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  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1267—Gram-positive bacteria
  • C07K16/1271—Micrococcaceae (F); Staphylococcaceae (F), e.g. Staphylococcus (G)
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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
  • C07K16/2809—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
• • 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
  • C07K16/2827—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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  • C07K16/46—Hybrid immunoglobulins
  • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• • 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
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • 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
  • C07K2317/524—CH2 domain
• • 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
  • C07K2317/526—CH3 domain
• • 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
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin

Definitions

• a method for producing a heterodimeric protein that comprises two distinct IgG CH3 domains that are capable of forming a CH3-CH3 interface.
• an isolated heterodimeric protein obtained by said method.
• an isolated heterodimeric antibody comprising a first IgG CH3 domain and a second IgG CH3 domain, wherein the first CH3 domain and the second CH3 domain are capable of forming a CH3-CH3 interface capable of generating multispecific binding domains, as well as a pharmaceutical composition comprising said isolated heterodimeric proteins having multiple binding specificities.
• Monospecific antibodies play an important role as therapeutic molecules of many diseases, in particular for the treatment of cancer.
• Monospecific antibodies bind to a single specific area, or epitope, of an antigen and, for use in therapy, are often selected for a desirable functional property (such as for example killing of tumour cells, blocking of receptor-ligand interactions or virus neutralization).
• Monospecific antibodies have many beneficial characteristics, such as they can be produced at large quantities, and their biophysical and biochemical characteristics can be analysed in great detail to ensure batch-to-batch consistency, which facilitates regulatory acceptability.
• a single cell may produce essentially a single antibody species through use of heterodimerization technology that pairs the constant region of the bispecific, to achieve this product requires use technology that limits the ability to combine different binding domains (e.g., a common light chain binding domain with a non-common light chain antibody).
• a common light chain binding domain with a non-common light chain antibody e.g., a common light chain binding domain with a non-common light chain antibody.
• the present disclosure in addition to allowing for Fab-arm exchange to be utilised with all IgG formats, is also associated with certain unexpected advantages. For example, it has now been found that upon exposure to reducing and reoxidising conditions half-antibodies comprising amino acid combinations 366K and 351 K, or 351 D and 368E, remain predominantly as half-antibodies, rather than to bind other half-antibodies with the same residues. As it will be clear to a person with skill in the art, this is advantageous in the context of producing bispecific antibodies, as the tendency of half-antibodies to form homodimers may hinder the efficient generation and purification of bispecific antibodies. Also, the presence of homodimers hampers screening large repertoires of multispecific antibodies for functional activities.
• the IgG CH3 domain is an lgG1 CH3 domain.
• the IgG CH3 domain of the first protein and of the second protein are an lgG1.
• the IgG CH3 domain is a human IgG CH3.
• the first protein and/or the second protein comprises or is selected from the group consisting of: an antibody and a half-antibody, or a fragment thereof.
• the first protein is an antibody comprising a binding specificity and the second protein may be an antibody comprising another distinct binding specificity.
• the first protein is a multispecific antibody (for instance a bispecific or trispecific antibody).
• the second protein is a multispecific antibody (for instance a bispecific or trispecific antibody).
• both the first and second proteins are a multispecific antibody (for instance a bispecific or trispecific antibody).
• the first protein and/or the second protein is a homodimeric antibody.
• the first protein and/or the second protein is a heterodimeric antibody comprising a common light chain.
• the method may further comprise the step of enriching for and/or isolating the heterodimeric protein as obtained after reoxidisation.
• the method is an in vitro method.
• CH3-CH3 interface refers to the association between the two distinct CH3 domains that is as a result of interacting amino acid residues, i.e. at least one interaction between an amino acid of a first CH3 domain and an amino acid of a second CH3 domain. Such interaction is for instance via Van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, the formation of disulphide bonds, or other forces known to one skilled in the art. It will be appreciated that when two distinct CH3 domains form a CH3-CH3 interface, they form a heterodimeric protein (heterodimeric by virtue of at least the distinct sequences of the CH3 domains).
