an Fv fragment in addition to the CHI and CL domains.
  • Fv Fv fragment
  • Fv region refers to a polypeptide that consists of a dimer of the VL and VH domain of an antibody.
  • Fc region refers to the two Fc region subunits being capable of stable association with each other thus forming the dimeric C-terminal region of an immunoglobulin. Accordingly, the two Fc region subunits (e.g. the first the second Fc region subunit) are complementary to each other.
  • the Fc region of a regular IgG molecule exists as a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains.
  • Fc region subunit 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 which form the dimeric Fc region are complementary to each other.
  • 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 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.
  • numbering of amino acid residues in the Fc region is according to the 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.
  • heavy chain constant region refers to the CHl-hinge-CH2-CH3 portion of an antibody (or fragments thereof), excluding the variable heavy domain; in EU numbering of human IgGl this is amino acids 118-447
  • heavy chain constant region fragment herein is meant a heavy chain constant region that contains fewer amino acids from either or both of the N- and C-termini but still retains the ability to form a dimer with another heavy chain constant region.
  • immunoglobulin (Ig) "hinge” refers to one of the two polypeptides forming the dimeric “hinge region” of an immunoglobulin.
  • the hinge includes the portion of an immunoglobulin heavy chain that joins the CHI domain to the CH2 domain.
  • a natural occurring immunoglobulin is composed of two identical hinges, which are linked via one or more disulfide bridges formed through interchain cysteins present in the two hinges.
  • a natural occurring immunoglobulin is composed of a dimeric disulfide stabilized hinge region, that joins the two Fab arms of an immunoglobulin to the Fc region.
  • a hinge can be subdivided into three distinct domains: upper, middle, and lower hinge (Roux et ah, J. Immunol. 1998 161:4083).
  • a “humanized antibody” or “humanized antibody fragment” is defined herein as an antibody molecule 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.
  • sequence identity in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithms or by visual inspection.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • sequence comparison algorithm test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • increase is meant the ability to cause an overall increase, for example, of 20% or greater, of 50% or greater, or of 75%, 85%, 90%, 95%, or greater.
  • the terms “inhibition” or “inhibit” or “reduction” or “reduce” or “neutralization” or “neutralize” and the like refer to a decrease or cessation of any phenotypic characteristic (such as binding, a biological activity or function) or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic.
  • the “inhibition”, “reduction” or “neutralization” and the like needs not to be complete as long as it is detectable using an appropriate assay.
  • by “reduce” or “inhibit” and the like is meant the ability to cause a decrease of 20% or greater. In another aspect, by “reduce” or “inhibit” is meant the ability to cause a decrease of 50% or greater. In yet another aspect, by “reduce” or “inhibit” and the like is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
  • an “isolated antibody” as used herein refers to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a given target is substantially free of antibodies that do not bind to that same target). In addition, an isolated antibody is substantially free of other cellular material and/or chemicals.
  • monoclonal antibody refers to an antibody 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 as disclosed herein may be made by the hybridoma method as described in Kohler et a/.; Nature, 256:495 (1975) or may be isolated from phage libraries using the techniques according to the present disclosure, for example.
  • multispecific antibody refers to an antibody that comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.
  • first and second epitopes do not overlap or do not substantially overlap.
  • the first and second epitopes are on different antigens, e.g., the different proteins (or different subunits of a multimeric protein).
  • subject means any animal, preferably a mammal, most preferably a human.
  • mammal as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc. In a specific embodiment, the subject is a human.
  • peptide can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art.
  • the conventional one-letter or three-letter code for amino acid residues is used herein.
  • peptide can be used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc. as well as other modifications known in the art.
  • nucleic acid molecule refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and doublestranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and doublestranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
  • amino acid sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.
  • recombinant antibody includes all antibodies or antigen-binding fragments that are prepared, expressed, created or segregated by means not existing in nature. For example antibodies isolated from a host cell transformed to express the antibody, 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 have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies 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 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 may be a monoclonal antibody.
  • T cell epitope refers to a specific lOmer peptide sequence within a given protein or polypeptide sequence which either binds with reasonable efficiency to MHC class II molecules or which in the form of peptide: MHC complexes bind strongly to the T cell receptors from the species to receive the therapeutic protein or which show the ability to stimulate T cells via presentation on MHC class II. Potential T cell epitopes can be measured by any computational or physical method to establish MHC binding.
  • the terms “treat,” “treatment,” and “treating” refer to the amelioration of at least one measurable physical parameter of a solid malignant tumor described herein, such as tumor size, rate of tumor growth, number of tumor cells, tumor invasiveness, presence of metastasis, or extent of metastasis.
  • the terms “treat,” “treatment,” and “treating” refer to the inhibition of the progression of a solid malignant tumor described herein, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both.
  • the terms “treat,” “treatment,” and “treating” refer to an increase in the immune system response of the human subject, such as increased T cell infiltration, increased T cell activation, upregulation of IFN pathways, upregulation of antigen presentation pathway, or increased Ki67+ induction in T cells following treatment with pembrolizumab or nivolumab.
  • treating a solid malignant tumor provides an improvement, or a lack of progression, in the disease associated with the tumor or the tumor condition, and/or an improvement, or a lack of progression, in the symptoms associated with the disease or condition.
  • treating a solid malignant tumor involves administering the bispecific antibody for a pre-specified period of time, discontinuing administration for another specific period of time, and resuming administration of the bispecific antibody for yet another specific period of time.
  • treating a solid malignant tumor involves administering the bispecific antibody until one of the treatment effects described herein is achieved, pausing administration of the bispecific antibody while this treatment effect continues to be observed, and resuming administration of the bispecific antibody if this treatment effect ceases to be observed.
  • tumor associated antigen refers to an antigen that is expressed or present on the surface of a tumor or of a cell of the tumor stroma.
  • Treatment according to the methods provided herein includes a “therapeutically effective amount” of the medicaments used.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the medicaments to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” for tumor therapy may also be measured by its ability to stabilize the progression of disease.
  • the ability of a compound to inhibit cancer may be evaluated in an animal model system predictive of efficacy in human tumors.
  • this property of a composition may be evaluated by examining the ability of the compound to inhibit cell growth or to induce apoptosis by in vitro assays known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject’s size, the severity of the subject’s symptoms, and the particular composition or route of administration selected.
  • variable region refers to the region of an immunoglobulin that comprises one or more Ig domains substantially encoded by any of the VK, VX, and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively, and contains the CDRs that confer antigen specificity.
  • VK, VX, and/or VH genes that make up the kappa, lambda, and heavy chain immunoglobulin genetic loci respectively, and contains the CDRs that confer antigen specificity.
  • a “variable heavy domain” pairs with a “variable light domain” to form an antigen binding domain (“ABD”).
  • each variable domain comprises three hypervariable regions (“complementary determining regions,” “CDRs”) (HCDR1, HCDR2 and HCDR3 for the variable heavy domain and LCDR1, LCDR2 and LCDR3 for the variable light domain) and four framework (FR) regions, arranged from amino-terminus to carboxy -terminus in the following order: FR1-CDR1-FR2- CDR2-FR3 -CDR3 -FR4.
  • CDRs composite determining regions
  • the present disclosure provides a novel bispecific antibody capable of simultaneously binding to CD3 and CD20.
  • the bispecific CD20xCD3 antibody of the present disclosure introduces a format that allows for bivalent high affinity binding to CD20 and monovalent low affinity binding to CD3, which reduces the risk for side effects associated with CD3 directed therapies, combined with all the desirable properties provided by a regular full-length immunoglobulin and which are easy to purify from the culture supernatant of respective production cell lines
  • the present disclosure pertains to a bispecific antibody which specifically binds to CD20 and CD3.
  • the antibody according to the present disclosure provides bivalent binding to CD20 and monovalent binding to CD3 (CD20xCD3 antibody).
  • CD20xCD3 antibody according to the present disclosure is a trivalent bispecific antibody.
  • the CD20xCD3 antibody according to the present disclosure is composed of two Fab fragments and one Fv fragment. This is achieved by using a regular IgG antibody structure (two heavy chains with associated two light chains) which incorporates one additional Fv fragment between the two Fab arms and the Fc portion of the IgG structure.
  • the CD20xCD3 antibody according to the present disclosure has a structure as depicted in FIGI
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody comprises a first binding domain and a second binding domain which specifically bind to CD20.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody comprises a third binding domain which specifically bind to CD3.
  • the present disclosure pertains to a bispecific antibody comprising a first and a second binding domain which specifically bind to CD20 and a third binding domain which specifically binds to CD3 (CD20xCD3 antibody).
  • the present disclosure pertains to a bispecific antibody comprising a first and a second binding domain which specifically bind to CD20 and a third binding domain which specifically binds to CD3 and further comprises a Fc region.
