JP2019048814A - Heterodimeric antibody fc-containing proteins and methods for producing such proteins - Google Patents

Abstract

To provide a protein comprising heterodimeric antibody Fc, pharmaceuticals comprising the same and methods for producing such a protein.SOLUTION: Disclosed herein is a heterodimeric protein comprising a first polypeptide comprising a first Fc region of an immunoglobulin, the first Fc region comprising a first CH3 region, and a second polypeptide comprising a second Fc region of an immunoglobulin, the second Fc region comprising a second CH3 region, where the sequences of the first and the second CH3 regions are different, and the heterodimeric interaction between the first and second CH3 regions is stronger than the interactions of homodimers each comprising the first or second CH regions, and the first homodimeric protein has an amino acid substitution in a specific amino acid sequence.SELECTED DRAWING: None

Description

FIELD OF THE INVENTION This invention relates to novel heterodimeric antibody Fc-containing proteins, such as bispecific antibodies, and novel methods for producing such proteins.

BACKGROUND OF THE INVENTION Monoclonal antibodies have recently become successful therapeutic molecules, particularly for the treatment of cancer. Unfortunately, however, monoclonal antibodies often can not cure the disease when used as a monotherapy. Bispecific antibodies can potentially overcome some of the limitations of monoclonal antibody therapy, for example, effectors to disease related sites as mediators to target drugs or toxic compounds to target cells It could be used as a mediator to retarget the mechanism, or as a mediator to increase the specificity for tumor cells, eg by binding to combinations of target molecules found exclusively on tumor cells .

  Recently, different formats and uses of bispecific antibodies have been reviewed by Chames and Baty (2009) Curr Opin Drug Disc 12: 276. One of the major obstacles in the development of bispecific antibodies has been the difficulty of producing materials of sufficient quality and quantity by conventional techniques, such as hybrid hybridomas and chemical conjugation methods ( Marvin and Zhu (2005) Acta Pharmacol Sin 26: 649). Host cell co-expression of two antibodies, consisting of different heavy and light chains, results in a mixture of possible antibody products in addition to the desired bispecific antibody.

  Several strategies have been described that favor the formation of heterodimeric, ie bispecific, products by co-expression of different antibody constructs.

  Lindhofer et al. (1995 J Immunol 155: 219) describe that fusion of rat and mouse hybridomas producing different antibodies is functional due to selective species-restricted heavy / light chain pairing. It is described that this results in the enrichment of various bispecific antibodies. Another strategy for promoting the formation of heterodimers as compared to homodimers is the "knob-in-hole" strategy, in which the bulge is the first The cavity is introduced at the interface of the heavy chain polypeptide and at the corresponding cavity at the interface of the second heavy chain polypeptide, so as to promote heterodimer formation and prevent homodimer formation. The ridge can be positioned on the "Bumps" are constructed by exchanging small amino acid side chains from the interface of the first polypeptide with larger side chains. Complementary "cavities" of the same or similar size to the protuberances are created at the interface of the second polypeptide by exchanging large amino acid side chains with smaller ones. EP 1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies to favor heterodimerization by co-expression of different antibody domains in host cells There is. In these methods, one or more residues constituting the CH3-CH3 interface in both CH3 domains are electrostatically disadvantageous for homodimerization and electrostatically for heterodimerization. It is replaced with a charged amino acid, which is advantageous. A further strategy is described in WO 2007 110 205 (Merck) (patent document 3), which utilizes the difference between the IgA CH3 domain and the IgG CH3 domain to promote heterodimerization. There is.

  Dall'acqua et al. (1998 Biochemistry 37: 9266) described five energetically important amino acid residues (366, 368), involved in the contact of CH3-CH3 at the interface of CH3 homodimers. 405, 407 and 409).

  In WO2008119353 (Genmab) (patent document 4), “Fab arm” or “half molecule” exchange (heavy and attached light chains) between two monospecific IgG4 antibodies or IgG4-like antibodies by incubation under reducing conditions In vitro methods for producing bispecific antibodies have been described in which bispecific antibodies are formed by swapping). In this Fab arm exchange reaction, the heavy chain disulfide bond in the hinge region of the parent (originally monospecific) antibody is reduced and the resulting free cysteine has a different parent antibody molecule (originally different) Of the disulfide bond of the parent antibody and the CH3 domain of the parent antibody are released and rearranged by dissociation / association, resulting in the isomerization reaction of disulfide bond and dissociation / association of CH3 domain It is. The resulting product is potentially a bispecific antibody with two Fab arms composed of different sequences. This process is, however, random, and Fab arm exchange may be performed between two molecules having the same sequence, or two bispecific molecules may be involved in Fab arm exchange to It should be noted that antibodies can be regenerated that contain the specificity of the monospecific parent antibody.

  Here, surprisingly, the introduction of an asymmetric mutation in the CH3 region of the two monospecific starting proteins leads to a directed, and thus an extremely stable heterodimeric protein It turned out that it is possible to force the exchange reaction.

EP1870459 WO2009089004 WO2007110205 WO2008119353

Chames and Baty (2009) Curr Opin Drug Disc Dev 12: 276 Marvin and Zhu (2005) Acta Pharmacol Sin 26: 649 Lindhofer et al. (1995 J Immunol 155: 219) Dall'acqua et al. (1998 Biochemistry 37: 9266)

  Thus, in one aspect, the present invention provides an efficient in vitro method for the production of highly stable heterodimeric Fc-containing proteins based on stable homodimeric Fc-containing starting materials. For example, extremely stable bispecific antibodies can be formed with high yield and purity based on two stable monospecific antibodies as starting materials.

Thus, in one aspect, the present invention relates to an in vitro method for producing a heterodimeric protein comprising the following steps:
a) providing a first homodimeric protein comprising an Fc region of an immunoglobulin, wherein the Fc region comprises a first CH3 region,
b) providing a second homodimeric protein comprising an immunoglobulin Fc region, wherein the Fc region comprises a second CH3 region,
Here, the sequences of the first CH3 region and the second CH3 region are different, and the heterodimeric interaction between the first CH3 region and the second CH3 region is the first CH3 region and Appear to be stronger than each of the homodimeric interactions of the two CH3 domains,
c) incubating the first protein with the second protein under reducing conditions sufficient to allow cysteines in the hinge region to cause disulfide bond isomerization, and
d) obtaining said heterodimeric protein.

