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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
  • C07K16/2803—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
  • C07K16/2827—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 B7 molecules, e.g. CD80, CD86
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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
  • C07K16/2887—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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
  • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• • C—CHEMISTRY; METALLURGY
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  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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  • C07K2317/41—Glycosylation, sialylation, or fucosylation
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  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/522—CH1 domain
• • C—CHEMISTRY; METALLURGY
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  • C07K2317/00—Immunoglobulins specific features
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  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/524—CH2 domain
• • C—CHEMISTRY; METALLURGY
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/526—CH3 domain
• • C—CHEMISTRY; METALLURGY
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
  • C07K2317/53—Hinge
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
• • 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
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/72—Increased effector function due to an Fc-modification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/734—Complement-dependent cytotoxicity [CDC]
• • 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

• compositions and methods for combining the target-specificity of an antigen binding domain with at least two Fc domains to generate new therapeutics with unique biological activity allow for the construction of proteins having multiple antigen binding domains and multiple Fc domains from multiple polypeptide chains.
• the number and spacing of antigen binding domains can be tuned to alter the binding properties (e.g., binding avidity) of the protein complexes for target antigens, and the number of Fc domains can be tuned to control the magnitude of effector functions to kill antigen-binding cells.
• Mutations are introduced into the polypeptides to reduce the number of undesired, alternatively assembled proteins that are produced.
• heterodimerizing and/or homodimerizing mutations are introduced into the Fc domain monomers, and differentially mutated Fc domain monomers are placed among the different polypeptide chains that assemble into the protein, so as to control the assembly of the polypeptide chains into the desired protein structure.
• Single-chain Fv or “scFv” antibody fragments include the V H and V L domains of antibody in a single polypeptide chain.
• the scFv polypeptide further includes a polypeptide linker between the V H and V L domains, which enables the scFv to form the desired structure for antigen binding.
• dimerization selectivity module refers to a sequence of the Fc domain monomer that facilitates the favored pairing between two Fc domain monomers.
• “Complementary” dimerization selectivity modules are dimerization selectivity modules that promote or favor the selective interaction of two Fc domain monomers with each other. Complementary dimerization selectivity modules can have the same or different sequences. Exemplary complementary dimerization selectivity modules are described herein, and can include complementary mutations selected from the engineered protuberance-forming and cavity-forming mutations of Table 3 or the electrostatic steering mutations of Table 4.
• the term“engineered cavity” refers to the substitution of at least one of the original amino acid residues in the CH3 antibody constant domain with a different amino acid residue having a smaller side chain volume than the original amino acid residue, thus creating a three dimensional cavity in the CH3 antibody constant domain.
• the term“original amino acid residue” refers to a naturally occurring amino acid residue encoded by the genetic code of a wild-type CH3 antibody constant domain.
• An engineered cavity can be formed by, e.g., any one or more of the cavity-forming substitution mutations of Table 3.
• albumin-binding peptide refers to an amino acid sequence of 12 to 16 amino acids that has affinity for and functions to bind serum albumin.
• An albumin-binding peptide can be of different origins, e.g., human, mouse, or rat.
• an albumin-binding peptide is fused to the C-terminus of an Fc domain monomer to increase the serum half-life of the Fc-antigen binding domain construct.
• An albumin binding peptide can be fused, either directly or through a linker, to the N- or C-terminus of an Fc domain monomer.
• polynucleotide refers to an oligonucleotide, or nucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single- or double-stranded, and represent the sense or anti-sense strand. A single polynucleotide is translated into a single polypeptide.
• polypeptide describes a single polymer in which the monomers are amino acid residues which are joined together through amide bonds.
• a polypeptide is intended to encompass any amino acid sequence, either naturally occurring, recombinant, or synthetically produced.
• the term“pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition.
• the pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient.
• the pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to the Fc-antigen binding domain construct.
• the nature of the carrier differs with the mode of administration. For example, for oral administration, a solid carrier is preferred; for intravenous administration, an aqueous solution carrier (e.g., WFI, and/or a buffered solution) is generally used.
• FIG. 7B depicts the amino acid sequence of a human lgG1 (SEQ ID NO: 45) with EU numbering.
• the hinge region which lacks E216-C220, inclusive, is indicated by a double underline, the CH2 domain is not underlined and the CH3 region is underlined and lacks K447.
• heterodimerizing mutations into the polypeptides that assemble into the same protein allow for the creation of a great diversity of protein configurations, including, e.g., antibody-like proteins with tandem Fc domains, symmetrically branched proteins, and asymmetrically branched proteins.
• the antigen binding domains of Fc-antigen binding domain construct 44 can include the three heavy chain and the three light chain CDR sequences of any one of the antibodies listed in Table 1A and 1 B.
• an Fc domain monomer containing a positively-charged amino acid substitution e.g., D356K or E357K
• an Fc domain monomer containing a negatively-charged amino acid substitution e.g., K370D or K370E
• an Fc domain monomer containing E357K and an Fc domain monomer containing K370D may selectively combine to form an Fc domain through favorable electrostatic steering of the charged amino acids.
