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  • 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
  • C07K16/2809—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 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
  • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/71—Decreased effector function due to an Fc-modification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• the present invention relates to fully human antibodies that bind to CD3 including structurally modified variants therefrom having a reduced potential to bind to HLA proteins and thus having a reduced risk in electing an immune response in human beings once administered.
• the present invention further provides bispecific antibodies employing the CD3 specific antibodies disclosed herein, methods for producing such antibodies, and methods of using them in the treatment of diseases, such as to treat cancer.
• the present disclosure provides an isolated human antibody or antigen- binding fragment thereof specific for CD3, wherein the VH and VL are selected from the group consisting of: i. the VH comprising the amino acid sequence of SEQ ID NO: 14 and the VL comprising the amino acid sequence of SEQ ID NO: 20, ii. the VH comprising the amino acid sequence of SEQ ID NO: 15 and the VL comprising the amino acid sequence of SEQ ID NO: 21, iii. the VH comprising the amino acid sequence of SEQ ID NO: 16 and the VL comprising the amino acid sequence of SEQ ID NO: 21, iv. the VH comprising the amino acid sequence of SEQ ID NO: 17 and the VL comprising the amino acid sequence of SEQ ID NO: 21, v.
• Figure 4 Cytotoxicity assay for mammalian produced bispecific antibodies with specify for HER2 and CD3 according to Example 2.4 comprising variable domains of affinity matured or cross-cloned CD3 specific antibodies of to the present disclosure on either HER2 expressing SKBR3 cells ( Figure 4A) or HER2 positive MCF-7 cells ( Figure 4B) in the presence of human T cells derived from one donor. Cytotoxic activity of human T cells is assessed by measuring incorporated CellToxGreen fluorescence. The graph shows the relative fluorescence levels of HER2 expressing SKBR3 or MCF-7 cells as a function of bispecific antibody concentration.
• Figure 7B is a graph showing the average percentage of CD69+ activated CD8+ T cells derived from 3 different donors as a function of bispecific antibody concentration.
• Figure 8 Epibase TM (Lonza, Epibase Version: v3.0) in silico screening results for the HCDR3 region of the cross-cloned antibody CD3-MABopt-cc from Example 2 for identification of potential T cell epitopes. Human antibody germline encoded sequence regions were excluded from analysis. For analysis, the whole VH sequence is splitted into overlapping 10mer peptides, each of which is shifted by one amino acid.
• FIG. 10 Summary of biophysical und functional properties of 27 preferred CDR single point variants of CD3-MAB opt-cc. after characterization in the bispecific Fab 2 -Fv-Fc antibody format of Example 4. For each substitution, the reduction of T cell epitopes in the VH or VL of CD3-MAB opt-cc is shown, as well as monomer content and yield of purified bispecific antibodies preparations, ELISA binding and affinities on recombinant human CD3epsilon.
• PTM motifs posttranslational modification sites
• K D refers to the dissociation constant, which is obtained from the ratio of K d to K a (i.e. K d /K a ) and is expressed as a molar concentration (M).
• K D values for antigen binding moieties like e.g. monoclonal antibodies can be determined using methods well established in the art. Methods for determining the K D of an antigen binding moiety like e.g. a monoclonal antibody are SET (soluble equilibrium titration) or surface plasmon resonance using a biosensor system such as a Biacore ® system.
• the present disclosure provides an isolated human antibody or antigen- binding fragment thereof specific for CD3, comprising a VH comprising a) an HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1, b) an HCDR2 region comprising the amino acid sequence of SEQ lD NO: 2, and c) an HCDR3 region comprising the amino acid sequence of SEQ lD NO: 5, and a VL comprising d) a LCDR1 region comprising the amino acid sequence of SEQ ID NO: 10, e) a LCDR2 region comprising the amino acid sequence of SEQ ID NO: 11, and f) a LCDR3 region comprising the amino acid sequence of SEQ ID NO: 24.
• the present disclosure pertains to an isolated human antibody or antigen- binding fragment thereof specific for human CD3, wherein said antibody or antigen- binding fragment thereof has a monovalent affinity for a human CD3 epsilon peptide comprising SEQ ID NO: 41 and/or SEQ ID NO: 43 with a lower KD compared to the KD of an antibody comprising a VH and VL comprising the amin acid sequence of SEQ ID NO: 25 and SEQ ID NO: 26, respectively.