  This method can be used, for example, for the in vitro production of heterodimeric proteins, such as bispecific antibodies, for various uses, such as therapeutic or diagnostic. The advantage of this in vitro method is that heavy chain / light chain pairings remain intact during the reaction so that undesired combinations of heavy and light chains are not obtained in the product. This is a common light chain that can form functional antibodies with both heavy chains in order to avoid the formation of non-functional heavy / light chain products by random heavy / light chain pairing in cells In contrast to some of the co-expression methods described in the prior art (see above) where it is necessary to find In addition, in vitro processes can be performed in the laboratory that allow for greater control, flexibility and yield of heterodimeric proteins than those enabled by coexpression.

The in vitro methods of the invention can also be used, for example, in screening methods to identify advantageous combinations of specificities, to generate larger sized compound libraries. For example, in the case of some combinations of antibody targets, neither bispecific antibody is functional, ie, it can not simultaneously bind both targets and mediate the desired functional effect . In such cases, bispecific antibodies having the desired properties, such as optimal target binding or cell killing,
a) providing a first set of homodimeric antibodies with different variable regions, wherein the first set of antibodies comprises a first CH3 region,
b) providing a second set of homodimeric antibodies with different variable regions, wherein the second set of antibodies comprises a second CH3 region,
Here, the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is the first CH3 region and the second CH3 region. Seems to be stronger than each of the domain's homodimer interactions,
c) incubating the combination of the first and second set of antibodies under reducing conditions sufficient to allow cysteines in the hinge region to cause isomerization of the disulfide bond, thus Creating a set of bispecific antibodies,
d) optionally returning the conditions to non-reducing conditions,
e) assaying the resulting set of bispecific antibodies for a given desired property, and
f) can be identified by selecting bispecific antibodies with the desired properties.

  In a further aspect, the present invention relates to heterodimeric proteins obtained or obtainable by the method of the present invention and methods for producing the heterodimeric proteins of the present invention by coexpression in suitable host cells About.