• each CH3 domain comprises a positively charged amino acid residue at position 351 and 366
• an IgG heterodimeric antibody that comprises two distinct IgG CH3 domains that are capable of forming a CH3-CH3 interface, said method comprising the steps of:
• each CH3 domain comprises a positively charged amino acid residue at position 351 and 366
• each CH3 domain comprises a negatively charged amino acid residue at position 351 and 368, wherein the numbering is according to Ell numbering,
• a method for producing a DEKK heterodimeric IgG antibody that comprises two distinct IgG CH3 domains that are capable of forming a CH3-CH3 interface comprising the steps of:
• each CH3 domain comprises a positively charged amino acid residue at position 351 and 366, wherein the positively charged amino acid residues at position 351 and 366 are K and K, respectively, and
• each CH3 domain comprises a negatively charged amino acid residue at position 351 and 368, wherein the negatively charged amino acid residues at position 351 and 368 are D and E, respectively wherein the numbering is according to Ell numbering,
• the method of the present disclosure comprises the step of providing: (a) a first protein comprising a CH3 domain which comprises a positively charged amino acid residue at position 351 and 366 (also referred to herein as a 351/366 positive CH3 domain), and (b) a second protein comprising a CH3 domain which comprises a negatively charged amino acid residue at position 351 and 368 (also referred to herein as a 351/368 negative CH3 domain), wherein the numbering is according to Ell numbering.
• the first and/or second proteins may comprise or is selected from the group consisting of: a monomeric protein, a homodimeric protein and a heterodimeric protein.
• a monomeric protein and “monomer”, which are used interchangeably herein, generally refer to a single, non-aggregated protein or polypeptide molecule.
• the term monomer may also refer to a half-body.
• an IgG monomer is an IgG half-body, i.e. a molecule which comprises or consists of one IgG heavy chain linked to one IgG light chain.
• the term monomer means a single, non-aggregated protein or polypeptide molecule (such as a single IgG CH3 domain or a monomeric IgG Fc region, for example), or a half-body (e.g. IgG half-body).
• homodimeric protein or “homodimer” generally refer to a dimer formed from two identical polypeptides (for example two 351/368 negative CH3 domains or two 351/366 positive CH3 domains) that are linked, either covalently or non-covalently.
• homodimer may also refer to an antibody (such as an IgG antibody) with two identical half-bodies. Therefore, in the context of the present disclosure, the term homodimer, depending on the context, means a dimer formed from two identical polypeptides (for example two identical IgG CH3 domains), linked either covalently or non- covalently, or a homodimeric antibody (e.g. homodimeric IgG antibody).
• the method of the present disclosure comprises the step of providing: (a) a first protein (e.g. a homodimeric IgG antibody or a fragment thereof) comprising two (identical) CH3 domains which each comprise a positively charged amino acid residue at position 351 and 366 (also referred to herein as a 351/366 positive CH3 domain).
• a first protein e.g. a homodimeric IgG antibody or a fragment thereof
• the method of the present disclosure comprises the step of providing (b) a second protein (e.g. a homodimeric IgG antibody or a fragment thereof) comprising two (identical) CH3 domains which each comprise a negatively charged amino acid residue at position 351 and 368 (also referred to herein as a 351/368 negative CH3 domain).
• the two IgG CH3 domains of the two monomers forming the heterodimer of the first protein have the same amino acids at positions 351 and 366, and similarly, the two IgG CH3 domains of the two monomers forming the heterodimer of the second protein have the same amino acids at positions 351 and 368.
• the IgG CH3 domains of the two monomers forming the heterodimer is identical (i.e. the polypeptide sequences of the IgG CH3 domains is the same along their entire lengths).
• heterodimer may also refer to an antibody (such as an IgG antibody) comprising or consisting of non-identical half-bodies. Therefore, in the context of the present disclosure, the term heterodimeric protein, may also mean a heterodimeric antibody.
• a heterodimeric antibody is made from two non-identical half-bodies, wherein the two halfbodies comprise IgG CH3 domains that have the same charge (i.e. 351/366 positive or 351/368 negative IgG CH3 domains).