  • first binding domain and the second binding that bind to CD20 are identical. In one embodiment, the first binding domain and the second binding domain that bind to CD20 have the identical amino acid sequence.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody is composed of a first Fab and a second Fab which specifically bind to CD20.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody is composed of a first Fab comprising the first binding domain that binds to CD20 according to the present disclosure and a second Fab comprising the second binding domain that binds to CD20 according to the present disclosure.
  • the first Fab and the second Fab are identical.
  • the first Fab and the second Fab have the identical amino acid sequence.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody is composed of a Fv fragment which specifically bind to CD3.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody is composed of a Fv fragment comprising the third binding domain that binds to CD3 according to the present disclosure.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody is composed of a first Fab and a second Fab which specifically bind to CD20 and of a Fv fragment which specifically bind to CD3.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody is composed of:
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody is composed of a first Fab and a second Fab which specifically bind to CD20 and of a Fv fragment which specifically bind to CD3 and a Fc region.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody is composed of:
  • the Fc region is composed of a first and a second Fc region subunit.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein said bispecific antibody is composed of:
  • the first Fab is fused to the Fv fragment.
  • the C-terminus of the heavy chain of the first Fab is fused to the Fv fragment.
  • the C-terminus of the of the heavy chain of the first Fab is fused to the N-terminus of the VH or the VL domain of the Fv fragment.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VH domain of the Fv fragment.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VL domain of the Fv fragment.
  • the first Fab is fused to the Fv fragment via a peptide linker.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VH or VL domain of the Fv fragment via a peptide linker.
  • the Fv fragment is fused to the Fc region.
  • the C-terminus of the VH or VL domain of the Fv fragment is fused to the N-terminus of the first Fc region subunit.
  • the C-terminus of the VH or VL domain of the Fv fragment is fused to the N- terminus of the second Fc region subunit.
  • the C-terminus of the VH domain of the Fv fragment is fused to the N-terminus of the first Fc region subunit and the C-terminus of the VL domain of the Fv fragment is fused to the N-terminus of the second Fc region subunit.
  • the C-terminus of the VL domain of the Fv fragment is fused to the N-terminus of the first Fc region subunit and the C-terminus of the VH domain of the Fv fragment is fused to the N-terminus of the second Fc region subunit.
  • the second Fab is fused to the Fv fragment.
  • the C-terminus of the heavy chain of the second Fab is fused to the Fv fragment.
  • the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VH or VL domain of the Fv fragment.
  • the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VH domain of the Fv fragment.
  • the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VL domain of the Fv fragment.
  • the second Fab is fused to the Fv fragment via a peptide linker.
  • the first Fab and the second Fab are fused to distinct variable domains of the Fv fragment. In an embodiment of the present disclosure, each fusion occurs via a peptide linker.
  • the bispecific antibody according to the present disclosure does not comprise more than two Fab fragments with specificity for CD20. In an embodiment, the bispecific antibody according to the present disclosure does not comprise more than one Fv fragment with specificity for CD3.
  • the heavy chain of the first Fab is composed in order from N- to C-terminus of a VH domain and a CHI domain.
  • the heavy chain of the second Fab is composed in order from N- to C-terminus of a VH domain and a CHI domain.
  • the C-terminus of the CHI domain of the first Fab is fused to the N-terminus of the VH or VL domain of the Fv fragment.
  • the C-terminus of the CHI domain of the second Fab is fused to the N-terminus of the VH or VL domain of the Fv fragment.
  • the C-terminus of the CHI domain of the second Fab is fused to the N-terminus of the VH or VL domain of the Fv fragment with the proviso that first Fab and second Fab are fused to distinct variable domains of the Fv fragment.
  • the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VL of the second Fv fragment with the proviso that first and second Fab are fused to distinct variable domains of the second Fv fragment.
  • the fusion occurs via a peptide linker.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VL of the Fv fragment and the C- terminus of the heavy chain of the second Fab is fused to the N-terminus of the VH domain of the Fv fragment.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VH domain of the Fv fragment and the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VL of the Fv fragment.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VH or VL domain of the Fv fragment and the N-terminus of the first Fc region subunit is fused to the C-terminus of the VH or VL domain of the Fv fragment.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VH domain of the Fv fragment and the N-terminus of the first Fc region subunit is fused to the C-terminus of the VH domain of the Fv fragment.
  • the C-terminus of the heavy chain of the first Fab is fused to the N- terminus of the VL domain of the Fv fragment and the N-terminus of the first Fc region subunit is fused to the C-terminus of the VL domain of the Fv fragment.
  • the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VH domain of the second Fv fragment and the N-terminus of the second Fc region subunit is fused to the C-terminus of the VH domain of the Fv fragment.
  • the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VL domain of the Fv fragment and the N-terminus of the second Fc region subunit is fused to the C-terminus of the VL domain of the Fv fragment.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VH or VL domain of the Fv fragment and the N-terminus of the first Fc region subunit is fused to the C-terminus of the VH or VL domain of the Fv fragment
  • the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VH or VL domain of the Fv fragment and the N-terminus of the first Fc region subunit is fused to the C-terminus of the VH or VL domain of the Fv fragment
  • the N-terminus of the second Fc region subunit is fused to the C-terminus of the VH or VL domain of the Fv fragment
  • the heavy chain of the first Fab and the heavy chain of the second Fab are fused to distinct variable domains of the Fv fragment
  • the first Fc region subunit an the second Fc region subunit are fused to distinct variable domains
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VH domain of the first Fv fragment and the N-terminus of the first Fc region subunit is fused to the C-terminus of the VH domain of the Fv fragment and the C- terminus of the heavy chain of the second Fab is fused to the N-terminus of the VL domain of the second Fv fragment subunit and the N-terminus of the second Fc region subunit is fused to the C- terminus of the VL domain of the Fv fragment.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VL domain of the first Fv fragment and the N-terminus of the first Fc region subunit is fused to the C-terminus of the VL domain of the Fv fragment and the heavy chain of the second Fab is fused to the N-terminus of the VH domain of the Fv fragment and the N-terminus of the second Fc region subunit is fused to the C-terminus of the VH domain of the Fv fragment.
  • each fusion occurs via a peptide linker.
  • the present disclosure pertains to a bispecific antibody according to the present disclosure, wherein said bispecific antibody is composed of 4 polypeptides.
  • the first polypeptide comprises the light chain of the first Fab.
  • the first polypeptide comprises the light chain of the first Fab composed of a VL and a CL domain.
  • the second polypeptide comprises from its N- to C- terminus:
  • the second polypeptide comprises from its N- to C- terminus:
  • the second polypeptide comprises from its N- to C- terminus:
  • the heavy chain for the first Fab is composed of a VH and a CHI domain.
  • the second polypeptide comprises from its N- to C- terminus:
  • the second polypeptide comprises from its N- to C- terminus:
  • the second polypeptide comprises from its N- to C- terminus:
  • the third polypeptide comprises from its N- to C- terminus
  • the third polypeptide comprises from its N- to C- terminus:
  • the third polypeptide comprises from its N- to C- terminus:
  • the heavy chain of the second Fab is composed of a VH and a CHI domain.
  • the third polypeptide comprises from its N- to C- terminus:
  • the third polypeptide comprises from its N- to C- terminus:
  • the third polypeptide comprises from its N- to C- terminus:
  • the fourth polypeptide comprises the light chain of the second Fab. In an embodiment of the present disclosure, the fourth polypeptide comprises the light chain of the second Fab composed of a VL and a CL domain. In an embodiment, the light chain of the first Fab comprises a VL and CL domain. In an embodiment, the light chain of the second Fab comprises a VL and CL domain. In an embodiment, the heavy chain of the first Fab comprises a VH and CHI domain. In an embodiment, the heavy chain of the second Fab comprises a VH and CHI domain.
  • the present disclosure pertains to a bispecific antibody according to the present disclosure, wherein said bispecific antibody is composed of 4 polypeptides, wherein:
  • the first polypeptide comprises the light chain of the first Fab
  • the second polypeptide comprises from its N- to C-terminus i. the heavy chain of the first Fab; ii. the VL or VH domain of the third binding domain, and iii. the first Fc region subunit;
  • the third polypeptide comprises from its N- to C-terminus i. the heavy chain of the second Fab, ii. the complementary VL or VH domain of the third binding domain, and iii. the second Fc region subunit; and (d) the fourth polypeptide comprises the light chain of the second Fab.