Generation of bispecific antibodies by interspecies Fab arm exchange. The generation of bispecific antibodies after GSH-induced in vitro Fab-arm exchange between the indicated EGFR (2F8) and CD20 (7D8) IgG4 antibodies was determined by ELISA. Concentration series (total antibody) of 0-1 μg / mL were analyzed in ELISA. The bispecific binding was higher after Fab arm exchange between the rhesus (Rh) IgG4 and human (Hu) IgG4 antibodies than between two antibodies of the same species. Core hinges of human and rhesus monkey antibody isotypes (ie CPPC sequences in human IgG1 containing two cysteine residues potentially forming an inter-heavy chain disulfide bond and corresponding residues in other human isotypes or other species) and CH3- Alignment of the amino acid sequence at the CH3 interface. Generation of bispecific antibodies using mutant human IgG1 involved in Fab arm exchange. Production of bispecific antibodies after GSH-induced in vitro Fab-arm exchange between human CD20 (7D8) IgG4 antibody and the indicated human EGFR (2F8) IgG1 antibody was determined by ELISA. The graph presented shows the average number of 3 independent Fab arm exchange experiments, in which a total antibody concentration of 1 μg / mL was used in the ELISA. The bispecific binding was higher after Fab arm exchange between IgG1-2F8-CPSC-ITL and IgG4-7D8 than between the two IgG4 antibodies. Combining IgG4-7D8 with either IgG1-2F8-CPSC or IgG1-2F8-ITL did not yield a bispecific antibody under the conditions used. Generation of bispecific antibodies by in vivo Fab arm exchange of human and variant IgG1 antibodies. Production of bispecific antibodies after Fab arm exchange in vivo in immunodeficient mice between human CD20 (7D8) IgG4 antibody and the indicated human EGFR (2F8) IgG1 and IgG4 variant antibodies was determined by ELISA. The graph presented shows the average number (n = 4). Bispecific reactivity is presented as the concentration (ratio) of bispecific antibody to total IgG concentration. Human IgG4 with R409K mutation or stabilized hinge (CPPC) in the CH3 domain can not participate in Fab arm exchange. IgG1 with CPSC sequence in the hinge and R409K mutation in the CH3 domain is involved in Fab arm exchange. ( ^* ) Bispecific binding to mixtures containing either IgG1-2F8, IgG4-2F8-CPPC or IgG4-2F8-R409K was below the detection limit and was therefore optionally set to zero. Generation of bispecific antibodies using 2-mercapto-ethylamine.HCl (2-MEA) induced Fab arm exchange between human IgG1 and IgG4 antibodies. The generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between the indicated human EGFR (2F8) and CD20 (7D8) antibodies was determined by ELISA. A dilution series 0-40 mM 2-MEA was tested. The graph presented shows the results of the ELISA, where a total antibody concentration of 20 μg / mL was used. 2-MEA efficiently induced Fab arm exchange even between antibodies containing stabilized hinges (CPPC). With respect to the CH3 domain, the combination of human IgG1 × human IgG4 with triple mutation T350I-K370T-F405L resulted in higher levels of bispecific reactivity compared to the two wild type IgG4 antibodies. Generation of bispecific antibodies using 2-MEA induced Fab arm exchange between human IgG1 and IgG4 antibodies. Generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between the indicated human EGFR (2F8) and CD20 (7D8) antibodies, at concentrations of 0-40 mM 2-MEA All samples in the series were determined by mass spectrometry. (A) A representative example of mass spectrometry profile for a sample of Fab arm exchange reaction between IgG1-2F8-ITL × IgG4-7D8-CPPC at 0 mM, 7 mM and 40 mM 2-MEA is shown. IgG4-2F8 × IgG4-7D8 yielded approximately 50% bispecific antibodies. IgG1-2F8-ITL × IgG4-7D8-CPPC yielded approximately 95% bispecific antibodies. Generation of bispecific antibodies using 2-MEA induced Fab arm exchange between human IgG1 and IgG4 antibodies. Generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between the indicated human EGFR (2F8) and CD20 (7D8) antibodies, at concentrations of 0-40 mM 2-MEA All samples in the series were determined by mass spectrometry. (B) After quantification of mass spectrometry data, the percentage of bispecific antibody was calculated and plotted against the concentration of 2-MEA in the Fab arm exchange reaction. IgG4-2F8 × IgG4-7D8 yielded approximately 50% bispecific antibodies. IgG1-2F8-ITL × IgG4-7D8-CPPC yielded approximately 95% bispecific antibodies. Stability analysis of heterodimeric bispecific antibodies obtained by 2-MEA induced Fab arm exchange. Bispecificity created by 2-MEA-induced Fab arm exchange by combining either IgG1-2F8-ITL × IgG4-7D8-CPPC (A) or IgG4-2F8 × IgG4-7D8 (B) The stability of the samples was tested by measuring EGFR / CD20 bispecific binding in an ELISA following GSH-induced Fab arm exchange reaction in the presence of the indicated concentration of irrelevant IgG4. Bispecific binding is presented relative to the bispecific binding of the starting material (control) set at 100%. (A) Fab arm exchange under GSH conditions, as the bispecific binding of the 2-MEA-induced bispecific product derived from IgG1-2F8-ITL × IgG4-7D8-CPPC was conserved It was suggested that it was a stable product that was not involved in (B) The bispecific EGFR / CD20 binding of the 2-MEA-induced bispecific product derived from IgG4-2F8 × IgG4-7D8 is impaired so that it is not associated with irrelevant IgG4 under GSH conditions It was suggested that it was a product involved in Fab arm exchange. Plasma clearance rates of heterodimeric bispecific antibodies generated by 2-MEA induced Fab arm exchange. Three groups of mice (3 mice per group) were injected with the indicated antibodies: (1) Generation by in vitro 2-MEA-induced Fab-arm exchange between IgG1-2F8-ITL × IgG4-7D8-CPPC (2) 100 μg of bispecific antibody + 1,000 μg of irrelevant IgG4; (3) 50 μg of IgG1-2F8-ITL + 50 μg of IgG4-7D8-CPPC. (A) Total antibody concentration over time as determined by ELISA. The curve of plasma concentration of total antibody was the same for all antibodies. (B) Bispecific antibody concentrations determined by ELISA. The bispecificity of the injected antibody was the same with or without the addition of excess irrelevant IgG4. ( ^* ) The binding of the bispecificity to the IgG1-2F8-ITL + IgG4-7D8-CPPC mixture was below the limit of detection, so the corresponding symbols could not be plotted in this graph. Average values of 2 ELISA experiments are shown. Purity of bispecific antibodies generated by Fab arm exchange between human IgG1-2F8 and IgG4-7D8-CPPC. (A) Reduced SDS-PAGE (a) shows heavy and light chain bands for both bispecific and IgG1 control samples. Non-reducing SDS-PAGE (b). Purity of bispecific antibodies generated by Fab arm exchange between human IgG1-2F8 and IgG4-7D8-CPPC. (B) The peak results from the HP-SEC analysis indicate that> 98% of the bispecific samples were homogeneous and, among other things, failed to detect antibody aggregates. Purity of bispecific antibodies generated by Fab arm exchange between human IgG1-2F8 and IgG4-7D8-CPPC. (C) Mass spectrometry shows that Fab arm exchange resulted in approximately 100% bispecific product. It is a continuation of FIG. 9C-1. Between triple mutants (ITL), double mutants (IT, IL, TL) and single mutants (L) human IgG1-2F8 in the production of