• the computational screening method as those provided by Lonza (EpibaseTM, Epibase Version: v3.0, WO 2003/105058).
• the present disclosure provides deimmunized isolated human antibodies or antigen-binding fragment thereof specific for CD3.
• the present disclosure provides an antibody or antigen-binding fragment thereof specific for CD3, which is a deimmunized isolated human antibody or antigen-binding fragment thereof specific for CD3.
• the risk is determined by excluding human antibody germline sequences present in the VH and/or the VL of an human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure. In an aspect, the risk is determined for the HCDR1, HCDR2, HCDR3 and LCDR3 region of an human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure. In an aspect, said risk excludes the risk for human antibody germline sequences in eliciting an immune response in human beings. In an aspect, the selected population is Caucasian.
• the isolated human antibody or antigen-binding fragment thereof specific for CD3 according to the present disclosure is used in a format as described herein in Example 3 or Example 5 or in WO 2020/115115.
• the present disclosure provides a bispecific or multispecific antibody comprising an antigen-binding fragment of an human antibody specific for CD3 according to the present disclosure and a second antigen-binding fragment of a second antibody which binds to a different antigen than said first antigen-binding fragment thereof.
• said second antigen-binding fragment binds to a cell surface antigen.
• said cell surface target antigen is a tumor associated antigen.
• the first and third peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31), the second peptide linker comprises the amino acid sequence of PKAAP (SEQ ID NO: 36), and the fourth peptide linker comprises the amino acid sequence of ASTKGP (SEQ ID NO: 37), or b) the first and third peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31) and the second and fourth peptide linker comprises the amino acid sequence of AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 33), or c) the first and third peptide linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 31), the second peptide linker comprises the amino acid sequence of PKAAPSVTLFPPSSEELQAN (SEQ ID NO: 34), and the fourth peptide linker comprises the amino acid sequence of ASTKGPSVFPL
• target cell killing is determined by the method as described herein in Example 4.13.
• the present disclosure provides an isolated human antibody or antigen- binding fragment thereof specific for CD3 or a bispecific antibody according to the present disclosure for use as a medicament.
• the present disclosure refers to an isolated human antibody or antigen-binding fragment thereof specific for CD3 or a bispecific antibody according to the present disclosure for use in the preparation or manufacture of a medicament.
• Panning stringency was increased by lowering the CD3 antigen concentration or cell count of CD3 expressing cells in each panning round (Low et al. 1996).
• off-rate selections were performed (Hawkins et al. 1992) using excesses of unbiotinylated CD3epsilon antigens as competitor to further increase selection stringency. All strategies were combined with prolonged washing steps.
• Example 1.2 Conversion into and production of a bispecific 2+1 Fab2-Fc-scFv antibody format
• VH and VL of CD3-MABGP of 48x sequence unique HCDR1-3 and 3x sequence unique LCDR3 derivatives with SET KD estimates of ⁇ 10nM on human CD3epsilon were converted into a bispecific 2+1 Fab2-Fc-scFv antibody format as shown in Figure 1A.
• Example 1.5 Jurkat NFAT Reporter Gene Cell Assay
• Jurkat cells ATCC #TIB-152 transiently transfected with an NFAT reporter gene construct were used as surrogate effector cells.
• target cells the HER2 positive human adenocarcinoma SKBR-3 (ATCC® HTB-30TM) cell line was used.
• Table 15 ELISA binding of affinity improved CD3 specific antibodies of the present disclosure to recombinant human or cynomolgus CD3epsilon antigens when tested in the bispecific antibody format Fab2-Fc-scFv of Example 1 n. a.: not applicable Summary functional characterization of affinity improved antibody Affinity maturation of grandparent antibody CD3-MAB GP resulted in the identification of 5 affinity optimized HCDR1-2 variants and 1 affinity optimized LCDR3 variant of CD3- MABGP with preferred characteristics and suited to be used in bispecific antibody formats Table 16 provides an overview of the favorable functional and biophysical properties of these antibodies compared to the grandparent antibody when tested in the bispecific antibody format of Example 1.