  • the present disclosure pertains to a bispecific antibody according to the present disclosure, wherein said bispecific antibody is composed of 4 polypeptides, wherein:
  • the first polypeptide comprises the light chain of the first Fab
  • the second polypeptide comprises from its N- to C-terminus i. the heavy chain of the first Fab; ii. the first peptide linker iii. the VL or VH domain of the third binding domain, iv. the second peptide linker, and v. the first Fc region subunit;
  • the third polypeptide comprises from its N- to C-terminus i. the heavy chain of the second Fab, ii. the third peptide linker, iii. the complementary VL or VH domain of the third binding domain, iv. the fourth peptide linker, and v. the second Fc region subunit;
  • the fourth polypeptide comprises the light chain of the second Fab.
  • the present disclosure pertains to a bispecific antibody according to the present disclosure, wherein said bispecific antibody is composed of 4 polypeptides, wherein:
  • the first polypeptide comprises the light chain of the first Fab
  • the second polypeptide comprises from its N- to C-terminus, i. the heavy chain of the first Fab, ii. the VL domain of the third binding domain, and iii. the first Fc region subunit;
  • the third polypeptide comprises from its N- to C-terminus i. the heavy chain of the second Fab, ii. the VH domain of the third binding domain, and iii. the second Fc region subunit;
  • the fourth polypeptide comprises the light chain of the second Fab.
  • the present disclosure pertains to a bispecific antibody according to the present disclosure, wherein said bispecific antibody is composed of 4 polypeptides, wherein:
  • the first polypeptide comprises the light chain of the first Fab
  • the second polypeptide comprises from its N- to C-terminus i. the heavy chain of the first Fab; ii. the first peptide linker iii. the VL domain of the third binding domain, iv. the second peptide linker, and v. the first Fc region subunit;
  • the third polypeptide comprises from its N- to C-terminus i. the heavy chain of the second Fab, ii. the third peptide linker. iii. the VH domain of the third binding domain, iv. the fourth peptide linker and v. the second Fc region subunit; and (d) the fourth polypeptide comprises the light chain of the second Fab.
  • the present disclosure pertains to a bispecific antibody according to the present disclosure, wherein said bispecific antibody is composed of 4 polypeptides, wherein:
  • the first polypeptide comprises the light chain of the first Fab
  • the second polypeptide comprises from its N- to C-terminus i. the heavy chain of the first Fab; ii. the VH domain of the third binding domain, and iii. the first Fc region subunit;
  • the third polypeptide comprises from its N- to C-terminus i. the heavy chain of the second Fab, ii. the VL domain of the third binding domain, and iii. the second Fc region subunit;
  • the fourth polypeptide comprises the light chain of the second Fab.
  • the present disclosure pertains to a bispecific antibody according to the present disclosure, wherein said bispecific antibody is composed of 4 polypeptides, wherein:
  • the first polypeptide comprises the light chain of the first Fab
  • the second polypeptide comprises from its N- to C-terminus i. the heavy chain of the first Fab, ii. the first peptide linker, iii. the VH domain of the third binding domain, iv. the second peptide linker, and V. the first Fc region subunit;
  • the third polypeptide comprises from its N- to C-terminus i. the heavy chain of the second Fab, ii. the third peptide linker. iii. the VL domain of the third binding domain, iv. the fourth peptide linker and v. the second Fc region subunit;
  • the fourth polypeptide comprises the light chain of the second Fab.
  • the first polypeptide and the second polypeptide form the first Fab fragment of the CD20xCD3 antibody according to the present disclosure.
  • the third polypeptide and the fourth polypeptide form the second Fab fragment of the CD20xCD3 antibody according to the present disclosure.
  • the second polypeptide and the third polypeptide form the Fv fragment of the CD20xCD3 antibody according to the present disclosure.
  • first and the second polypeptide are linked to each other via at least one disulfide bond. In one embodiment, the second and the third polypeptide are linked to each other via at least one disulfide bond. In one embodiment, the third and the fourth polypeptide are linked to each other via at least one disulfide bond.
  • said peptide linker is a peptide linker according to the present disclosure.
  • said first, second, third and/or fourth peptide linker are identical.
  • said first, second, third and/or fourth peptide linker are different.
  • said first, second, third and/or fourth peptide linker have the same length.
  • said first, second, third and/or fourth peptide linker have a different length.
  • said first and third peptide linker are identical or have the same length.
  • said second and fourth peptide linker are identical or have the same length.
  • said first and third peptide linker are identical and said second and fourth peptide linker are identical.
  • said first and third peptide linker are identical and said second and fourth peptide linker are identical.
  • said first and third peptide linker have the same length and said second and fourth peptide linker have he same length.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VL domain of the Fv fragment via a first peptide linker and the N- terminus of the first Fc region subunit is fused to the C-terminus of the VL domain of the Fv fragment via a second peptide linker.
  • the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VH domain of the Fv fragment via a third peptide linker and the N-terminus of the second Fc region subunit is fused to the C-terminus of the VH domain of the Fv fragment via a fourth peptide linker.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VL domain of the Fv fragment via a first peptide linker and the N- terminus of the first Fc region subunit is fused to the C-terminus of the VL domain of the Fv fragment via a second peptide linker and the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VH domain of the Fv fragment via a third peptide linker and the N- terminus of the second Fc region subunit is fused to the C-terminus of the VH domain of the Fv fragment via a fourth peptide linker.
  • the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the VH domain of the first Fv fragment via a first peptide linker and the N-terminus of the first Fc region subunit is fused to the C-terminus of the VH domain of the Fv fragment via a second peptide linker and the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the VL domain of the Fv fragment via a third peptide linker and the N- terminus of the second Fc region subunit is fused to the C-terminus of the VH domain of the Fv fragment via a fourth peptide linker.
  • the bispecific CD20xCD3 antibody according to the present disclosure may be designed such that its individual components (e.g. Fab, Fv fragment, Fc region, variable domains, constant domains) are fused directly to each other or indirectly through a linker.
  • the individual components or domains of the bispecific antibody according to the present disclosure are genetically fused to each other. Such fusion may be achieved by a number of strategies, which include, but are not limited to polypeptide fusion between the N- and C-terminus of polypeptides, fusion via disulfide bonds, and fusion via chemical cross-linking reagents.
  • linkers may be used in the embodiments described herein to covalently fuse the individual components or domains of an bispecific antibody according to the present disclosure to its intended fusion partner.
  • the composition and length of a linker may be determined in accordance with methods well known in the art and may be tested for efficacy.
  • the linker is non-immunogenic.
  • 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 peptide linker should have a length that is adequate to fuse two polypeptides (or components or domains) 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.
  • the 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.
  • said peptide linker is a flexible peptide linker.
  • said peptide linker does not comprise a protease cleavage site.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein the peptide linker is comprised of G-A polymers, A-S polymers, P-A polymers or P-A-S polymers.
  • the peptide linker according to the present disclosure is selected from the group of: (GS) n (SEQ ID NO: 28), (G 4 S) n (SEQ ID NO: 29), (SG 4 )n (SEQ ID NO: 30), (GSGGS) n (SEQ ID NO: 31), (GGGS)n (SEQ ID NO: 32), G 4 (SG 4 ) n (SEQ ID NO: 33), (GGSG) n (SEQ ID NO: 34), (GGSGG) n (SEQ ID NO: 35), (GSGSG) n (SEQ ID NO: 36),(GSGGG) n (SEQ ID NO: 37), (GGGSG) n (SEQ ID NO: 38), and (GSSSG) n (SEQ ID NO: 39), 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 CLK or CLZ. domains or the CHI 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: 40) or ASTKGP (SEQ ID NO: 41).
  • a peptide linker may also comprise an immunoglobulin hinge (e.g. a human IgGl hinge or part thereof) or any peptide derived from such hinge.
  • an immunoglobulin hinge e.g. a human IgGl hinge or part thereof
  • 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 bispecific antibody according to the present disclosure.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein the 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.
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein the peptide linker according to the present disclosure comprises an amino acid sequence selected from the group of:
  • KTHT (SEQ ID NO: 42), KTHTCPPCP (SEQ ID NO: 43), DKTHTCPPCP (SEQ ID NO: 44), EPKSCDKTHTCPPCP (SEQ ID NO: 45), EPKSC (SEQ ID NO: 46), GQPSG (SEQ ID NO: 47), EPKSCGQPSG (SEQ ID NO: 48) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 49), AQPAAPDAHEAPAPAQGS (SEQ ID NO: 20), AQPAAPAPDAHEAPAPAQGSKTHT (SEQ ID NO: 50), AQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 51), AQPAAPAPAE (SEQ ID NO: 52), AQPAAPAPAEKTHTCPPCP (SEQ ID NO: 53), AQPAAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 54), QPKAAPDKTHTCPPCP (SEQ ID NO: 55),
  • the peptide linker according to the present disclosure comprises the amino acid sequence selected from the group of:
  • AQPAAPAPDAHEAPAPAQGS SEQ ID NO: 20
  • EPKSCAQPAAPAPDAHEAPAPAQGS SEQ ID NO: 21
  • DQPAAPAPDAHEAPAPAQGS SEQ ID NO: 22
  • DQPAAPDAHEAPAPAQGSKTHTCPPCP SEQ ID NO: 23
  • the present disclosure pertains to a bispecific antibody which specifically bind to CD20 and CD3, wherein the peptide linker according to the present disclosure has a length of between 1 to 50 amino residues, such as 1 to 45 amino acid residues, 1 to 40 amino acid residues, 1 to 35 amino acid residues, 1 to 30 amino acid residues, 1 to 25 amino acid residues, 1 to 20 amino acid residues, 1 to 15 amino acid residues, 1 to 10 amino acid residues, 1 to 5 amino acid residues.