bispecific antibodies by Fab arm exchange with human IgG4-7D8 comparison. Between human IgG1-2F8 triple mutant and double mutant and wild type IgG4-7D8 with CPSC hinge (A) or mutant IgG4-7D8-CPPC with stabilized hinge (B ) Or after a 2-MEA-induced in vitro Fab-arm exchange of (C) between or single mutant IgG1-2F8-F405L and IgG4-7D8 with wild type CPSC hinge or stabilized CPPC hinge Production of bispecific antibodies was determined by ELISA. Concentration series (total antibody) of 0-20 μg / mL or 0-10 μg / mL were analyzed by ELISA for experiments involving double mutants and single mutants, respectively. The combination with the double mutant IgG1-2F8-IL and IgG1-2F8-TL results in bispecific EGFR / CD20 binding similar to the triple mutant IgG1-ITL. In combination with IgG1-2F8-IT, no bispecific product is obtained. In combination with the single mutant IgG1-2F8-F405L, bispecific EGFR / CD20 binding is obtained. Generation of bispecific antibodies using 2-MEA induced Fab arm exchange at different temperatures. Generation of bispecific antibodies by combining the indicated human EGFR (2F8) and CD20 (7D8) antibodies in 2-MEA induced in vitro Fab arm exchange reactions at 0 ° C, 20 ° C and 37 ° C , Followed by ELISA for a defined time. Bispecific binding was most efficient at 37 ° C and more slowly at 20 ° C. At 0 ° C., production of bispecific binding was not measured. Generation of bispecific antibodies by in vitro Fab arm exchange induced by different reducing agents. An ELISA was used to measure the production of bispecific antibodies by combining human IgG1-2F8-ITL and IgG4-7D8-CPPC in the reduction reactions with the indicated concentration of reducing agent. Bispecific binding was measured after reaction with DTT (which obtained a maximum at 2.5 mM DTT) and 2-MEA (which obtained a maximum at 25 mM 2-MEA) but not GSH. ( ^* ) Data of GSH concentrations> 10 mM were excluded due to the formation of antibody aggregates. 2-MEA induced Fab arm exchange between IgG1-2F8-ITL and IgG1-7D8-K409X variants. The generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between IgG1-2F8-ITL and the indicated IgG1-7D8-K409X variants was determined by ELISA. (A) A concentration series (total antibody) of 0-20 μg / mL was analyzed. The positive control is a purified batch of bispecific antibodies derived from IgG1-2F8-ITL × IgG4-7D8-CPPC. (B) Exchange is presented as bispecific binding at 20 μg / mL to positive control (black bars). Thick gray bars indicate dual specificity between IgG4 controls (IgG4-7D8 × IgG4-2F8), negative controls (IgG1-2F8 × IgG1-7D8-K409R) and between IgG1-2F8-ITL and IgG4-7D8-CPPC Represents a sex bond. White gray bars represent the results of Fab arm exchange reactions performed simultaneously between the indicated IgG1-7D8-K409X variants and IgG1-2F8-ITL. Antibody deglycosylation does not affect the generation of bispecific antibodies by 2-MEA induced Fab arm exchange. The generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between the indicated EGFR (2F8) and CD20 (7D8) antibodies was determined by ELISA. Exchange with the 7D8 antibody was compared to its enzymatically deglycosylated variant. Concentration series (total antibody) of 0-20 μg / mL were analyzed in ELISA. Fab arm exchange reactions with deglycosylated (deglyc) antibodies showed a bispecific binding curve identical to the glycosylation variants from which they were derived. The ability to participate in Fab arm exchange correlates with CH3-CH3 interaction strength. (A) Double by GSH-induced Fab arm exchange between IgG1-2F8 and IgG1-7D8 construct (A) with the indicated mutations, presented as bispecific binding in ELISA over time Production of specific antibodies. Bispecificity is presented relative to the IgG4-2F8 × IgG4-7D8 control after 24 hours. The ability to participate in Fab arm exchange correlates with CH3-CH3 interaction strength. (B) Double by GSH-induced Fab arm exchange between IgG4-2F8 and IgG4-7D8 construct (B) with the indicated mutations, presented as bispecific binding in ELISA over time Production of specific antibodies. Bispecificity is presented relative to the IgG4-2F8 × IgG4-7D8 control after 24 hours. The ability to participate in Fab arm exchange correlates with CH3-CH3 interaction strength. (C) Double by GSH-induced Fab arm exchange between IgG4-2F8 and IgG4-7D8 construct (C) with the indicated mutations, presented as bispecific binding in ELISA over time Production of specific antibodies. Bispecificity is presented relative to the IgG4-2F8 × IgG4-7D8 control after 24 hours. The ability to participate in Fab arm exchange correlates with CH3-CH3 interaction strength. (D) Relationship between apparent K _D (Table 2) and the generation of bispecific antibodies after 24 hours (FIG. 15A / B / C) on IgG1-based molecules (D). The ability to participate in Fab arm exchange correlates with CH3-CH3 interaction strength. (E) Relationship between the apparent K _D (Table 2) and the generation of bispecific antibodies after 24 hours (FIG. 15A / B / C) against an IgG4-based molecule (E). Sequence alignment of anti-EGFr antibody 2F8 in the IgG1, IgG4 and (partial) IgG3 backbone. Amino acid numbering by Kabat and amino acid numbering by EU index (both described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) Has been Generation of bispecific antibodies by in vitro Fab arm exchange induced by different reducing agents. An ELISA was used to measure the production of bispecific antibodies by combining human IgG1-2F8-F405L and IgG1-7D8-K409R in the reduction reactions at the indicated concentration series of reducing agents. Measured OD value against 100% of the signal of bispecific control sample derived from 2-MEA induced Fab arm exchange between IgG1-2F8-ITL × IgG4-7D8-CPPC, set to 100% Turned Maximum bispecific binding is obtained after reaction with DTT in the concentration range 0.5-50 mM, 2-MEA in the concentration range 25-50 mM 2-MEA and tris (2-carboxyethyl) phosphine (TCEP) in the concentration range 0.5-5.0 mM It was measured but not by GSH. ( ^* ) Data for GSH concentrations above 25 mM were excluded due to the formation of antibody aggregates. Generation of bispecific antibodies using 2-MEA induced Fab arm exchange between human IgG1-2F8-F405L and IgG1-7D8-K409R. (A) Production of bispecific antibodies after 2-MEA induced in vitro Fab arm exchange was determined by ELISA. The graph presented shows the results of ELISA using a total antibody concentration of 20 μg / mL. 2-MEA efficiently induced Fab arm exchange. (B) The production of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange was determined by mass spectrometry for all samples in the concentration series 0-40 mM 2-MEA. After quantification of mass spectrometry data, the percentage of bispecific antibody was calculated and plotted against the concentration of 2-MEA in the Fab arm exchange reaction. IgG1-2F8-F405L × IgG1-7D8-K409R yielded approximately 100% bispecific antibodies, confirming the data of the ELISA. Purity of bispecific antibodies generated by Fab arm exchange between human IgG1-2F8-F405L × IgG1-7D8-K409R. Mass spectrometry shows that Fab arm exchange yielded approximately 100% bispecific product. It is a continuation of FIG. 19-1. Plasma clearance of bispecific antibodies generated by 2-MEA induced Fab arm exchange. Two groups of mice (3 mice per group) were injected