Classifications

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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/283—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2833—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
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  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2851—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2875—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2893—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD52
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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  • 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
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the antibody constructs of the invention can thus be useful for tumor therapy, because they are not only capable of removing cells in a tumor that have high expression of the target antigen, but also those cells that have low or very low expression of the target antigen, including tumor stem cells Thus, the antibody construct can target the entire tumor
• the antibody constructs of the invention can thus be useful for targeting tumors with heterologous expression of the target antigen.
• Figure 1 Schematic representation of antibody constructs at least four first binding domains (A-E) and reference antibody constructs (F-H).
• Calcein-labeled MCF-7 cells were co-cultured with enriched primary human NK cells as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the indicated antibodies. After 4 h incubation the fluorescence of the calcein released from lysed target cells into the supernatant was quantified and used for calculation of % specific lysis. Mean and SD of duplicate values are plotted
• Calcein-labeled Daudi cells were co-cultured with enriched primary human NK cells as effector cells at an E:T ratio of 5:1 in the presence of serial dilutions of the indicated antibodies. After 4 h incubation the fluorescence of the calcein released from lysed target cells into the supernatant was quantified and used for calculation of % specific lysis. Mean and SD of duplicate values are plotted.
• FIG. 5 Concentration-dependent induction of NK cell fratricide by various antibody constructs. Calcein-labeled enriched primary human NK cells were co-cultured with autologous NK cells as effector cells at an E:T ratio of 1:1 in the presence of serial dilutions of the indicated antibodies. After 4 h incubation the fluorescence of the calcein released from lysed target cells into the supernatant was quantified and used for calculation of % specific lysis. Anti-CD38 IgGl (IgAb_51) was used as a positive control. Mean and SD of duplicate values are plotted.
• Antibody constructs as defined in the context of the invention may also comprise modified fragments of antibodies, also called antibody variants, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single chain diabodies (scDb), tandem diabodies (TandAb's), tandem di-scFv, tandem tri-scFv, "multibodies” such as triabodies or tetrabodies, and single domain antibodies such as nanobodies or single variable domain antibodies comprising merely one variable domain, which might be VHH, VH or VL, that specifically bind an antigen or epitope independently of other V regions or domains.
• antibody variants such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3,
• antibody construct generally includes multivalent constructs, including bispecific constructs, specifically binding to only two antigenic structures, as well as polyspecific/multispecific constructs, which specifically bind more than two antigenic structures, e.g. three, four or more, through distinct binding domains.
• Antibody constructs of the present invention are multivalent (e g. pentavalent or hexavalent) antibody constructs that are at least bispecific (such as bispecific or trispecific).
• target cell surface antigen or “antigen on the surface of a target cell”, which are used interchangeably, refers to an antigenic structure expressed by a cell and which is present at the cell surface such that it is accessible for an antibody construct as described herein. It may be a protein, preferably the extracellular portion of a protein, a peptide that is presented on the cell surface in an MHC context (including HLA-A2, HLA-A11, HLA-A24, HLA-B44, HLA-C4) or a carbohydrate structure, preferably a carbohydrate structure of a protein, such as a glycoprotein. It is preferably a tumor associated or tumor restricted antigen.
• CD 16 A, CD56, NKG2A, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1, CD 89, CD96, CD160, TIGIT, TIM-3, KIR2DL1-5, KIR3DL1-3, and KIR2DS1-5 are not target cell surface antigens according to the present invention.
• bispecific antibody constructs are (at least) bispecific, they do not occur naturally and they are markedly different from naturally occurring products.
• a bispecific antibody construct is hence an artificial hybrid antibody having at least two distinct binding sides with different specificities.
• Bispecific antibody constructs can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315- 321 (1990).
• humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also CDR) of the recipient are replaced by residues from a hypervariable region of a non- human (e.g., rodent) species (donor antibody) such as mouse, rat, hamster or rabbit having the desired specificity, affinity, and capacity.
• donor antibody e.g., rodent
• Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
• "humanized antibodies” as used herein may also comprise residues which are found neither in the recipient antibody nor the donor antibody. These modifications are made to further refine and optimize antibody performance.