  • a peptide linker according to the present disclosure has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 30, 35, 40, 45, 50, 60, or 70 amino acids residues.
  • a peptide linker according to the present disclosure has a length of 15 - 35 amino acids residues.
  • the different components of a bispecific antibody to the present disclosure are fused in a “non-cleavable” fashion.
  • non-cleavable is meant the inability to be cleaved and released from a bispecific antibody according to the present disclosure through the activity of an enzyme expressed by a cancer cell to which such bispecific antibody binds to.
  • a “non cleavable peptide linker” as used 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. Accordingly, a peptide linker according to the present disclosure is not cleavable by a protease.
  • said protease comprises a tumor specific protease.
  • said protease comprises a matrix metalloprotease (MMP) or a serine protease.
  • MMP matrix metalloprotease
  • 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: 69) or Ala-Val-Arg-Trp-Leu-Leu-Thr- Ala (SEQ ID NO: 70), which can be cleaved by metalloproteinases, or Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 71), which is cleaved by a furin.
  • a peptide linker according to the present disclosure 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 my 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 glycineserine polymer and an immunoglobulin hinge derived sequence in order to retain or obtain the desired activity.
  • the Fc region of the CD20xCD3 antibody according to the present disclosure is composed of a pair of polypeptides comprising heavy chain 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 the Fc region of the bispecific antibody according to the present disclosure are capable of stable association with each other.
  • the Fc region of the bispecific antibody according to the present disclosure is an IgG Fc region.
  • the Fc region is an IgGl Fc region.
  • the Fc region is human Fc region.
  • the Fc region is a human IgGl Fc region.
  • the two Fc region subunits of the CD20xCD3 antibody according to the present disclosure are comprised in two non-identical polypeptide chains.
  • the present disclosure provides a heterodimeric CD20xCD3 antibody.
  • the Fc region of the CD20xCD3 antibody according to the present disclosure 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 the bispecific antibody according to the present disclosure comprises one or more amino acid substitutions promoting the association of the first and the second Fc region subunit.
  • the first Fc region subunit and second Fc region subunit of the CD20xCD3 antibody according to the present disclosure comprises one or more modifications that reduce homodimerization or reduce homodimer formation between two identical polypeptide chains comprising the same Fc region subunit.
  • the modification is present in the first and second Fc region subunit.
  • the modification is present in the CH3 domain of each Fc region subunit.
  • One heterodimerization approach known in the art is the so-called "knobs-into-holes" technology, which is described in detail providing several examples in e.g. 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 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 the CD20xCD3 antibody 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 Fc region subunit.
  • 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 the CD20xCD3 antibody according to the present disclosure, 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 Fc region of the CD3xCD20 antibody according to the present disclosure is mutated to have an abolished or reduced binding affinity to an Fc receptor and/or to Clq or to have an abolished or reduced effector function, as compared to a wild- type or nonengineered Fc region.
  • Altered effector functions are typically achieved by mutating at least one, preferably both, of the parental Fc region subunits.
  • non-conservative amino acid substitutions i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties, are preferred.
  • the reduced or abolished effector function is one or more selected from the group consisting of CDC, ADCC and ADCP. In an embodiment, the reduced or abolished effector function is ADCC. In an embodiment, the reduced or abolished effector function is CDC. In an embodiment, the reduced or abolished effector function is ADCP. In an embodiment, the reduced or abolished effector function is CDC, ADCC and ADCP.
  • the Fc region of the CD20xCD3 antibody according to the present disclosure is mutated to have a reduced binding affinity to an Fc receptor and/or to Clq and/or to have reduced effector function when compared to a non-engineered Fc region. In an embodiment, the Fc region of the CD20xCD3 antibody according to the present disclosure is mutated to have reduced effector function when compared to a non-engineered Fc region.
  • the Fc region of the CD20xCD3 antibody according to the present disclosure comprises one or more amino acid substitutions that reduces the binding affinity of the Fc region to an Fc receptor and/or to Clq and/or reduces the effector function.
  • the same one or more amino acid mutation(s) is present in each 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 mutated Fc region does substantially not bind to an Fc receptor and/or Clq 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 Fey receptor.
  • the Fc receptor is an activating human Fey receptor, more specifically human FcyRIIIa, FcyRI or FcyRIIa, most specifically human FcyRIIIa.
  • the binding affinity of the Fc region to a complement component in particular the binding affinity to Clq, is reduced or abolished.
  • the Fc region of the CD20xCD3 antibody according to the present disclosure comprises one or more amino acid mutations that reduce(s) the binding affinity of the Fc region to an Fc receptor and/or to Clq and/or reduces the effector function.
  • the amino acid mutation is an amino acid substitution.
  • the Fc region of the CD20xCD3 antibody 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 Cl q and/or reduces the effector function.
  • each Fc region subunit comprises at least one 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 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 embodiment, 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 embodiment, each Fc region subunit comprises an amino acid substitution at the positions 330 and 331 with numbering according EU index. In an embodiment, the amino acid substitution is A33 OS or P33 IS.
  • the Fc region of the bispecific antibody according to the present disclosure comprises one or more amino acid substitutions in each Fc region subunit, wherein said amino acid substitutions reduces the binding affinity of the Fc region to an Fc receptor and/or to Clq 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 the CD20xCD3 antibody according to the present disclosure consists of one or more amino acid mutation in each Fc region subunit wherein said amino acid substitutions reduces the binding affinity of the Fc region to an Fc receptor and/or to Clq and/or reduces the effector function, wherein the one or more amino acid mutations are L234A, L235E, G237A, A330S and P331S.
  • the present disclosure provides the CD20xCD3 antibody according to the present disclosure, wherein in the first and the second Fc region subunit at least 1, 2, 3, 4, or 5 amino acid residue in the positions corresponding to positions L234, L235, G237, A330, or P331 with numbering according EU index are mutated to A, E, A, S, and S, respectively.
  • the present disclosure provides an CD20xCD3 antibody according to the present disclosure, wherein in the first and the second Fc region subunit at least 5 amino acid residues in the positions corresponding to positions L234, L235, G237, A330, P331 with numbering according EU index are mutated to A, E, A, S, and S, respectively.
  • the Fc region is an IgGl Fc region, particularly a human IgGl Fc region.
  • the Fc region subunit according to the present disclosure comprises the amino acid sequence of (Fc-AEASS-Knob):
  • the Fc region subunit according to the present disclosure comprises the amino acid sequence of (Fc-AEASS-hole): PSVFLFPPI ⁇ PI ⁇ DTLMISRTPEVTCVVVDVSHEDPEVI ⁇ FNWYVDGVEVHNAI ⁇ TI ⁇ PREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSRE EMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 73).
  • Binding of the Fc region to Fc receptors can be easily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors may be obtained by recombinant expression.
  • the binding affinity of Fc regions may be evaluated using cell lines known to express particular Fc receptors, such as NK cells expressing Fcyllla receptor.
  • Effector function of an Fc region can be measured by methods known in the art. Suitable in vitro assays to assess ADCC activity of a molecule of interest are for instance described in WQ2012130831. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738- 2743 (2004)).
  • Clq binding assays (such as ELISA) may be carried out to determine whether an bispecific antibody is able to bind Clq and hence has CDC activity (WO 2006/029879 and WO 2005/100402).
  • the Fab and Fv fragments used in the CD20xCD3 antibody according to the present disclosure can be of any animal species origin, such as murine, rat, human or non-human primate.
  • the Fab and/or Fv fragments used in the CD20xCD3 antibody according to the present disclosure are human or humanized Fab and/or Fv fragments.
  • the first and second binding domain that bind to CD20 of the CD20xCD3 antibody according to the present disclosure are humanized binding domains.
  • the first and second Fab fragment that bind to CD20 of the CD20xCD3 antibody are humanized Fab fragments.
  • the third binding domain that binds to CD3 of the CD20xCD3 antibody according to the present disclosure is a human binding domain.