with the indicated antibodies: (1) Generation by in vitro 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R (2) 100 μg of bispecific antibody + 1,000 μg of irrelevant IgG4. (A) Total antibody concentration over time as determined by ELISA. The curve of plasma concentration of total antibody was the same for all antibodies. (B) Bispecific antibody concentrations determined by ELISA. The bispecificity of the injected antibody was the same with or without the addition of excess irrelevant IgG4. CDC-mediated cell killing of CD20 expressing cells by bispecific antibodies generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R. The indicated antibody concentration series were used to test its ability to mediate CDC on Daudi cells (A) and Raji cells (B). Both cell lines express CD20 but not EGFR. Introduction of K409R in IgG1-7D8 did not affect its ability to induce CDC. Bispecific antibodies derived from 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R were still able to induce CDC. ADCC-mediated cell killing of EGFR-expressing cells by bispecific antibodies generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R. The indicated antibody concentration series were used to test its ability to mediate ADCC on A431 cells. IgG1-7D8 failed to bind to CD20 negative A431 cells and consequently did not induce ADCC. ADCC was also induced by the EGFR antibody IgG1-2F8 after introduction of the F405L mutation in the CH3 domain. The ADCC effector function of IgG1-2F8-F405L was retained in the form of bispecificity obtained by Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R. 2-MEA induced Fab arm exchange between IgG1-2F8-F405X mutant and IgG1-7D8-K409R. The generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between the indicated IgG1-2F8-F405X variants and IgG1-7D8-K409R was determined by ELISA. (A) A concentration series (total antibody) of 0-20 μg / mL was analyzed in ELISA. The positive control is a purified batch of bispecific antibodies derived from IgG1-2F8-F405L × IgG1-7D8-K409R. (B) Exchange is presented as bispecific binding at an antibody concentration of 20 μg / mL against a positive control (black bar). Heavy gray bars represent bispecific binding between IgG4 controls (IgG4-7D8 × IgG4-2F8) and between negative controls (IgG1-2F8 × IgG1-7D8-K409R). White gray bars represent the results of Fab arm exchange reactions performed simultaneously between the indicated IgG1-2F8-F405X variants and IgG1-7D8-K409R or controls. 2-MEA induced Fab arm exchange between IgG1-2F8-Y407X mutant and IgG1-7D8-K409R. The generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between the indicated IgG1-2F8-Y407X variants and IgG1-7D8-K409R was determined by ELISA. (A) A concentration series (total antibody) of 0-20 μg / mL was analyzed in ELISA. The positive control is a purified batch of bispecific antibodies derived from IgG1-2F8-F405L × IgG1-7D8-K409R. (B) Exchange is presented as bispecific binding at an antibody concentration of 20 μg / mL against a positive control (black bar). Heavy gray bars represent bispecific binding between IgG4 controls (IgG4-7D8 × IgG4-2F8) and between negative controls (IgG1-2F8 × IgG1-7D8-K409R). White gray bars represent the results of Fab arm exchange reactions performed simultaneously between the indicated IgG1-2F8-Y407X variants and IgG1-7D8-K409R or controls. Analysis by SDS-PAGE under non-reducing conditions (FIG. 25 (A)) and reducing conditions (FIG. 25 (B)) of bispecific antibodies generated by 2-MEA induced Fab arm exchange. Homodimeric Starting Material IgG1-2F8-F405L (FIG. 26 (B)), Homodimeric Starting Material IgG1-7D8-K409R (FIG. 26A), Mixture of Both Dimers (1: 1) (FIG. 26 (C)), and HP- of a bispecific antibody (FIG. 26 (D)) generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R SEC profile. Homodimeric Starting Material IgG1-2F8-F405L (FIG. 27 (B)), Homodimeric Starting Material IgG1-7D8-K409R (FIG. 27A), Mixture of Both Dimers (1: 1) (FIG. 27 (C)) and mass spectrometry of bispecific antibodies (FIG. 27 (D)) generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R (ESI-MS). Capillary isoelectric focusing (cIEF) profile of homodimeric starting material IgG1-2F8-F405L (FIG. 28 (A)). Capillary isoelectric focusing (cIEF) profile of homodimeric starting material IgG1-7D8-K409R (FIG. 28 (B)). Capillary isoelectric focusing (cIEF) profile of a mixture of both dimers (1: 1) (FIG. 28 (C)). Capillary isoelectric focusing (cIEF) profile of bispecific antibody (FIG. 28 (D)) generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R. Homodimeric Starting Material IgG1-2F8-F405L (FIG. 29 (A)), Homodimeric Starting Material IgG1-7D8-K409R (FIG. 29 (B)), Mixture of Both Dimers (1: 1) (FIG. 29 (C)), and HPLC of a bispecific antibody (FIG. 29 (D)) generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R CIEX profile. Electrospray ionization mass spectrometry of IgG obtained by cotransfection of expression vectors encoding heavy and light chains of IgG1-7D8-K409R or IgG1-2F8-F405. The heterodimer peak is indicated by ^* . The peak of the homodimer is indicated by †. Exchange reactions of homodimeric IgG1-2F8-F405L and IgG1-7D8-K409R as monitored by high pressure liquid chromatography cation exchange (HPLC-CIEX) after injection at different intervals. The remaining homodimers (indicated by arrows) of the exchange reaction shown in FIG. 32 as detected by the CIEX method. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as determined by ELISA. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as determined by ELISA. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as determined by ELISA. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as determined by ELISA. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as determined by ELISA. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as determined by ELISA. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as determined by ELISA and compared to controls arbitrarily set at 100%. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as determined by ELISA and compared to controls arbitrarily set at 100%. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as determined by ELISA and compared to controls arbitrarily set at 100%. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as determined by ELISA and compared to controls arbitrarily set at 100%. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as analyzed by HPLC-CIEX. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as analyzed by HPLC-CIEX. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as analyzed by HPLC-CIEX. Generation of bispecific antibodies at various IgG concentrations, 2-MEA concentrations, incubation temperature and time as analyzed by HPLC-CIEX. Generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between the indicated IgG1-2F8-L368X variants and IgG1-7D8-K409R, in a concentration series of 0-20 μg / mL It was judged by ELISA using (total antibody) (FIG. 37 (A)). The positive control is a purified batch of bispecific antibodies derived from IgG1-2F8-F405L × IgG1-7D8-K409R. FIG. 37 (B) shows bispecific binding at 20 μg / mL to a positive control (black bar). Heavy gray bars represent bispecific binding between IgG4 controls (IgG4-7D8 × IgG4-2F8) and between negative controls (IgG1-2F8 × IgG1-7D8-K409R). White gray bars represent the results of Fab arm exchange reactions performed simultaneously between the indicated IgG1-2F8-L368X variants and IgG1-7D8-K409R. Generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between the indicated IgG1-2F8-K370X variants and IgG1-7D8-K409R, in a concentration series of 0-20 μg / mL It was judged by ELISA using (total antibody) (FIG. 37 (A)). The positive control is a purified batch of bispecific antibodies derived from IgG1-2F8-F405L × IgG1-7D8-K409R. FIG. 37 (B) shows bispecific binding at 20 μg / mL to a positive control (black bar). Heavy gray bars represent bispecific binding between IgG4 controls (IgG4-7D8 × IgG4-2F8) and between negative controls (IgG1-2F8 × IgG1-7D8-K409R). White gray bars represent the results of Fab arm exchange reactions performed simultaneously between the indicated IgG1-2F8-D370X variants and IgG1-7D8-K409R. Generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between the indicated IgG1-2F8-D399X variants and IgG1-7D8-K409R, concentration series 0-20 μg / mL It was determined by ELISA using (total antibody) (FIG. 38 (A)). FIG. 38 (B) shows bispecific binding at an antibody concentration of 20 μg / mL to a positive control (black bar). Heavy gray bars represent bispecific binding between IgG4 controls (IgG4-7D8 × IgG4-2F8) and between negative controls (IgG1-2F8 × IgG1-7D8-K409R). White gray bars represent the results of Fab arm exchange reactions performed simultaneously between the indicated IgG1-2F8-D399X variants and IgG1-7D8-K409R. 2-MEA induced Fab arm exchange between combinations of 4 different IgG1 variants at 15 ° C. after 0, 30, 60, 105 and 200 minutes incubation as determined by sandwich ELISA. 2-MEA induced Fab arm exchange between combinations of different IgG1 variants after antibody incubation for 90 minutes at 15 ° C. as determined by sandwich ELISA. Phosphorylation of c-Met by c-Met specific antibodies. A549 cells are incubated for 15 minutes with HGF or a panel of different antibodies. Proteins are separated by SDS-page gel electrophoresis and transferred to membranes by western blotting. Phosphorylated c-Met, total c-Met and β-actin are detected by antibodies against phosphorylated c-Met, total c-Met or β-actin. Proliferation assay on NCI-H441 cells. NCI-H441 cells were incubated for 7 days with monovalent bispecific IgG1069 / b12, control antibodies (IgG1-069, UniBody-069, IgG1-b12) and left untreated. Cell clumps were determined and plotted as a percentage of untreated samples (set at 100%). CDC-mediated cell killing of CD20 expressing cells by bispecific antibodies generated by 2-MEA induced Fab arm exchange between IgG1-7D8-F405L or IgG1-2F8-F405L and IgG1-7D8-K409R. The indicated antibody concentration series were used to test its ability to mediate CDC on Daudi cells (A) and Raji cells (B). Both cell lines express CD20 but not EGFR. Bispecific antibodies generated by 2-MEA-induced Fab-arm exchange between IgG1-7D8-F405L x IgG1-7D8-K409R are as effective as IgG1-7D8 in inducing CDC-mediated cell killing Met. Bispecific antibodies derived from 2-MEA induced Fab arm exchange between IgG2-2F8-F405L × IgG1-7D8-K409R result in monovalent CD20 binding bispecific antibodies, which are CDC-mediated cells It slightly affected the induction of death. Killing of A431 cells induced by anti-κ-ETA 'preincubated HER2 × HER2 bispecific antibody. Viability of A431 cells after 3 days incubation with HER2 antibody preincubated with anti-κ-ETA '. Cell viability was quantified using alamar blue. The data shown is the fluorescence intensity (FI) of one experiment on A431 cells treated with anti-κ-ETA ′ conjugated HER2 antibody and HER2 × HER2 bispecific antibody. Staurosporine was used as a positive control, while an isotype control antibody was used as a negative control. The HER2 × HER2 bispecific molecule induced downregulation of the HER2 receptor. Relative percentage of HER2 expression levels in AU565 cell lysates after 3 days incubation with 10 μg / mL mAb. The amount of HER2 was quantified using a HER2-specific capture ELISA and plotted as% inhibition relative to untreated cells. Data shown are the mean of two experiments + standard deviation. Colocalization analysis of HER2 × HER2 bispecific antibody (FITC) with the lysosomal marker LAMP1 (Cy5). FITC pixel intensities overlapping with Cy5 for various monospecific HER2 and HER2 × HER2 bispecific antibodies (FIG. 46 (B)). FITC pixel intensities of LAMP1 / Cy5 positive pixels of three different images were plotted for each antibody tested. For monospecific antibodies, even lower FITC pixel intensities of LAMP1 / Cy5 positive pixels are evident as compared to bispecific antibodies. FIG. 46 (B) represents the mean value of FITC pixel intensity per LAMP1 / Cy5 positive pixel calculated from three different images. Taken together, these results suggest that after internalization, higher levels of bispecific antibodies localize to Lamp1 / Cy5 positive vesicles as compared to monospecific antibodies. Inhibition of proliferation by HER-2 monospecific and bispecific antibodies. AU565 cells were seeded in serum-free cell culture medium in the presence of 10 μg / mL of HER2 antibody or HER2 × HER2 bispecific antibody. After 3 days, the amount of live cells was quantified with Alamar Blue and cell viability was presented as a percentage of untreated cells. An isotype control antibody was used as a negative control. Data shown are% AU 565 viable cells measured as 5-fold ± standard deviation and compared to untreated cells. ^* Indicates that only one data point was drawn. Binding of monospecific and bispecific IgG1 antibodies and hinge deleted IgG1 antibodies to human and mouse FcRn at different pH. Plates with human and mouse FcRn were incubated with different monospecific and bispecific IgG1 antibodies or hinge deleted IgG1 molecules. Binding to FcRn was analyzed by ELISA at 405 nm. (A) Binding of monospecific and bispecific IgG1 antibodies and hinge deleted IgG1 (Uni-G1) molecules to human FcRn at pH 7.4 and 6.0. Binding to human FcRn is very low at neutral pH. At pH 6.0, (bispecific) antibodies bind efficiently to human FcRn if they do not contain the H435A mutation. The hinge-deleted IgG1 (Uni-G1) molecule binds human FcRn with low efficiency. Binding of monospecific and bispecific IgG1 antibodies and hinge deleted IgG1 antibodies to human and mouse FcRn at different pH. Plates with human and mouse FcRn were incubated with different monospecific and bispecific IgG1 antibodies or hinge deleted IgG1 molecules. Binding to FcRn was analyzed by ELISA at 405 nm. (B) Binding of monospecific and bispecific IgG1 antibodies and hinge deleted IgG1 (Uni-G1) molecules to mouse FcRn at pH 7.4 and 6.0. Binding to mouse FcRn is very low at neutral pH. At pH 6.0, the (bispecific) antibody binds very efficiently to mouse FcRn if it does not contain the H435A mutation in both Fab arms. Bispecific molecules with the H435A mutation in only one Fab arm can still bind mouse FcRn. The hinge-deleted IgG1 (Uni-G1) molecule binds mouse FcRn with moderate efficiency and the hinge-deleted IgG1 (Uni-G1) bispecific molecule with H435A mutation in only one Fab arm is slightly It is low efficiency. T cell-mediated cytotoxicity of AU565 cells by N297Q variants of Her2xCD3 and Her2xCD3 bispecific antibodies. T cell-mediated cytotoxicity of AU565 cells by N297Q variants of Her2xCD3 and Her2xCD3 bispecific antibodies.