• epitope refers to a side on an antigen to which a binding domain, such as an antibody or immunoglobulin, or a derivative, fragment or variant of an antibody or an immunoglobulin, specifically binds.
• a binding domain such as an antibody or immunoglobulin, or a derivative, fragment or variant of an antibody or an immunoglobulin, specifically binds.
• An “epitope” is antigenic and thus the term epitope is sometimes also referred to herein as “antigenic structure” or “antigenic determinant”.
• the binding domain is an "antigen interaction site”. Said binding/interaction is also understood to define a "specific recognition”.
• CDRs form a loop structure that can be classified as a canonical structure.
• canonical structure refers to the main chain conformation that is adopted by the antigen binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Correspondent loops between antibodies may, therefore, have very similar three dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia and Lesk, J. Mol.
• the CDR3 of the light chain and, particularly, the CDR3 of the heavy chain may constitute the most important determinants in antigen binding within the light and heavy chain variable regions.
• the heavy chain CDR3 appears to constitute the major area of contact between the antigen and the antibody.
• CDR3 is typically the greatest source of molecular diversity within the antibody-binding side.
• H3 for example, can be as short as two amino acid residues or greater than 26 amino acids.
• the sequence of antibody genes after assembly and somatic mutation is highly varied, and these varied genes are estimated to encode 10 10 different antibody molecules (Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press, San Diego, CA, 1995). Accordingly, the immune system provides a repertoire of immunoglobulins.
• the term "repertoire” refers to at least one nucleotide sequence derived wholly or partially from at least one sequence encoding at least one immunoglobulin.
• the sequence(s) may be generated by rearrangement in vivo of the V, D, and J segments of heavy chains, and the V and J segments of light chains.
• amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, lie, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged sidechain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gin, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).
• Amino acid modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequences of the antibody constructs. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
• the amino acid changes also may alter post-translational processes of the antibody constructs, such as changing the number or position of glycosylation sites.
• amino acid sequence insertions into the antibody construct include amino- and/or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues to polypeptides containing a hundred or more residues, as well as intra sequence insertions of single or multiple amino acid residues.
• amino acid sequence insertions into the antibody construct include amino- and/or carboxyl-terminal fusions ranging in length from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues to polypeptides containing a hundred or more residues, as well as intra sequence insertions of single or multiple amino acid residues.
• Corresponding modifications may also be performed within a third binding domain of the antibody construct defined in the context of the invention.
• An insertional variant of the antibody construct defined in the context of the invention includes the fusion to the N- terminus or to the C-terminus of the antibody construct of an enzyme or the fusion to a polypeptide.
• PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol. 35:351-360; the method is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151 -153.
• Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
• intracellular phospho-tyrosines serve as docking sites for various adaptor proteins or enzymes including SHC, GRB2, PLCg and PI(3)K/Akt, which simultaneously initiate many signaling cascades that influence cell proliferation, angiogenesis, apoptosis resistance, invasion and metastasis.
• T cells gaining innate immune functions by e.g. expressing immune regulatory receptors normally expressed by innate immune cells (e.g. CD16A. NKp46) (Quelle: (CD16A CAR T cells D'Aloia et al Chimeric Antigen Receptor T Cells, 18(2):278-290).
• immune regulatory receptors normally expressed by innate immune cells e.g. CD16A. NKp46
• Quelle CD16A CAR T cells D'Aloia et al Chimeric Antigen Receptor T Cells, 18(2):278-290.
• such a modification comprises one or both of Leu 234 -> Ala and Leu 235 -> Ala (also known as “LALA” mutation).
• such a modification further comprises a Pro 329 -> Gly mutation, also known as “LALA-PG” mutation (Leu 234 -> Ala, Leu 235 -> Ala, and Pro 329 -> Gly).
• such a modification comprises 1, 2, or 3 of the mutations Leu 234 -> Phe, Leu 235 -> Glu, and Asp 265 -> Ala, more preferably all three of these mutations.
• Fratricide describes in the context of the invention the reduction of effector cells by cytotoxic kill and, thereby the reduction of the available effector cell population/compartment.