  • the Fv fragment that bind to CD3 of the CD20xCD3 antibody according to the present disclosure is a human Fv fragment.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences are according to the Kabat numbering system. In some embodiments, the HCDR1, HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 sequences are according to the Chothia numbering system.
  • the CD3xCD20 antibody according to the present disclosure comprises a third binding domain that binds to CD3, wherein the third binding domain comprises a VH domain comprising a HCDR1 , HCDR2 and HCDR3 having an amino acid sequence of a HCDR1 , HCDR2 and HCDR3, respectively, of a VH domain having an amino acid sequence provided in Table 1.
  • the CD3xCD20 antibody according to the present disclosure comprises a third binding domain that binds to CD3, wherein the third binding domain comprises a VL domain comprising a LCDR1 , LCDR2 and LCDR3 having an amino acid sequence of a LCDR1 , LCDR2 and LCDR3, respectively, of a VL domain having an amino acid sequence provided in Table 1.
  • the CD3xCD20 antibody according to the present disclosure comprises a third binding domain that binds to CD3, wherein the third binding domain comprises a VH domain comprising a HCDR1 , HCDR2 and HCDR3 having an amino acid sequence of a HCDR1 , HCDR2 and HCDR3, respectively, of a VH domain having an amino acid sequence provided in Table 1 and a VL domain comprising a LCDR1, LCDR2 and LCDR3 having an amino acid sequence of a LCDR1, LCDR2 and LCDR3, respectively, of a VL domain having an amino acid sequence provided in Table 1.
  • the CD3xCD20 antibody according to the present disclosure comprises a third binding domain that binds to CD3, wherein the third binding domain comprises a VH domain comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.
  • the CD3xCD20 antibody according to the present disclosure comprises a third binding domain that binds to CD3, wherein the third binding domain comprises a VL domain comprising a LCDR1 , a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
  • the CD3xCD20 antibody according to the present disclosure comprises a third binding domain that binds to CD3, wherein the third binding domain comprises a VH domain comprising a HCDR1, a HCDR2, and a HCDR3 having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and a VL domain comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
  • the third binding domain of the CD3xCD20 antibody according to the present disclosure that binds to CD3 comprises a VH domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7.
  • the CD3xCD20 antibody according to the present disclosure comprises a third binding domain that binds to CD3, wherein the third binding domain comprises a VH domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7.
  • the third binding domain that binds to CD3 comprises a VH domain having an amino acid sequence that is about 90% identical to the amino acid sequence of SEQ ID NO: 7. In another embodiment, the third binding domain that binds to CD3 comprises a VH domain having an amino acid sequence that is about 95% identical to the amino acid sequence of SEQ ID NO: 7. In another embodiment, the third binding domain that binds to CD3 comprises a VH domain having an amino acid sequence that is about 98% identical to the amino acid sequence of SEQ ID NO: 7. In another embodiment, the third binding domain that binds to CD3 comprises a VH domain having an amino acid sequence that is about 99% identical to the amino acid sequence of SEQ ID NO: 7. In one embodiment, the third binding domain that binds to CD3 comprises a VH domain having an amino acid sequence that is about 100% identical to the amino acid sequence of SEQ ID NO: 7.
  • third binding domain of the CD3xCD20 antibody according to the present disclosure that binds to CD3 comprises a VL domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO 8.
  • the CD3xCD20 antibody comprises a third binding domain that binds to CD3, wherein the third binding domain comprises a VL domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.
  • the third binding domain that binds to CD3 comprises a VL domain having an amino acid sequence that is about 90% identical to the amino acid sequence of SEQ ID NO: 8.
  • the third binding domain that binds to CD3 comprises a VL domain having an amino acid sequence that is about 95% identical to the amino acid sequence of SEQ ID NO: 8. In another embodiment, the third binding domain that binds to CD3 comprises a VL domain having an amino acid sequence that is about 98% identical to the amino acid sequence of SEQ ID NO: 8. In another embodiment, the third binding domain that binds to CD3 comprises a VL domain having an amino acid sequence that is about 99% identical to the amino acid sequence of SEQ ID NO: 8.
  • the third binding domain that binds to CD3 comprises a VL domain having an amino acid sequence that is about 100% identical to the amino acid sequence of SEQ ID NO: 8.
  • the CD3xCD20 antibody according to the present disclosure comprises a third binding domain that binds to CD3, wherein the first binding domain comprises a VH domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.
  • the third binding domain that binds to CD3 comprises a VH domain having an amino acid sequence that is about 90%, identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 90% identical to the amino acid sequence of SEQ ID NO: 8.
  • the third binding domain that binds to CD3 comprises a VH domain having an amino acid sequence that is about 95%, identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 95% identical to the amino acid sequence of SEQ ID NO: 8.
  • the third binding domain that binds to CD3 comprises a VH domain having an amino acid sequence that is about 98%, identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 98% identical to the amino acid sequence of SEQ ID NO: 8.
  • the third binding domain that binds to CD3 comprises a VH domain having an amino acid sequence that is about 99%, identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 99% identical to the amino acid sequence of SEQ ID NO: 8.
  • the third binding domain that binds to CD3 comprises a VH domain having an amino acid sequence that is about 100%, identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 100% identical to the amino acid sequence of SEQ ID NO: 8.
  • said CD3 is human CD3.
  • said CD3 is cynomolgus CD3.
  • said CD3 is CD3 epsilon.
  • said CD3 is human CD3 epsilon.
  • said CD3 is cynomolgus CD3 epsilon.
  • the third binding domain that binds to CD3 binds to the extracellular region of CD3. In one embodiment, the third binding domain that binds to CD3 is a deimmunized binding domain. In one embodiment, the third binding domain that binds to CD3 has a reduced risk in electing an immune response in human beings.
  • the third binding domain that binds to CD3 is less- or non-immunogenic in human beings. In one embodiment, the third binding domain that binds to CD3 has a reduced risk in electing an immune response or an immunogenic reaction in human beings of a Caucasian population.
  • the first and second binding domain that bind to CD20 of the CD3xCD20 antibody according to the present disclosure each comprises a VH domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15. In one embodiment, the first and second binding domain that bind to CD20 of the CD3xCD20 antibody according to the present disclosure each comprises a VL domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.
  • the CD3xCD20 antibody according to the present disclosure comprises a first and second binding domain that bind to CD20, wherein the first and second binding domain each comprises a VH domain comprising a HCDR1, HCDR2 and HCDR3 having an amino acid sequence of a HCDR1, HCDR2 and HCDR3, respectively, of a VH domain having an amino acid sequence provided in Table 1.
  • the CD3xCD20 antibody according to the present disclosure comprises a first and a second binding domain that bind to CD20, wherein the first and second binding domain comprises a VL domain comprising a LCDR1 , LCDR2 and LCDR3 having an amino acid sequence of a LCDR1, LCDR2 and LCDR3, respectively, of a VL domain having an amino acid sequence provided in Table 1.
  • the CD3xCD20 antibody according to the present disclosure comprises a first and a second binding domain that bind to CD20, wherein the first and second binding domain each comprises a VH domain comprising a VCDR1, HCDR2 and HCDR3 having an amino acid sequence of a HCDR1, HCDR2 and HCDR3, respectively, of a VH domain having an amino acid sequence provided in Table 1, and a VL comprising a LCDR1, LCDR2 and LCDR3 having an amino acid sequence of a LCDR1, LCDR2 and LCDR3, respectively, of a VL having an amino acid sequence provided in Table 1.
  • the CD3xCD20 antibody according to the present disclosure comprises a first and second binding domain that bind to CD20, wherein the first and second binding domain each comprises a VH domain comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.
  • the CD3xCD20 antibody according to the present disclosure comprises a first and second binding domain that bind to CD20, wherein the first and second binding domain each comprises a VL domain comprising a LCDR1 , a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.
  • the CD3xCD20 antibody according to the present disclosure comprises a first and a second binding domain that bind to CD20, wherein the first and second binding domain comprises a VH domain comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 9, SEQ ID NO:10, and SEQ ID NO:11, respectively and a VL domain comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.
  • the CD3xCD20 antibody according to the present disclosure comprises a first and a second binding domain that binds to CD20, wherein the first and second binding domain each comprises a VH domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15.
  • the first and the second binding domain that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 90% identical to the amino acid sequence of SEQ ID NO: 15. In another embodiment, the first and the second binding domain that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 95% identical to the amino acid sequence of SEQ ID NO: 15. In another embodiment, the first and the second binding domain that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 98% identical to the amino acid sequence of SEQ ID NO: 15. In another embodiment, the first and the second binding domain that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 99% identical to the amino acid sequence of SEQ ID NO: 15.