Detailed Description of the Invention
Definitions The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of low molecular weight light (L) chains and one pair of heavy (H) chains, All four of these are linked to each other by disulfide bonds. The structure of immunoglobulins is well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain is typically comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is typically composed of three domains, CH1, CH2 and CH3. The heavy chains are linked to each other by disulfide bonds in the so-called "hinge region". Each light chain is typically composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is typically composed of one domain, CL. Typically, the numbering of amino acid residues in the constant region is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) As per the EU index. Figure 16 shows a schematic of EU and Kabat numbering for different isotype types of antibody 2F8 (WO 02/100348). The VH and VL regions are hypervariable regions (or sequences and / or structurally also referred to as complementarity determining regions (CDRs) mediated by more conserved regions referred to as framework regions (FR) It can be further subdivided into hypervariable regions that can be hypervariable in the form of defined loops. Each VH and VL is typically composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)).

  As used herein, the term "Fab arm" refers to one heavy chain-light chain pair.

  As used herein, the term "Fc region" refers to an antibody region comprising at least a hinge region, a CH2 domain and a CH3 domain.

  The term "antibody" (Ab) in the context of the present invention is at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours (h), for a significant period of time, such as about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, or any other relevant, functionally A defined period of time (such as sufficient time to induce, enhance, enhance and / or modulate the physiological response associated with antibody binding to antigen, and / or sufficient time for the antibody to mobilize effector activity, etc.) An immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any of them having a half life and having the ability to specifically bind an antigen under typical physiological conditions. The heavy and light chain variable regions of immunoglobulin molecules contain binding domains that interact with the antigen. The constant region of the antibody (Ab) contains various tissues of the immune system (such as effector cells) and components of the host system or components of the complement system such as C1q, which is the first component of the classical pathway of complement activation. It may mediate the binding of the immunoglobulin to the factor. The antibodies may also be bispecific antibodies, bispecific antibodies, or similar molecules. The term "bispecific antibody" refers to an antibody that has specificity for at least two different epitopes, typically non-overlapping epitopes. As mentioned above, the term antibody herein includes fragments of the antibody which retain the ability to specifically bind to the antigen unless otherwise stated or clearly not in context. Such fragments can be provided by any known technique, such as enzymatic cleavage, peptide synthesis and recombinant expression techniques. It has been shown that the antigen binding function of antibodies can be performed by fragments of full length antibodies, such as F (ab ') 2 fragments. The term antibody is also to be understood to include antibody-like polypeptides such as polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, unless otherwise stated. Antibodies generated in this manner can have any isotype.

  The term "full-length antibody" as used herein refers to an antibody comprising all heavy and light chain constant and variable domains normally found in antibodies of that isotype.

  As used herein, “isotype” refers to the immunoglobulin class (eg, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by heavy chain constant region genes.

  The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may be introduced amino acid residues not encoded by human germline immunoglobulin sequences (eg, introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo (Mutation) can be included. However, the term "human antibody" as used herein is intended to mean that the CDR sequences derived from the germline of another mammalian species, such as mouse, do not include antibodies grafted onto human framework sequences. Be done.

  As used herein, the term "heavy chain antibody" refers to an antibody consisting of only two heavy chains and lacking the two light chains normally found in antibodies. For example, heavy chain antibodies naturally occurring in camelids can bind antigens despite having only a VH domain.

  The term "epitope" refers to a protein determinant capable of specifically binding to an antibody. Epitopes usually consist of surface groups of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former is lost in the presence of denaturing solvents but binding to the latter is not. Epitopes are amino acid residues that are directly involved in binding (also referred to as immunodominant components of the epitope), and others that are not directly involved in binding, such as amino acid residues that are effectively blocked by specific antigen binding peptides. It may comprise an amino acid residue (ie, this amino acid residue is within the footprint of a specific antigen binding peptide).

As used herein, the term "binding" in the context of binding of an antibody to a given antigen typically refers to surface plasmons in a BIAcore 3000 device, for example, using the antigen as a ligand and the antibody as an analyte. About 10 ⁻⁶ M or less, such as about 10 ⁻⁷ M or less, such as about 10 ⁻⁸ M or less, such as about 10 ⁻⁹ M or less, as determined by resonance (SPR) techniques about 10 ^-10 M or less, or a bond of about 10 ^-11 M or even affinity corresponding to less K _D, nonspecific antigens other than the antigen of a given antigen or a closely related (e.g., At least 10 times lower, eg at least 100 times lower, eg at least 1000 times lower, eg at least 10,000 times lower, eg at least 100,000 times lower than the affinity of the binding for BSA, casein) With an affinity corresponding to have K _D, binding to a predetermined antigen. The amount with which the affinity is lower is dependent on K _D of an antibody, therefore a very low K _D of an antibody (i.e., the antibody is highly specific), the affinity affinity for antigen relative to non-specific antigen Amounts less than sex may be at least 10,000 times. The term "K _D " (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

  As used herein, the term "heterodimer interaction between the first CH3 region and the second CH3 region" refers to the first CH3 / second CH3 heterodimer protein. Refers to the interaction between the first CH3 region and the second CH3 region.

  As used herein, the term "homodimeric interaction of the first CH3 region and the second CH3 region" refers to the first in the first CH3 / first CH3 homodimeric protein. Interaction between the CH3 domain and another first CH3 domain, as well as between a second CH3 domain and another second CH3 domain in a second CH3 / second CH3 homodimer protein It is an interaction.

  An "isolated antibody" as used herein is that the material is removed from its original environment (eg, the natural environment if naturally occurring or the host cell if recombinantly expressed) Means It is also convenient for the antibody to be in purified form. The term "purified" does not require absolute purity, but rather is a relative definition indicating an increase in antibody concentration relative to the concentration of contaminants in the composition as compared to the starting material. Intended.

  The term "host cell" as used herein is intended to refer to an expression vector, eg, a cell into which an expression vector encoding an antibody of the present invention has been introduced. Recombinant host cells include transfectomas, such as, for example, CHO cells, HEK 293 cells, NS / 0 cells, and lymphocyte cells.

  As used herein, the term "co-expression" of two or more nucleic acid constructs refers to the expression of two constructs in a single host cell.

  The term "tumor cell protein" refers to a protein located on the cell surface of a tumor cell.

  As used herein, the term "effector cells" refers to immune cells involved in the effector phase of an immune response, as opposed to the recognition and activation phases of an immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (such as B cells and T cells including cytotoxic T cells (CTLs)), killer cells, natural killer cells, macrophages, single cells. Included are polymorphonuclear cells such as spheres, eosinophils, neutrophils, granulocytes, mast cells and basophils. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells can induce ADCC, such as natural killer cells that can induce antibody dependent cellular cytotoxicity (ADCC). In some embodiments, effector cells can phagocytose target antigen or target cells.

  The terms "reducing conditions" or "reducing environment" refer to conditions or environments in which cysteine residues in the hinge region of the substrate, as referred to herein, are more likely to be in a reduced state than oxidized.

  The term "disulfide bond isomerization" refers to the exchange of disulfide bonds between different cysteines, ie the reorganization of disulfide bonds.