• Fratricide can be caused by cross-linking of two immune cells.
• cross-linking of NK cells can cause the killing of either one or both of the NK cells.
• an antibody construct recruits two different types of effector cells, e.g. NK cells and macrophages or NK cells and T cells also the elimination of one type of effector cells by the other type of effector cells is understood as fratricide.
• Fratricide can be e.g. measured in an assay as essentially described in Example 7.
• NK cells with antigens selected from the group comprising e.g. CD16A, CD56, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1 (CD226), SLAMF7 (CD319), CD244 (2B4), 0X40, CD137, CD 160, KIR2DS1-5, NKG2A, TIGIT, PD-1, PD-L1, CD47, LAG-3, CTLA-4, CD96, TIM-3, CD137, KIR2DL1-5 and KIR3DL1-3; and/or monocytes, macrophages and/or neutrophils with antigens selected from the group comprising e.g.
• Such an antibody construct may comprise two polypeptide chains, one polypeptide chain in the arrangement VH(B)-VL(B)-hinge-CH2-CH3-VL(A2)-VH(Al)-VL(Al)-VH(A2) (or less preferred VH(B)-VL(B)-hinge-CH2-CH3-VH(A2)-VL(Al)-VH(Al)-VL(A2), VL(B)-VH(B)- hinge-CH2-CH3 - VL(A2)- VH(A 1 )- VL(A 1 )- VH(A2), or VL(B)-VH(B)-hinge-CH2-CH3 - VH(A2)-VL(A1)-VH(A1)-VL(A2)), and another polypeptide chain in the arrangement hinge- CH2-CH3-VL(A4)-VH(A3)-VL(A3)-VH(A4) (or less
• a preferred antibody construct of the invention is preferably in a format as essentially shown in Figure IB.
• Such an antibody construct comprises an immunoglobulin that has two scDb fragments fused to the C-termini of the heavy chains, optionally via a linker, which is preferably a glycine serine linker or a serine linker, preferably a glycine serine linker, which preferably comprise no more than about 75 amino acids, preferably not more than about 50 amino acids.
• a suitable linker comprises one or more (e.g.
• the first dimer (A1A2) and/or second dimer (A3A4) may be fused to a constant domain of an antibody via a linker disclosed herein (such as a glycine serine linker or a serine linker, preferably a glycine serine linker, which preferably comprise no more than about 75 amino acids, preferably not more than about 50 amino acids.
• a suitable linker comprises one or more (e g. 1, 2, 3, 4, 5, 6, 7, or 8) GGGGS sequences (SEQ ID NO: 6), such as (GGGGS) 2 (SEQ ID NO: 7), (GGGGS) 4 (SEQ ID NO: 8), or preferably (GGGGS) 6 (SEQ ID NO: 9).
• Other illustrative examples for linkers are shown in SEQ ID NOs: 2-5) or a hinge domain, with a hinge domain being preferred.
• a hinge domain showing reduced flexibility is preferred for some antibody constructs of the disclosure, in particular if the dimer (A1A2) and/or second dimer (A3 A4) are fused to the hinge domain.
• preferred hinge domains are characterized to consist of less than 25 aa residues. More preferably, the length of the hinge is 10 to 20 aa residues.
• a hinge domain comprised in an antibody construct of the disclosure may also comprise or consists of the IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 23), the IgG3 subtype hinge sequence ELKTPLDTTHT CPRCP (SEQ ID NO: 30) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 131), and/or the IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 132).
• Further hinge domains that can be used in the context of the present invention are known to the skilled person and are e.g. described in WO 2017/134140.
• Such an antibody construct may comprise two polypeptide chains, one polypeptide chain in the arrangement VL(A2)-VH(Al)-VL(Al)-VH(A2)-hinge-CH2-CH3- VH(B)-VL(B) (or less preferred VH(A2)-VL(Al)-VH(Al)-VL(A2)-hinge-CH2-CH3-VH(B)- VL(B), VL(A2)-VH(Al)-VL(Al)-VH(A2)-hinge-CH2-CH3-VL(B)-VH(B), or VH(A2)- VL(Al)-VH(Al)-VL(A2)-hinge-CH2-CH3-VL(B)-VH(B)), and another polypeptide chain in the arrangement VL(A4)-VH(A3)-VL(A3)-VH(A4)-hinge-CH2-CH3-VH
• VH(A4) or VL(B)-VH(B)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)).