  • the first and the second binding domain that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 100% identical to the amino acid sequence of SEQ ID NO: 15.
  • the CD3xCD20 antibody according to the present disclosure comprises a first and a second binding domain that bind to CD20, wherein the first and the second binding domain each comprises a VL domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.
  • the first and the second binding domain that bind to CD20 each comprises a VL domain having an amino acid sequence that is about 90% identical to the amino acid sequence of SEQ ID NO: 16.
  • first and the second binding domain that bind to CD20 each comprises a VL domain having an amino acid sequence that is about 95% identical to the amino acid sequence of SEQ ID NO: 16. In another embodiment, the first and the second binding domain that bind to CD20 each comprises a VL domain having an amino acid sequence that is about 98% identical to the amino acid sequence of SEQ ID NO: 16. In another embodiment, the first and the second binding domain that bind to CD20 each comprises a VL domain having an amino acid sequence that is about 99% identical to the amino acid sequence of SEQ ID NO: 16. In another embodiment, the first and the second binding domain that bind to CD20 each comprises a VL domain having an amino acid sequence that is about 100% identical to the amino acid sequence of SEQ ID NO: 16.
  • the CD3xCD20 antibody according to the present disclosure comprises a first and second binding domain that bind to CD20, wherein the first and the second binding domain each comprises a VH domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16.
  • the first and the second binding domain that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 90%, identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 90% identical to the amino acid sequence of SEQ ID NO: 16.
  • first and the second binding domain that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 95%, identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 95% identical to the amino acid sequence of SEQ ID NO: 16.
  • first and the second binding domain that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 98%, identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 98% identical to the amino acid sequence of SEQ ID NO: 16.
  • first and the second binding domain that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 99%, identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 99% identical to the amino acid sequence of SEQ ID NO: 16.
  • first and the second binding domain that bind to CD20 each comprises a VH domain having an amino acid sequence that is about 100%, identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 100% identical to the amino acid sequence of SEQ ID NO: 16.
  • said CD20 is human CD20. In one embodiment, the CD20 is cynomolgus monkey CD20. In one embodiment, said first and second binding domain bind to the extracellular region of CD20.
  • the bispecific CD20 + CD3 antibody The bispecific CD20 + CD3 antibody
  • the CD3xCD20 antibody according to the present disclosure comprises a first and a second binding domain that bind to CD20 and a third binding domain that bind to CD3, wherein the first and second binding domain that bind to CD20 each comprises a VH domain comprising a HCDR1 , HCDR2 and HCDR3 having an amino acid sequence of a HCDR1 , HCDR2 and HCDR3, respectively, of a VH domain having an amino acid sequence provided in Table 1, and a VL domain comprising a LCDR1 , LCDR2 and LCDR3 having an amino acid sequence of a LCDR1, LCDR2 and LCDR3, respectively, of a VL domain having an amino acid sequence provided in Table 1; and wherein the third binding domain that bind to CD3 comprises a VH domain comprising a HCDR1, HCDR2 and HCDR3 having an amino acid sequence of a HCDR1, HCDR2 and HCDR3, respectively, of a VH domain having an amino acid sequence provided in Table
  • the CD3xCD20 antibody according to the present disclosure comprises a first and a second binding domain that binds to CD20 and a third binding domain that binds to CD3, wherein the first and second binding domain comprises a VH domain comprising a HCDR1, a HCDR2, and a HCDR3 having an amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively and a VL domain comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and the third binding domain comprises a VH domain comprising a HCDR1, a HCDR2, and a HCDR3 having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and a VL domain comprising a LCDR1, a LCDR2, and a LCDR3 having an amino acid sequence of SEQ ID NO: 4, SEQ
  • the CD3xCD20 antibody according to the present disclosure comprises a first and a second binding domain that bind to CD20 and a third binding domain that binds to CD3, wherein the first and the second binding domain each comprises a VH domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 16, and the third binding domain comprises a VH domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.
  • the first and the second binding domain each comprises a VH domain having an amino acid sequence that is about 90%, identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 90% identical to the amino acid sequence of SEQ ID NO: 16 and the third binding domain comprises a VH domain having an amino acid sequence that is about 90%, identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 90% identical to the amino acid sequence of SEQ ID NO: 8.
  • the first and the second binding domain each comprises a VH domain having an amino acid sequence that is about 95%, identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 95% identical to the amino acid sequence of SEQ ID NO: 16, and the third binding domain comprises a VH domain having an amino acid sequence that is about 95%, identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 95% identical to the amino acid sequence of SEQ ID NO: 8.
  • the first and the second binding domain each comprises a VH domain having an amino acid sequence that is about 98%, identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 90% identical to the amino acid sequence of SEQ ID NO: 16 and the third binding domain comprises a VH domain having an amino acid sequence that is about 98%, identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 98% identical to the amino acid sequence of SEQ ID NO: 8.
  • the first and the second binding domain each comprises a VH domain having an amino acid sequence that is about 99%, identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 99% identical to the amino acid sequence of SEQ ID NO: 16, and the third binding domain comprises a VH domain having an amino acid sequence that is about 99%, identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 99% identical to the amino acid sequence of SEQ ID NO: 8.
  • the first and the second binding domain each comprises a VH domain having an amino acid sequence that is about 10%, identical to the amino acid sequence of SEQ ID NO: 15 and a VL domain having an amino acid sequence that is about 100% identical to the amino acid sequence of SEQ ID NO: 16, and the third binding domain comprises a VH domain having an amino acid sequence that is about 100%, identical to the amino acid sequence of SEQ ID NO: 7 and a VL domain having an amino acid sequence that is about 100% identical to the amino acid sequence of SEQ ID NO: 8.
  • the present disclosure pertains to the CD20xCD3 antibody according to the present disclosure, wherein the CD20xCD3 antibody is composed of 4 polypeptides, wherein
  • the first polypeptide comprises the light chain of the first Fab
  • the second polypeptide comprises from its N- to C-terminus i. the heavy chain of the first Fab; ii. the VL or VH domain of the third binding domain, and iii. the first Fc region subunit;
  • the third polypeptide comprises from its N- to C-terminus i. the heavy chain of the second Fab, ii. the complementary VL or VH domain of the third binding domain, and iii. the second Fc region subunit;
  • the fourth polypeptide comprises the light chain of the second Fab.
  • the first and the fourth polypeptide each comprises an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17.
  • the first and the fourth polypeptide each comprises an amino acid sequence that is about 90%, identical to the amino acid sequence of SEQ ID NO: 17. In one embodiment, the first and the fourth polypeptide each comprises an amino acid sequence that is about 95%, identical to the amino acid sequence of SEQ ID NO: 17. In one embodiment, the first and the fourth polypeptide each comprises an amino acid sequence that is about 98%, identical to the amino acid sequence of SEQ ID NO: 17. In one embodiment, the first and the fourth polypeptide each comprises an amino acid sequence that is about 99%, identical to the amino acid sequence of SEQ ID NO: 17. In one embodiment, the first and the fourth polypeptide each comprises an amino acid sequence that is about 100%, identical to the amino acid sequence of SEQ ID NO: 17. In one embodiment, the first and the fourth polypeptide each comprises an amino acid sequence of SEQ ID NO: 17.
  • the second polypeptide comprises an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the second polypeptide comprises an amino acid sequence that is about 90%, identical to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the second polypeptide comprises an amino acid sequence that is about 95% identical to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the second polypeptide comprises an amino acid sequence that is about 98% identical to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the second polypeptide comprises an amino acid sequence that is about 99% identical to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the second polypeptide comprises an amino acid sequence that is about 100%, identical to the amino acid sequence of SEQ ID NO: 18. In one embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 18.
  • the third polypeptide comprises an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19. In one embodiment, the third polypeptide comprises an amino acid sequence that is about 90% identical to the amino acid sequence of SEQ ID NO: 19. In one embodiment, the third polypeptide comprises an amino acid sequence that is about 95%, identical to the amino acid sequence of SEQ ID NO: 19. In one embodiment, the third polypeptide comprises an amino acid sequence that is about 98% identical to the amino acid sequence of SEQ ID NO: 19. In one embodiment, the third polypeptide comprises an amino acid sequence that is about 99% identical to the amino acid sequence of SEQ ID NO: 19. In one embodiment, the third polypeptide comprises an amino acid sequence that is about 100% identical to the amino acid sequence of SEQ ID NO: 19. In one embodiment, the third polypeptide comprises an amino acid sequence of SEQ ID NO: 19.
