Method for generating bispecific proteins
OwnerCornelis Adriaan DE KRUIF, Franziska MORTENSENinventors
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Abstract
Provided herein is a method for producing a heterodimeric protein that comprises two distinct IgG CH3 domains that are capable of forming a CH3-CH3 interface. Further, provided herein is an isolated heterodimeric protein obtained by said method. Provided herein is also 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, as well as a pharmaceutical composition comprising said isolated heterodimeric proteins.
Claims26
A method for producing a heterodimeric antibody that comprises two distinct IgG CH3 domains that are capable of forming a CH3-CH3 interface, said method comprising the steps of:
- providing:
- (a) a first antibody or half-antibody comprising a first CH3 domain which comprises a positively charged amino acid residue at position 351 and 366, and
- (b) a second antibody or half-antibody comprising a second CH3 domain which comprises a negatively charged amino acid residue at position 351 and 368,
- wherein the numbering is according to EU numbering,
- incubating the antibodies and/or half-antibodies of (a) and (b) together under reducing conditions to provide a reduced first antibody or half-antibody and a reduced second antibody or half-antibody;
- and
- reoxidising the reduced antibodies and/or half-antibodies to obtain the heterodimeric antibody, wherein the IgG CH3 domains of the heterodimeric antibody do not comprise an arginine at position 409 wherein the heterodimeric antibody comprises an IgG Fc region, and wherein the IgG Fc region comprises the IgG1 or IgG2 core hinge region CPPC.
The method of any one of the preceding claims, wherein the first antibody or half-antibody and/or the second antibody or half-antibody is a human antibody or half-antibody.
The method of any one of the preceding claims, wherein the first antibody or half-antibody is an antibody having a first binding specificity and the second antibody or half-antibody is an antibody have a second distinct binding specificity.
The method of any one of the preceding claims, wherein the first antibody and/or the second antibody is a homodimeric antibody.
The method of any one of the preceding claims, wherein the first antibody and/or the second antibody is a heterodimeric antibody.
The method of any one of the preceding claims, wherein the first CH3 domain comprises:
- 351K and 366R, 351R and 366K, 351K and 366K, or 351R and 366R.
The method of any one of the preceding claims, wherein the second CH3 domain comprises:
- 351D and 368E, 351E and 368D, 351D and 368D, or 351E and 368E.
The method of any one of the preceding claims, wherein the first CH3 domain comprises 351K and 366K and the second CH3 domain comprises 351D and 368E.
The method of any one of the preceding claims, wherein the first and/or second antibody or half-antibody of a) and b) are Fc engineered.
The method of any one of the preceding claims, wherein the first and/or second antibody or half-antibody of a) and b) are Fc silenced or Fc enhanced.
The method of any one of the preceding claims, wherein the first and/or second antibody or half-antibody of a) and b) comprise a CH2 domain having an ADCC-affecting mutation at position 235 and/or 236.
The method of any one of the preceding claims, wherein the second antibody or half-antibody is obtained independently from the first antibody or half-antibody.
The method of any one of the preceding claims, wherein the reducing conditions comprise:
- (a) incubating the antibodies or half-antibodies in the presence of a reducing agent, optionally wherein the reducing agent comprises or is selected from the group consisting of:
- 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, beta-mercapto-ethanol, thioglycolate, cysteamine, homocysteine, penicillamine, and sodium borohydride;
- and/or
- (b) incubating the antibodies or half-antibodies at a pH of between 6.0 and 12.0;
- and/or
- (c) incubating the antibodies or half-antibodies at a redox potential of between −150 and −600 mV, optionally wherein the redox potential is between −250 and −400 mV.
The method of any one of the preceding claims, wherein the method further comprises the step of enriching for and/or isolating the heterodimeric antibody obtained after reoxidisation.
An isolated heterodimeric antibody obtainable by the methods according to any one of the preceding claims.
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,
- wherein the first CH3 domain comprises one or more of the amino acid variants 366K, 366R, 351K or 351R, and
- wherein the second CH3 domain comprises one or more of the amino acid variants 351E, 351D, 368E or 368D,
- said heterodimeric antibody further comprising two light chains that have non-identical sequences, wherein the IgG CH3 domains of the heterodimeric antibody do not comprise an arginine at position 409, wherein the heterodimeric antibody comprises and IgG Fc region, and wherein the IgG Fc region comprises the IgG1 or IgG2 core hinge region CPPC.
Dependencies are inferred from the claim text.