• VL(A3 )- VH(A4) or VL(B)-VH(B)-hinge-CH2-CH3-VH(A4)-VL(A3)-VH(A3)-VL(A4)).
• antibody constructs described herein having two scDb fragments comprising the four first binding domains (A1-A4) that are fused to the C-termini of the two heavy chains are preferred.
• those that comprise one or two second binding domains (B) in form of an scFv that are fused to one or two N termini of the Fc region are most preferred.
• the distance between the binding site of the first binding domains are short. It is thus preferred that the two binding domains are within the distance of about 30 or less, preferably about 25 nm or less, more preferably about 22 nm or less, more preferably about 20 nm or less, more preferably about 19 nm or less, more preferably about 18 nm or less, more preferably about 17 nm or less, more preferably about 16 nm or less, more preferably about 15 nm or less, more preferably about 14 nm or less, more preferably about 13 nm or less, more preferably about 12 nm or less, more preferably about 11 nm or less, more preferably about 10 nm or less, more preferably about 9 nm or less, more preferably about 8 nm or less, more preferably about 7 nm or less, more preferably about 6 nm or less, more preferably about 5 nm or less.
• the antibody construct comprises a T366W mutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations in the CH3 domain of the “hole chain”.
• An additional interchain disulfide bridge between the CH3 domains can also be used (Merchant, A. M, et al., Nature Biotech 16 (1998) 677-681) e g. by introducing a Y349C mutation into the CH3 domain of the “knobs chain” and a E356C mutation or a S354C mutation into the CH3 domain of the “hole chain”.
• one or more, preferably all, of the at least four first binding domain (A) comprises a pair of VH- and VL-chains having a sequence as depicted in the pairs of sequences selected form the group consisting of SEQ ID NOs: 60 and 69.
• the second binding domain (B) is specific for a second target (B’) that is a tumor associated antigen.
• the second target (B’) is preferably selected from the group consisting of CD19, CD20, CD22, CD30, CD33, CD52, CD70, CD74, CD79b, CD123, CLL1, BCMA, FCRH5, EGFR, EGFRvlll, HER2, GD2.
• the antibody construct of the invention is characterized by inducing a low degree of fratricide, which is also referred to as a “reduced” degree of fratricide.
• the degree of fratricide can be measured in a cytotoxicity assay, such as an assay as essentially described in Example 7. Such an assay is preferably conducted as follows.
• SABC specific antibody binding capacity
• SACB assays are known in the art (Serke et al., 1998, Cytometry, 33(2): 179-87). Such an assay may be conducted as essentially described in Example 5.
• the density of an antigen on the surface of one or more cell line can be determined using QIFIKIT (Dako) and suitable antibodies, such as anti-HER2 mAb MAB 1129 (RnD Systems) or anti-EGFR mAb Hll (Dianova), according to the manufacturer’s instructions.
• a cell line is preferably classified as high expressing cell line, if it has at least 50 % of the SABC score for the respective second target (B’) as compared with the reference high expressing cell line.
• a cell line is preferably classified as low expressing cell line, if it has 15 % or less of the SABC score for the respective second target (B’) as compared with the reference high expressing cell line.
• a cell line is preferably classified as very low expressing cell line, if it has 5 % or less of the SABC score for the respective second target (B’) as compared with the reference high expressing cell line.
• Very low expressing cell lines are to be understood as a subgroup of low expressing cell lines.
• a nucleic acid molecule disclosed in this application may be "operably linked" to a regulatory sequence (or regulatory sequences) to allow expression of this nucleic acid molecule.
• promoter regions normally include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35/- 10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5 '-capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell.
• a nucleic acid molecule of the disclosure can include a regulatory sequence, such as a promoter sequence.
• a nucleic acid molecule of the disclosure includes a promoter sequence and a transcriptional termination sequence. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.