  • the present disclosure pertains to the CD20xCD3 antibody according to the present disclosure, wherein the CD20xCD3 antibody is composed of 4 polypeptides, wherein: (a) the first polypeptide comprises the light chain of the first Fab;
  • the second polypeptide comprises from its N- to C-terminus: the heavy chain of the first Fab, the VL domain of the third binding domain, and the first Fc region subunit;
  • the third polypeptide comprises from its N- to C-terminus: the heavy chain of the second Fab, the VH domain of the third binding domain, and the second Fc region subunit;
  • the fourth polypeptide comprises the light chain of the second Fab, and wherein the first and the fourth polypeptide each comprises an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 17, the second polypeptide comprises an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 18, and third polypeptide comprises an amino acid sequence that is about 90%, 95%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 19.
  • the CD20xCD3 antibody according to the present disclosure may be used for the prevention and treatment of diseases which are mediated by biological pathways in which the CD20 is involved.
  • the biological activity of the CD20xCD3 antibody according to the present disclosure can be measured by various assays known in the art, including those described in Examples 2 -3 described herein. Methods for assaying functional activity may utilize binding assays, such as the enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS) and other methods that are well known in the art (see Hampton, R. et al. (1990; Serological Methods a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. etal. (1983; J.
  • ELISA enzyme- linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • assays may test the ability of the bispecific antibody in eliciting a biological response either in vivo or in vitro.
  • Biological activities may for example include the induction of proliferation of T cells, the induction of signaling in T cells, the induction of expression of activation markers in T cells, the induction of cytokine secretion by T cells, the inhibition of signaling in CD20 expressing cells such as tumor cells or cells of the tumor stroma, the inhibition of proliferation of CD20 expressing cells, the induction of lysis of CD20 expressing cells, and the induction of tumor regression and/or the improvement of survival.
  • the present disclosure provides a method for inducing lysis of a cancer cell expressing CD20, comprising contacting said cell in the presence of a cytotoxic T-cell with the CD20xCD3 antibody according to the present disclosure.
  • the present disclosure provides a method for inhibition of signaling in a cancer cell expressing CD20 comprising contacting said cancer cell in the presence of a cytotoxic T-cell with the CD20xCD3 antibody according to the present disclosure.
  • the present disclosure provides a method for inhibition of proliferation of cancer cell expressing CD20, comprising contacting said cell in the presence of a cytotoxic T-cell with the CD20xCD3 antibody according to the present disclosure.
  • the present disclosure provides a method for inducing a cellular response in a cytotoxic T-cell, comprising contacting said cytotoxic T-cell in the presence of a cancer cell expressing CD20 with the CD20xCD3 antibody according to the present disclosure.
  • said cellular response is selected from the group of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.
  • the present disclosure provides a method for inducing human T-cell proliferation in the presence of a cancer cell expressing CD20, comprising contacting said cancer cell in the presence of a T-cell with the CD20xCD3 antibody according to the present disclosure.
  • the present disclosure provides a method for stimulating a primary T-cell response in the presence of a cancer cell expressing CD20, comprising contacting the cancer cell in the presence of the T-cell with the CD20xCD3 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 CD20, comprising contacting the cancer cell in the presence of the T-cell with the CD20xCD3 antibody according to the present disclosure.
  • said T-cell is a CD3 expressing T-cell.
  • the present disclosure provides the use of the CD20xCD3 antibody according to the present disclosure for the treatment of cancer expressing CD20, said use comprising: (a) selecting a subject who is afflicted with a cancer,
  • the cancer cell is a cancer cell line. In an embodiment, the cancer cell is a lymphoma cell or a lymphoma cell line. In one embodiment, the lymphoma cell line is Raji cell line (ATCC CCL-213). In one embodiment, the cell proliferative disease is a cancer.
  • the cancer is selected from the group consisting but not limited to: esophageal cancer, stomach cancer, small intestine cancer, large intestine cancer, colorectal cancer, breast cancer, non-small cell lung cancer.
  • the cancer is a B-cell related cancer.
  • the cancer is a lymphoma.
  • the cancer is a B-cell related disease, such as non-Hodgkin's lymphoma (NHL), B cell lymphoma, B cell leukemia, multiple myeloma, renal cancer, prostate cancer, liver cancer, head and neck cancer, melanoma, ovarian cancer, mesothelioma, glioblastoma, germinal-center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), chronic lymphoid leukemia (CLL), marginal zone lymphoma (MZL), small lymphocytic leukemia (SLL), lymphoplasmacytic lymphoma (LL), Waldenstrom macroglobulinemia (WM), central nervous system lymphoma (CNSL), Burkitt's lymphoma (BL), B-cell prolymphocytic leuk
  • NHL
  • the CD20xCD3 antibody according to the present disclosure may be used in therapeutic methods.
  • the CD20xCD3 antibody according to the present disclosure may be used for the treatment of cancer.
  • the present disclosure provides a method for the treatment of a disease.
  • the present disclosure provides the CD20xCD3 antibody according to the present disclosure for use in the treatment of a disease.
  • the present disclosure provides the CD20xCD3 antibody according to the present disclosure for use in the treatment of a disease in an individual in need thereof.
  • the present disclosure provides the use of the CD20xCD3 antibody according to the present disclosure for the manufacture of a medicament.
  • the present disclosure provides the CD20xCD3 antibody according to the present disclosure for use as a medicament.
  • the present disclosure provides the CD20xCD3 antibody according to the present disclosure for use as a medicament for the treatment of a disease in an individual in need thereof.
  • the disease is associated with the undesired presence of CD20.
  • the disease to be treated is a proliferative disease.
  • the disease is cancer.
  • the present disclosure provides the CD20xCD3 antibody according to the present disclosure for use in a method of treating a subject or individual having a disease comprising administering to the subject a therapeutically effective amount of the CD20xCD3 antibody. In an embodiment, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent.
  • the subject or individual in need of treatment is typically a mammal, more specifically a human.
  • the CD20xCD3 antibody would be formulated, dosed, and administered in a way consistent with good medical practice.
  • 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 the CD20xCD3 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 the CD20xCD3 antibody according to the present disclosure. In one embodiment, the present disclosure provides a method for eliciting, stimulating and/or inducing an immune response in a subject who has cancer, comprising administering to said subject a therapeutically effective amount of the CD20xCD3 antibody of the present disclosure. In an embodiment, 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 the CD20xCD3 antibody of the present disclosure.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the CD20xCD3 antibody 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.
  • the pharmaceutical composition can be used in the diagnosis, prevention and/or treatment of diseases associated with a target antigen of interest.
  • the present disclosure provides a pharmaceutical compositions comprising the CD20xCD3 antibody according to the present disclosure that is suitable for prophylactic, therapeutic and/or diagnostic use in a mammal, more particular in a human.
  • the CD20xCD3 antibody according to the present disclosure may be formulated as a pharmaceutical composition comprising the CD20xCD3 antibody and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active compounds.
  • Such a formulation may be suitable for oral, parenteral, topical administration or for administration by inhalation.
  • the CD20xCD3 antibody according to the present disclosure may be used in combination with one or more pharmaceutically active compounds that are or can be used for the prevention and/or treatment of the diseases in which CD20 is involved, as a result of which a synergistic effect may or may not be obtained.
  • pharmaceutically active compounds that are or can be used for the prevention and/or treatment of the diseases in which CD20 is involved, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.
  • the present disclosure provides a pharmaceutical composition comprising the CD20xCD3 antibody according to the present disclosure for use in the prevention and/or treatment of a disease associated with the undesired presence of CD20. In one embodiment, the present disclosure provides a pharmaceutical composition comprising the CD20xCD3 antibody according to the present disclosure for the use as a medicament. In one embodiment, the present disclosure provides a pharmaceutical composition comprising the CD20xCD3 antibody according to the present disclosure for use in the prevention and/or treatment of autoimmune diseases or cancer
  • the present disclosure provides a method for the treatment of autoimmune diseases or cancer in a subject in need thereof using a pharmaceutical composition comprising the CD20xCD3 antibody according to the present disclosure. Further provided is a method of producing the CD20xCD3 antibody according to the present disclosure in a form suitable for administration in vivo, the method comprising
  • compositions according to the present disclosure comprise a therapeutically effective amount of the CD20xCD3 antibody dissolved in a pharmaceutically acceptable carrier.
  • the present disclosure provides a kit comprising the CD20xCD3 antibody according to the present disclosure or a pharmaceutical composition comprising the CD20xCD3 antibody according to the present disclosure.
  • the present disclosure provides a kit comprising the CD20xCD3 antibody according to the present disclosure or a pharmaceutical composition comprising the CD20xCD3 antibody according to the present disclosure, and a package insert comprising instructions for administration of the CD20xCD3 antibody for treating or delaying progression of cancer or reducing or inhibiting tumor growth in a subject in need thereof.