• the heterodimeric protein for example a heterodimeric antibody
• the monomeric protein which is a half-body
• one of the monomers of the homodimeric protein for example one of the half-bodies of the homodimeric antibody. Therefore, the produced heterodimeric protein (for example heterodimeric antibody) will comprise a 351/366 positive IgG CH3 domain and a 351/368 negative IgG CH3 domain.
• the first protein provided in (a) of the method of the disclosure is a homodimeric protein (for example, a first homodimeric antibody) and the second protein provided in (b) may also be a homodimeric protein (for example, a second homodimeric antibody).
• the first homodimeric protein (for example the first homodimeric antibody) may comprise two 351/366 positive IgG CH3 domains
• the second protein for example the second homodimeric antibody
• the heterodimeric protein (for example a heterodimeric antibody) produced will comprise one of the monomers of the first homodimeric protein (for example one of the half-bodies of the first homodimeric antibody) and one of the monomers of the second heterodimeric protein (for example one of the half-bodies of the second heterodimeric antibody). Therefore, the produced heterodimeric protein (for example heterodimeric antibody) will comprise a 351/366 positive IgG CH3 domain and a 351/368 negative CH3 domain.
• first and/or second protein provided in (a) of the method is a monomeric protein, it is a monomeric Fc region.
• the first protein and/or the second protein may comprise or is selected from the group consisting of: an antibody and a half-antibody, or a fragment thereof. Antibodies, half- bodies and fragments thereof are described elsewhere herein. In certain aspects, the fragment is a monomeric or dimeric Fc region.
• the first and/or second proteins can have sequences of any origin, for example murine and human sequences.
• the first protein and/or the second protein can consist of sequences from one origin only, such as fully human antibodies, or they can have sequences of more than one origin, resulting for instance in chimeric or humanized antibodies.
• the antibody, half-antibody, or fragment thereof is a human antibody, human half-antibody, or fragment thereof. It will be appreciated that the fragment must at least comprise an IgG CH3 domain, a hinge region and a Fab arm.
• the first protein is an antibody having a first binding specificity and the second protein is an antibody have a second distinct binding specificity.
• said ratio is between 10:1 and 1 :10. In certain aspects, said ratio is between 5:1 and 1 :5. In certain aspects, said ratio is at least 1 :1 (w/w), for example at least 1.2:1 (w/w), for example, at least 1.5:1 (w/w) or at least 2:1 (w/w). In certain aspects, the ratio of the first protein (comprising a 351/366 positive IgG CH3 domain) to the second protein (comprising a 351/368 negative IgG CH3 domain) is at between 1 :1 (w/w) and 2:1 (w/w).
• the ratio of a KKKK first protein (comprising a 351/366 positive IgG CH3 domain) to a DEDE second protein (comprising a 351/368 negative IgG CH3 domain) is at least 1 :1 (w/w), for example at least 1.2:1 (w/w), for example, at least 1.5:1 (w/w) or at least 2:1 (w/w).
• the ratio of the KKKK first protein (comprising a 351/366 positive IgG CH3 domain) to the DEDE second protein (comprising a 351/368 negative IgG CH3 domain) is at between 1 :1 (w/w) and 2:1 (w/w).
• first protein and second protein is used when incubating said first and second proteins under reducing conditions, followed by reoxidising them.
• Appropriate concentrations is determined by a person of skill in the art, e.g. using the methodology described in the examples section below.
• each of the first and second proteins are used under said reducing conditions at a concentration of at least 50 pg/ml, for example at least 0.1 mg/ml, at least 1.0 mg/ml, at least 10 mg/ml or at least 25 mg/ml but not more than 100 mg/ml.
• the first and second proteins are obtained independently from each other.
• the first and second proteins are produced by different host cells. Exemplary method of obtaining a first and second protein are provided in the Examples section below. However, other methods will be known to those skilled in the art.