• HeLa cells e g., HeLa cells or CHO cells
• primary mammalian cells e g., HeLa cells or CHO cells
• amino acids such as glycine, alanine, glutamine, asparagine, threonine, proline, 2- phenylalanine, including charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and/or histidine
• buffers buffer systems and buffering agents which are used to maintain the composition at physiological pH or at a slightly lower pH
• examples of buffers are borate, bicarbonate,
• chelating agents such as ethylenediamine tetraacetic acid (EDTA);
• sulfur containing reducing agents such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha] -monothioglycerol, and sodium thio sulfate
• surfactants or wetting agents such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal
• surfactants may be detergents, preferably with a molecular weight of >1.2 KD and/or a polyether, preferably with a molecular weight of >3 KD
• non-limiting examples for preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 and Tween 85
• non-limiting examples for preferred polyethers are PEG 3000, PEG 3350, PEG 4000 and PEG 5000;
• stability enhancing agents such as sucrose or sorbitol
• intravenous delivery vehicles including fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose).
• electrolyte replenishers such as those based on Ringer's dextrose.
• amino acid can act as a buffer, a stabilizer and/or an antioxidant
• mannitol can act as a bulking agent and/or a tonicity enhancing agent
• sodium chloride can act as delivery vehicle and/or tonicity enhancing agent; etc.
• Item 2 The antibody construct of item 1, wherein the antibody construct binds to a target cell and an immune effector cell simultaneously.
• Item 6 The antibody construct of any one of items 1-4, wherein the antibody construct comprises a third binding domain (C), which is capable of specifically binding to a third target (C’) that is an antigen on the surface of a target cell that is other than the second target
• Item 27 The antibody construct of any one of items 7 to 24, wherein a third first binding domain and a fourth first binding domain that are fused to each other (A3A4) is fused to the C terminus of a CH3 domain of a fourth domain (D).
• Item 29 The antibody construct of any one of items 7 to 24, wherein a first first binding domain and a second first binding domain that are fused to each other (A1A2) is fused to the C terminus of a first CH3 domain of a fourth domain (D), and wherein a third first binding domain and a fourth first binding domain that are fused to each other (A3A4) is fused to the C terminus of a second CH3 domain of a fourth domain (D).
• Item 32 The antibody construct of any one of items 7 to 29 and 31, wherein a second binding domain (B) is fused to the N terminus of a hinge of a fourth domain (D).
• Item 33 The antibody construct of any one of items 7 to 28 and 30 to 31, wherein a second binding domain (B) is fused to the C terminus of a CH3 domain of a fourth domain
• Item 36 The antibody construct of any one of items 7 to 28 and 31, wherein a second binding domain (B) is fused to the N terminus of a hinge of a fourth domain (D), and wherein another second binding domain (B) is fused to the C terminus of a CH3 domain of a fourth domain (D).
• Item 43 The antibody construct of any one of items 7 to 24, wherein a first first binding domain and a second first binding domain that are fused to each other (A1A2) is fused to the C terminus of a first CH3 domain of a fourth domain (D), and wherein a third first binding domain and a fourth first binding domain that are fused to each other (A3A4) is fused to the C terminus of a second CH3 domain of a fourth domain (D), and wherein a second binding domain (B) is fused to the N terminus of a hinge of a fourth domain (D).
• Item 45 The antibody construct of item 43, wherein a third binding domain (C) is fused to the N terminus of another hinge of a fourth domain (D).
• Item 46 The antibody construct of any one of items 7 to 24, wherein a first first binding domain and a second first binding domain that are fused to each other (A1A2) is fused to the N terminus of a first hinge of a fourth domain (D), and wherein a third first binding domain and a fourth first binding domain that are fused to each other (A3A4) is fused to the N terminus of a second hinge of a fourth domain (D), and wherein a second binding domain (B) is fused to the C terminus of a CH3 domain of a fourth domain (D).