• Eukaryotic HEK293-6E cells were transfected with mammalian expression vector DNA encoding the heavy and light chain of the source IgG molecules.
• Cell culture supernatants were harvested on day 3 or 6 post transfection and subjected to standard Protein A affinity chromatography (MabSelect SURE ⁇ GE Healthcare). Buffer exchange was performed to 1x Dulbcecco ⁇ s PBS (pH 7.2 ⁇ Invitrogen) and samples were sterile filtered (0.2 ⁇ m pore size).
• Example 2.1 KD Determination via Antibody Capture Setup Affinity determination by determining kinetic rate constants was performed on an Octet HTX (FortéBIO, Sartorius AG ) instrument. Bispecific antibody preparations of Example 2 diluted in assay buffer (DPBS (GIBCO, no Ca 2+ , no Mg 2+ ; Thermo Fisher Cat. No.14190) supplemented with 0.05% (v/v) Polysorbate 20 (Merck, 8.22184.0500) and 0.1% (w/v) bovine serum albumin (Sigma, A7906) were captured onto IgG-specific BLI sensors with a loading level of approx. 2 nm.
• DPBS GIBCO, no Ca 2+ , no Mg 2+ ; Thermo Fisher Cat. No.14190
• Polysorbate 20 Merck, 8.22184.0500
• bovine serum albumin Sigma, A7906
• Bispecific antibodies were serially diluted in Jurkat growth medium.50 ⁇ l antibody dilution was added to each well resulting in a final concentration range of 50 nM to 0.01 nM (4 step dilution). Assay plates were incubated for 5 h in a humidified incubator at 37°C and 5%CO2. Bright-Glo TM Reagent (Promega, #E2620) was reconstituted according to manufacturer’s instructions. Assay plates and reagent were equilibrated at room temperature. 100 ⁇ l of the Bright-Glo TM reagent was added to each well of the assay plate and mixed. Luminescence was measured using an InfiniteM1000 Pro plate reader (Tecan).
• Example 2.4 Cytotoxicity assays with bispecific antibodies. Bispecific antibodies according Example 2 were tested for their ability to mediate T cell dependent killing of the HER2 expressing tumor cell lines SKBR3, MCF-7 and the HER2 negative cell line MDA-MB-468. The assay was conducted as described in Example 1.6. As effector cells, either human PBMCs (for SKBR3) cells or purified human T cells (for MCF-7 cells) were used.
• Table 24 summarizes monomer content and volumetric yields of the different preparations obtained for the produced bispecific antibodies. All produced bispecific antibodies revealed comparable production characteristics. However, all bispecific antibodies using CH1- or C ⁇ -derived peptide linkers revealed a very heterogenous mass pattern indicating potential O-linked glycosylation of these linkers. Only bispecific antibody BissIg_21#2 using Linker Combination 2, i.e. a 20mer glycine-serine linker (SEQ ID NO: 31) in combination with the 20mer PAPDA linker (SEQ ID NO: 33) showed a homogenous mass pattern and no potential signs of O- linked glycosylation.
• Linker Combination 2 i.e. a 20mer glycine-serine linker (SEQ ID NO: 31) in combination with the 20mer PAPDA linker (SEQ ID NO: 33) showed a homogenous mass pattern and no potential signs of O- linked glycosylation.
• FIG. 12 summarizes ELISA EC 50 values for 33 preferred bispecific combined variants determined on human CD3epsilon antigen.
• Example 4.8 KD Determination via Antibody Capture Setup Affinity determination by determining kinetic rate constants for 46 bispecific combined variants of Example 4.6 which revealed better ELISA binding to CD3 (Example 4.7) was done as previously described in Example 4.4.
• Example 4.12 Re-directed T cell cytotoxicity mediated by bispecific antibodies comprising 5 preferred deimmunized CD3 specific antibodies.
• Bispecific combined variant antibodies BissIg_21#CD3-MABdeimm_1, BissIg_21#CD3- MABdeimm_2, BissIg_21#CD3-MABdeimm_3, BissIg_21#CD3-MABdeimm_4 and BissIg_21#CD3-MABdeimm_5 were tested for their potential to induce T cell mediated killing of tumor cells upon binding to CD3 and HER2. The method was carried out as described in Example 3.6.

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