  • the appropriate dosage of the CD20xCD3 antibody according to the present disclosure will depend on the type of disease to be treated, the route of administration, the body weight of the individual, the severity and course of the disease, whether the CD20xCD3 antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the individual's clinical history and response to the CD20xCD3 antibody, and the discretion of the attending physician.
  • the CD20xCD3 antibody according to the present disclosure is suitably administered to the patient at one time or over a series of treatments.
  • 1 pg/kg to 15 mg/kg (e.g. 0.1 mg/kg - 10 mg/kg) of the CD20xCD3 antibody according to the present disclosure can be an initial dosage for administration to the individual, whether, for example, by one or more separate administrations, or by continuous infusion.
  • One typical daily dosage might range from 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • a dose may also comprise 1 pg/kg body weight, 5 pg/kg body weight, 10 pg/kg body weight, 50 pg/kg body weight, 100 pg/kg body weight, 200 pg/kg body weight, 350 pg/kg body weight, 500 pg/kg body weight, 1 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight, 50 mg/kg body weight, 100 mg/kg body weight, 200 mg/kg body weight, 350 mg/kg body weight, 500 mg/kg body weight, to 1000 mg/kg body weight or more per administration, and any range derivable therein.
  • a range of 5 mg/kg body weight to 100 mg/kg body weight, 5 pg/kg body weight to 500 mg/kg body weight, etc. can be administered, based on the numbers described above.
  • one or more doses of 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the individual.
  • Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the individual receives from two to twenty, or e.g. six doses of the CD20xCD3 antibody).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • An bispecific antibody according to the present disclosure will generally be used in a therapeutically amount effective to achieve the intended purpose.
  • the CD20xCD3 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 an individual 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 the CD20xCD3 antibody according to the present disclosure can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent.
  • the CD20xCD3 antibody according to the present disclosure can also be used in combination with radiation therapy.
  • the present disclosure also provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the CD20xCD3 antibody according to the present disclosure.
  • the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the CD20xCD3 antibody according to the present disclosure.
  • the present disclosure provides a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the CD20xCD3 antibody described in Tables 1.
  • the nucleic acid composition is an isolated nucleic acid composition.
  • the present disclosure is directed to a nucleic acid sequence or a plurality of nucleic acid sequences encoding the CD20xCD3 antibody according to the present disclosure, wherein the nucleic acid sequence or the plurality of nucleic acid sequences encodes for the individual polypeptides of the CD20xCD3 antibody.
  • Polypeptides forming the CD20xCD3 antibody according to the present disclosure are described in Table 1.
  • the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid sequence composition according to the present disclosure. In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding the CD20xCD3 antibody according to the present disclosure. In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding the CD20xCD3 antibody as described in Table 1. In certain embodiments, the vector is an expression vector.
  • 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 plurality of nucleic acid sequences encoding the CD20xCD3 antibody according to the present disclosure.
  • the present disclosure refers to a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising the nucleic acid sequence or plurality of nucleic acid sequences encoding the CD20xCD3 antibody as described in Table 1.
  • the vectors can be introduced into the appropriate host cells such as prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or mammalian) cells by methods well known in the art (see, e.g., "Current Protocol in Molecular Biology", Ausubel et al. (eds.), Greene Publishing Assoc, and John Wiley Interscience, New York, 1989 and 1992).
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • a method of producing the CD20xCD3 antibody according to the present disclosure comprises culturing a host cell comprising vector composition comprising a vector or a plurality of vectors comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding the CD20xCD3 antibody according to the present disclosure, under conditions suitable for expression of the CD20xCD3 antibody, and recovering the CD20xCD3 antibody from the host cell or host cell culture medium.
  • the methods for the production of the CD20xCD3 antibody according to the present disclosure further comprise the step of isolating the produced CD20xCD3 antibody from the host cells or medium.
  • the CD20xCD3 antibody recovered according to the present disclosure may be purified techniques know in the art, such as high performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • HPLC high performance liquid chromatography
  • ion exchange chromatography gel electrophoresis
  • affinity chromatography size exclusion chromatography
  • size exclusion chromatography and the like.
  • the present disclosure pertains to the CD20xCD3 antibody according to the present disclosure comprising a first binding domain and a second binding domain that both bind to CD20 and a third binding domain that bind to CD3 (the “CD3xCD20 antibody”).
  • Amino acid sequences forming the CD3xCD20 antibody according to the present disclosure are shown in Table 1.
  • the bispecific CD20xCD3 antibody according to the present was generated in vitro using the bispecific antibody platform technology as described in W02020/115115 and WO2023/180346 which are both incorporated herein in their entirety.
  • This bispecific antibody in the 2+1 Fab2-Fv-Fc format is built from an aglycosylated monoclonal human IgGl antibody backbone incorporating one additional antibody Fv fragment inserted between the Fc region and the two Fab arms of a regular human IgGl molecule.
  • a basic structure of such a bispecific antibody is provided in FIG. 1.
  • This format provides the advantage of bivalent binding to CD20 and monovalent binding to CD3.
  • This format also allows for a short distance between a target cell and a cytotoxic T cell once bridged via the bispecific antibody. This narrow immunological synapse leads to an efficient killing of the target cell by the recruited cytotoxic T cell.
  • the Fc region was modified by introducing mutations into the CH3 domain of each Fc region subunit according to the “knob-into-holes” technology. Thereby, the polypeptide comprising one mutated CH3 domain is forced to heterodimerize with the other polypeptide comprising the other CH3 domain, which is engineered in a complementary manner.
  • VH and VL domain of a phage display derived fully human antibody with specificity for CD3epsilon was used (SEQ ID NO: 7 and SEQ ID NO: 8, respectively).
  • SEQ ID NO: 7 and SEQ ID NO: 8 The generation of this antibody is described in WO2023/180346.
  • Example 2 In vivo efficacy of the CD3xaCD20 antibody
  • a Raji tumor cell model with human T-cell co-engraftment was performed (EPO Berlin) On day 0 of the study, T-cells were isolated from the whole blood of four healthy donors. 2x10 A 6 Raji cells (Burkitt lymphoma cell line) were mixed 1:1 with 2xlO A 6 human T-cells of donor 1, 2, 3 or 4 in matrigel and implanted subcutaneously (s.c.) in the left flank of NOD-SCID mice (three mice per donor per group). Treatment with CD20xCD3 antibody started 1-2 h after cell inoculation at day 0 and was administered i.v. twice per week for five weeks in total with an application volume of 5
  • CD20xCD3 and isotype control (non-targeted CD3) antibody were applied in concentrations of 0.001 to 1 mg/kg, Lymphoma growth and body weight were monitored for up to 100 days. Animals were individually sacrificed at a tumor volume >1.5 cm A 3. Mean tumor growth curves were calculated using GraphPad Prism software (version 8.4.3).
  • FIG. 2A & B Treatment with CD20xCD3 antibody showed statistically significant differences in tumor growth (FIG. 2A & B) and progression-free survival (FIG. 2C & D) relative to vehicle control or isotype control treated animals at antibody concentrations of greater 0.1 mg/kg.
  • Example 3 In vivo efficacy of the CD3xCD20 antibody
  • a Raji tumor cell model in CD34 hematopoietic stem cell humanized NCG mice boosted with human IL- 15 plasmid DNA was carried out (Transcure Bioservices) .
  • 2x10 A 6 Raji cells per animal were implanted s.c. into the right flank of 30-31 weeks old female CD34 humanized NCG mice with IL- 15 boost.
  • mice were randomized into study groups of nine mice each according to their tumor volume, humanization rate, and human cord blood donor (three CD34 cell donors per group). Treatments were initiated on day 7 and applied twice per week i.v. at dosages of 0.01 to 1.0 mg/kg of aCD20xaCD3 antibody.
  • Tumor volume and body weight were measured at least three times per week. Mice were individually sacrificed when reaching a tumor volume >1500 mm A 3 or latest at study end on day 77 after tumor cell implantation. Mean tumor growth curves were calculated using GraphPad Prism software (version 8.4.3).

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Abstract

La présente invention concerne des anticorps bispécifiques comprenant un premier et un deuxième domaine de liaison ayant une spécificité pour CD20 et un troisième domaine de liaison ayant une spécificité pour CD3 humain, en particulier pour CD3epsilon. Les anticorps bispécifiques de la présente invention sont capables d'inhiber la croissance de tumeurs à cellules B exprimant CD20 et sont ainsi particulièrement utiles pour traiter des maladies associées à CD20 dans lesquelles une réponse des cellules T ciblée induite est thérapeutiquement bénéfique.
PCT/EP2024/078149 2023-10-26 2024-10-07 Anticorps bispécifiques contre cd3 et cd20 Pending WO2025087681A1 (fr)

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