• Item 49 The antibody construct of any one of items 7 to 24, wherein a first first binding domain and a second first binding domain that are fused to each other (A1A2) is fused to the N terminus of a a first hinge-CH2-CH3 element of a fourth domain (D), and wherein a third first binding domain and a fourth first binding domain that are fused to each other (A3 A4) is fused to the C terminus of a second hinge-CH2-CH3 element a fourth domain (D).
• Item 50 The antibody construct of item 49, wherein a second binding domain (B) is fused to the C terminus of the first hinge-CH2-CH3 element of a fourth domain (D).
• Item 55 The antibody construct of any one of the preceding items, wherein the first binding domain (A) comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3 and a VL region comprising CDR-L1, CDR-L2 and CDR-L3 selected from:
• Item 56 The antibody construct of any one of the preceding items, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 148, 149, 150 and 151, 152 and 153, 154 and 155, 156 and 157, 158 and 159, 160 and 161, 162 and 163, 180-183, 190, and 191 and 192.
• Item 60 The antibody construct of item 59, wherein the EC50 of the antibody construct has a numerical value that is about 0.5 times or lower as compared to the EC50 the reference antibody, preferably determined with a target cell having high expression of the second target (B’).
• Item 73 A method of treatment or amelioration of a proliferative disease, a tumorous disease, a viral disease or an immunological disorder, comprising the step of administering to a subject in need thereof the antibody construct of any one of items 1 to 62, or produced by the method of item 66.
• Eluate fractions containing purified duplex body constructs were pooled and subjected to buffer exchange using Sephadex G-25 column against 10 mM sodium acetate, 4.5% sorbitol pH 5.0, and concentrated by ultrafiltration. Final samples were assessed by SDS-PAGE under reducing and non-reducing conditions (see Figure 2). The samples were mixed with nonreducing 2> ⁇ SDS-PAGE sample buffer or reducing 2> ⁇ SDS-PAGE sample buffer containing dithiothreitol (DTT) as reducing agent. All samples were heated at 95°C. for 5 min prior to loading on 4-20% Criterion TGX Precast SDS Page Gel. 2 pg of purified protein sample per lane were used.
• the MM. IS cell line was purchased from ATCC (cat.: CRL-2974), and cultured under standard conditions in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL penicillin G sodium, and 100 pg/mL streptomycin sulfate as recommended by the supplier at 37°C and 5% CO2 in a humidified atmosphere.
• HER2 and EGFR on the surface of different cell lines was determined using QIFIKIT (Dako) and anti-HER2 mAb MAB 1129 (RnD Systems) or anti-EGFR mAb Hll (Dianova) according to the manufacturer’s instructions.
• QIFIKIT Dako
• anti-HER2 mAb MAB 1129 RnD Systems
• anti-EGFR mAb Hll Dianova
• SABC HER2 and EGFR densities
• Table 1 The HER2 and EGFR densities (SABC) depicted in Table 1 represent mean values of at least 2 independent experiments. SABC values determined with anti-HER2 mAb MAB 1129 and HER2 IHC score were used to generate an artificial EGFR score based on the SABC values determined with anti-EGFR mAb HI 1 (Table 1). Scoring of tumor cell lines regarding HER2 and EGFR expression based on specific antibody binding capacity (SABC) is illustrated in Figure 10.
• target cells were labeled with 10 mM of the fluorescent dye calcein AM (Invitrogen, cat.: C3100MP) for 30 min, and aliquots of lxlO 4 labeled target cells were seeded in individual wells of a round-bottom 96-well micro plate together with freshly isolated and enriched primary human NK cells at an effectontarget (E:T) ratio of 5: 1 in the presence of 12 serial 1:5 dilutions of the indicated antibodies usually starting at 30 pg/rnL, if not otherwise indicated, in duplicates. Control samples to measure spontaneous release, maximal release and antibody-independent lysis by effector cells were tested in 4 replicates.
• E:T effectontarget
• phagocytosis assays macrophages were seeded in 96-well UpCell plates and cultured overnight. Target cells were labeled with 0.5 pM CellTrackerTM Green CMFDA Dye at 37°C for 30 min, washed, and cultured overnight. Target cells were seeded on top of the macrophages (E:T ratio of 5:1), and the indicated antibodies were added at serial concentrations (0.3 pg/mL - 30 pg/mL) in duplicates. After 4 hours incubation, cells were detached from the culture plate by incubation on ice and stained with A700-labeled anti- CD1 lb and fixable viability dye eF780 for 30 min at 4°C.