• Said incubation under reducing and reoxidising conditions of the method facilitate the production of the heterodimeric proteins by recombination of the first and second proteins (such as antibodies, or dimeric fragments thereof), or binding of the first and second proteins (such as half-bodies or monomeric fragments thereof).
• Recombination of the first and second protein occurs when the first and second proteins exchange monomers (for example half-bodies or dimeric fragments thereof) to produce a heterodimeric protein comprising two distinct IgG CH3 domains that are capable of forming a CH3-CH3 interface.
• reducing conditions refers to an environment in which the first and/or second protein is more likely to become reduced than oxidised.
• the reducing conditions may result in “disulphide bond reduction” (i.e. the process of cleaving a disulphide bond, thereby resulting in two thiol groups (-SH groups)).
• disulphide bond reduction i.e. the process of cleaving a disulphide bond, thereby resulting in two thiol groups (-SH groups)
• reducing agents examples include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, beta- mercapto-ethanol, thioglycolate, cysteamine, homocysteine, penicillamine, and/or sodium borohydride.
• 2-mercaptoethylamine 2-MEA
• DTT dithiothreitol
• DTE dithioerythritol
• glutathione glutathione
• tris(2-carboxyethyl)phosphine (TCEP) tris(2-carboxyethyl)phosphine
• L-cysteine beta- mercapto-ethanol
• cysteamine homocysteine
• penicillamine and/or sodium borohydride.
• the reducing agent when it is 2-MEA, it is at a concentration of from about 25 mM to about 100 mM, for example from about 50 mM to about 75 mM. In certain aspects, the concentration of 2-MEA is about 75 mM.
• the time of incubation with a reducing agent may depend upon the concentration and/or temperature at which the incubation occurs. For example, a higher concentration of the reducing agent may allow for a shorter incubation time and/or lower incubation temperature.
• the incubation when the concentration of 2-MEA is about 75 mM, the incubation is at about 31°C and last for at least 300 minutes. In certain aspects, when the concentration of 2-MEA is about 75 mM, the incubation is at about 31°C and last about 300 minutes.
• the reducing agent does not comprise an enzyme.
• the method further comprises reoxidising the reduced proteins to obtain the heterodimeric protein.
• reoxidising or “oxidise” as used means to undergo or cause to undergo a reaction in which electrons are lost to another species.
• the step of reoxidising may allow the reduced first and reduced second proteins as obtained to bind together to form heterodimeric proteins comprising two distinct IgG CH3 domains that are capable of forming a CH3-CH3 interface.
• Fresh MEA stock solution (750mM) was prepared by dissolving 852 mg Cysteamine hydrochloride (Sigma cat# 30078) in 5-6 mL PBS (pH7.4). pH was adjusted to pH 7.3 - 7.5 by adding NaOH (5M) to the solution at room temperature and the solution was filled with PBS pH 7.4 up till the final volume of 10 mL to achieve 750mM stock solution. The solution was filtered through 0.2 pm filter before use.
• IgG molecules were incubated in the presence of p-Mercapto-ethylamine hydrochloride without shaking.
• samples were buffer exchanged (Bex) to PBS using the AKTA Pure 25 system equipped with a Spark ALIAS autosampler and a single HiPrep 26/10 desalting column, Cytiva.
• samples were kept at 4°C for a sufficient time to allow full reoxidation to occur.
• the FAE reaction was performed on IgG molecules produced in Example 1 as, listed in Table 3.
• IgG mixtures listed in Table 3 were prepared at 1.1 mg/mL final concentration, 3.15 mL final volume adjusted by adding PBS (diluted from 10 x stock Gibco Cat# 70011-051 pH 7.4 using Fresenius Versylene sterile endotoxin-free water, Cat# B230531)). 100 uL of this mixture was withdrawn and kept at 4 degree as non-reacted control i.e. not exposed to MEA or Bex and stored at 4°C in the dark until further use. To start the FAE, to the remaining 3.05 mL, 350 uL of 75 mM MEA was added and solutions were mixed gently and kept at 31°C for 5 hours without shaking. Buffer exchange:

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