• an Fc domain includes Fc domain monomers including quadruple mutants combining any pair of the double mutants, e.g., K409D/D399K/E357K/K370E. Examples of homodimerizing selectivity modules are further shown in Tables 5 and 6. Homodimerizing Fc domains can be used to create symmetrical branch points on an Fc- antigen binding domain construct. In one embodiment, an Fc-antigen binding domain construct described herein has one homodimerizing Fc domain.
• an Fc domain monomer can be modified to include one of the following positively-charged and negatively-charged amino acid substitutions: D356K, D356R, E357K, E357R, K370D, K370E, K392D, K392E, D399K, K409D, K409E, K439D, and K439E.
• one Fc domain monomer for example, an Fc domain monomer having a cavity (Y349C, T366S, L368A and Y407V), can also include K370D mutation and the other Fc domain monomer, for example, an Fc domain monomer having a protuberance (S354C and T366W) can include E357K.
• Electrostatic steering is the utilization of favorable electrostatic interactions between oppositely charged amino acids in peptides, protein domains, and proteins to control the formation of higher ordered protein molecules.
• a method of using electrostatic steering effects to alter the interaction of antibody domains to reduce for formation of homodimer in favor of heterodimer formation in the generation of bi-specific antibodies is disclosed in U.S. Patent Application Publication No. 2014-00241 1 1.
• the two Fc domain monomers may be selectively formed through heterodimerization or homodimerization.
• albumin-binding peptides may be attached to the N- or C-terminus of certain polypeptides in the Fc-antigen binding domain construct.
• an albumin-binding peptide may be attached to the C-terminus of one or more polypeptides in Fc constructs containing an antigen binding domain.
• an albumin-binding peptide can be fused to the C- terminus of the polypeptide encoding two Fc domain monomers linked in tandem series in Fc constructs containing an antigen binding domain.
• an albumin-binding peptide can be attached to the C-terminus of Fc domain monomer (e.g., Fc domain monomers 1 14 and 1 16 in FIG.
• the Fc-antigen binding domain constructs can be assembled to form bispecific constructs using long and short chains with different antigen binding domain sequences.
• the Fc-antigen binding domain constructs can be assembled to form bispecific and trispecific constructs using chains with different sets of heterodimerization mutations and different antigen binding domains.
• a bispecific Fc-antigen binding domain construct includes two different antigen biding domains.
• a trispecific Fc-antigen binding domain construct includes three different antigen binding domains.
• a hexa-histidine peptide (HHHHHH (SEQ ID NO: 38)) binds to nickel- functionalized agarose affinity column with micromolar affinity.
• a FLAG peptide includes the sequence DYKDDDDK (SEQ ID NO: 39).
• Fc-antigen binding domain constructs described in this disclosure are able to activate various Fc receptor mediated effector functions.
• One component of the immune system is the complement- dependent cytotoxicity (CDC) system, a part of the innate immune system that enhances the ability of antibodies and phagocytic cells to clear foreign pathogens.
• CDC complement- dependent cytotoxicity
• Three biochemical pathways activate the complement system: the classical complement pathway, the alternative complement pathway, and the lectin pathway, all of which entail a set of complex activation and signaling cascades.
• the first short Fc chain contains an Fc domain monomer with a first set of cavity-forming mutations selected from Table 3 and/or one or more reverse charge mutation selected from Table 4 (wherein the mutations are different from mutations in the second short Fc chain).
• the second short Fc chain contains an Fc domain monomer with a second set of cavity-forming mutations selected from Table 3 and/or one or more reverse charge mutation selected from Table 4 (wherein the mutations are different from the first set off mutations in the first short Fc chain), and an antigen binding domain at the N-terminus.
• DNA sequences were optimized for expression in mammalian cells and cloned into the pcDNA3.4 mammalian expression vector.
• the DNA plasmid constructs were transfected via liposomes into human embryonic kidney (HEK) 293 cells.
• the amino acid sequences for the short and long Fc chains were encoded by multiple plasmids.
• anti-PD-L1 Fc constructs induced FcyRIla signaling in an ADCP reporter assay.
• Target HEK-PD-L1 cells (1 .25 x 10 4 cells/well) and effector Jurkat/FcyRII la cells (Promega) (7.45 x 10 4 cells/well) were resuspended in RPMI 1640 Medium supplemented with 4% low IgG serum (Promega) and seeded in a 96-well plate with serially diluted anti-PD-L1 constructs. After incubation for 6 hours at 37°C in 5% C02, the luminescence was measured using the Bio-Glo Luciferase Assay Reagent (Promega) according to the manufacturer’s protocol using a PHERAstar FS luminometer (BMG
• the target cells used in the anti-CD20 CDC assay are the Daudi lymphoblastoid human B cell line. Daudi cells were removed from suspension culture by centrifugation and resuspended in X-VIVO 15 media at 6 x 105 cells/ml. Daudi cells were transferred to a 96 well flat-bottom assay plate in a volume of 100 m I per well (6 x104 cells/well). Each of the anti-CD20 monoclonal antibodies (mAbs) and SIF Bodies were diluted to 3.33 mM in XVIV015 media.
• mAbs monoclonal antibodies
• SIF Bodies were diluted to 3.33 mM in XVIV015 media.

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• 2019
  • 2019-07-11 US US17/259,480 patent/US20220267460A1/en not_active Abandoned
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• 2021
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