• Phagocytosis of labeled target cells was quantified by analyzing CMFDA + /CD1 lb + cells in % of viable cells by flow cytometry. ADCP in absence of antibodies was used for normalization. Additionally, loss of labeled target cells was quantified by analyzing CMFDA + /CD1 lb cells in % of viable cells by flow cytometry. Loss of target cells in absence of antibodies was used for normalization.
• the assays performed as described in Example 6 were used to compare multivalent anti-CD 16A constructs (Bi-scDb- Fc_02 (SEQ ID NOs: 149), aBi-scDb-Fc_05 (SEQ ID NOs: 158 and 159), Bi-scDb-IgAb_06 (SEQ ID NOs: 162 and 163)) with constructs comprising two anti-CD 16A domains (scFv- IgAb_43 (SEQ ID NOs: 174 and 175) and scFv-IgAb_167 (SEQ ID NOs: 178 and 179)), and with Fc-enhanced (S239D/I332E) anti-EGFR IgGl (IgAb_53 (SEQ ID NOs: 168 and 169)) in three independent assays using NK cells from different blood donors.
• scFv- IgAb_43 SEQ ID NOs: 174 and 175
• Table 3 Potency and efficacy of anti-EGFR antibody constructs determined in cytotoxicity assays on Daudi target cells. Potency (EC 50 ) and efficacy (E maX ) were determined in 4 calcein-release cytotoxicity assays on Daudi target cells using NK cells as effector cells at an E:T ratio of 5:1. Mean values of three independent experiments are shown.
• Example 12 4h phagocytosis assay on DK-MG cells
• a representative exemplary graph in Figure 7 demonstrates phagocytosis induction of multivalent anti-CD 16A constructs.
• scFv- IgAb_43, IgAb_49 and IgAb_53 did not induce phagocytosis of MCF-7 target cells by macrophages.
• Example 15 ELISA investigation of binding of EGFR/CD16A engagers or BCMA/CD16A engagers to CD16A
• 96-well ELISA plates (Immuno Maxisorp, Nunc) were coated overnight at 4°C with different multivalent bispecific engagers at concentrations of 3.5-5 pg/mL (equalizing 24-27 nM) in 100 mM Carbonate-bicarbonate buffer.
• 96-well ELISA plates (Immuno Maxisorp, Nunc) were coated overnight at 4°C with His-tagged human EGFR or BCMA extracellular domain at concentrations of 3.0 or 0.4 pg/mL, respectively.
• Table 5 Half maximal binding values for CD16 binding of tetravalent or bivalent CD16- binding constructs targeting EGFR or BCMA analyzed in ELISA. Concentration dependent binding of antibody constructs to coated recombinant CD 16A antigen, or of soluble monomeric or dimeric CD 16 antigen variants to coated or target antigen captured antibody constructs was analyzed in ELISA. Half maximal binding concentrations (EC50) were determined by fitting a nonlinear regression model to sigmoidal dose-response curves (four parameters logistic fit) using GraphPad Prism software.
• the multivalent anti-CD16A constructs (Bi-scDb-Fc_02 (SEQ ID NOs: 149), aBi-scDb-Fc_05 (SEQ ID NOs: 158 and 159), Bi-scDb-IgAb_06 (SEQ ID NOs: 162 and 163)) were compared with constructs comprising two anti-CD16A domains (scFv- IgAb_43 (SEQ ID NOs: 174 and 175)), with Fc-wildtype anti-EGFR IgGl (IgAb_49 (SEQ ID NOs: 164 and 165)) and with Fc-enhanced (S239D/I332E) anti-EGFR IgGl (IgAb_53 (SEQ ID NOs: 168 and 169)) in three independent assays using macrophages generated from monocytes of different blood donors.
• Example 19 Efficacy of CD 19-targeting innate cell engagers in 4 h cytotoxicity assays on Daudi target cells

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