Further Aspects and Embodiments of the Invention As mentioned above, in a first aspect, the invention relates to an in vitro method for producing a heterodimeric protein comprising the steps of:
a) providing a first homodimeric protein comprising an Fc region of an immunoglobulin, wherein the Fc region comprises a first CH3 region,
b) providing a second homodimeric protein comprising an immunoglobulin Fc region, wherein the Fc region comprises a second CH3 region,
Here, the sequences of the first CH3 region and the second CH3 region are different, and the heterodimeric interaction between the first CH3 region and the second CH3 region is the first CH3 region and Appear to be stronger than each of the homodimeric interactions of the two CH3 domains,
c) incubating the first protein with the second protein under reducing conditions sufficient to allow cysteines in the hinge region to cause disulfide bond isomerization, and
d) obtaining said heterodimeric protein.

  The bispecific format can be used in many ways to generate the desired combination of bispecific antibodies. In addition to being able to combine antibodies that target different antigens in a very selective manner, it is used to alter the desired properties by combining two different antibodies that target the same antigen, eg CDC Can be increased. In addition, it can be used to eliminate the partial agonist activity of an antagonist antibody, or convert the agonist antibody to an antagonist antibody by producing the bispecific antibody with an unrelated (inactive) antibody be able to.

  In one embodiment, the homodimeric protein comprises a fusion protein comprising an Fc region, such as (i) an Fc region, (ii) an antibody, (iii) a receptor, an Fc region fused to a cytokine or a hormone, and (iv) selected from the group consisting of Fc regions conjugated to prodrugs, peptides, drugs or toxins.

  In some embodiments, the first and / or second homodimeric protein comprises, in addition to the Fc region, one of the other regions of the antibody, ie the CH1 region, the VH region, the CL region and / or the VL region. Or include multiple or all. Thus, in one embodiment, the first homodimeric protein is a full-length antibody. In another embodiment, the second homodimeric protein is a full-length antibody.

  In important embodiments, the first and second homodimeric proteins are both antibodies, preferably full length antibodies, which bind to different epitopes. In such embodiments, the heterodimeric protein produced is a bispecific antibody. The epitopes can be located on different antigens or on the same antigen.

  However, in other embodiments, only one of the homodimeric proteins is a full-length antibody and the other homodimeric protein is not a full-length antibody, eg, another protein such as a receptor, a cytokine or a hormone or A variable region-free Fc region that is expressed with the peptide sequence or conjugated to a prodrug, peptide, drug or toxin. In further embodiments, none of the homodimeric proteins are full-length antibodies. For example, either homodimeric protein may be an Fc region fused to another protein or peptide sequence such as a receptor, cytokine or hormone, or bound to a prodrug, peptide, drug or toxin Can.

  In one embodiment, the Fc region of the first homodimeric protein is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 and the Fc region of the second homodimeric protein Is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. In a preferred embodiment, the Fc regions of both the first homodimeric protein and the second homodimeric protein are of the IgG1 isotype. In another preferred embodiment, one of the Fc regions of the homodimeric protein is of the IgG1 isotype and the other is of the IgG4 isotype. In the latter embodiment, the resulting heterodimer comprises an Fc region of IgG1 and an Fc region of IgG4 and thus may have interesting intermediate properties with respect to activating effector function. If the first and / or second homodimeric protein contains a mutation that removes the acceptor site for Asn-linked glycosylation, or is otherwise engineered to alter glycosylation properties, similar The product of

  In a further embodiment, for example by the addition of a compound to the medium during antibody production, as described for example in US2009317869 or in van Berkel et al. (2010) Biotechnol. Bioeng. 105: 350, or for example for example Yamane-Ohnuki et al (2004) Biotechnol. Bioeng 87: 614, use FUT8 knockout cells to glycoengineer one or both of the homodimeric proteins to reduce fucose and thus enhance ADCC. . Alternatively, ADCC can be optimized using the method described by Umana et al. (1999) Nature Biotech 17: 176.

  In a further embodiment, one or both of the homodimeric proteins are engineered to enhance complement activation, as described, for example, in Natsume et al. (2009) Cancer Sci. 100: 2411.

  In a further embodiment, one or both of the homodimeric proteins are engineered to reduce or increase binding to the neonatal Fc receptor (FcRn) to manipulate the serum half-life of the heterodimeric protein .

  In a further embodiment, one of the homodimeric starting proteins is engineered such that it does not bind to protein A, thus passing a product through a protein A column to obtain a heterodimeric amount from said homodimeric starting protein It becomes possible to separate body proteins. This may be particularly useful in embodiments where an excess of one homodimeric protein is used relative to the other homodimeric protein as a starting material. In such embodiments, it may be useful to engineer the homodimeric protein in excess to loosen its ability to bind protein A. After the heterodimerization reaction, the heterodimeric protein can then be separated from excess non-exchanged homodimeric protein by passing over a protein A column.

  In a further embodiment, one of the homodimeric proteins is a full-length antibody that recognizes an Fc region or an irrelevant epitope or a full-length antibody that does not receive somatic hypermutation and contains germline derived sequences that do not bind self-antigens. . In such embodiments, the heterodimeric protein functions as a monovalent antibody. In another embodiment, both homodimeric proteins contain the same heavy chain, but only one of the homodimeric proteins contains a light chain which forms a functional antigen binding site with said heavy chain, One homodimeric protein contains a nonfunctional light chain that does not bind to any antigen in combination with the heavy chain. In such embodiments, the heterodimeric protein functions as a monovalent antibody. Such nonfunctional light chains can be, for example, germline derived sequences that have not undergone somatic hypermutation and do not bind to self antigens.

  The antibodies used as homodimeric starting material of the invention may be produced, for example, by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or produced by recombinant DNA methods May be Monoclonal antibodies may also be phage antibody libraries, for example, using the techniques described in Clackson et al., Nature 352, 624 628 (1991) and Marks et al., J. Mol. Biol. 222, 581 597 (1991). It may be isolated from Monoclonal antibodies may be obtained from any suitable source. Thus, for example, the monoclonal antibody may be from a hybridoma prepared from murine spleen B cells obtained from a mouse immunized with the antigen of interest, eg in the form of cells expressing the antigen on the surface, or of interest It may be obtained in the form of a nucleic acid encoding an antigen of Monoclonal antibodies may also be obtained from immunized human or hybridomas derived from antibody-expressing cells of non-human mammals such as rats, dogs, primates and the like.

  The antibodies used as homodimeric starting material of the invention may be, for example, chimeric antibodies or humanized antibodies. In another embodiment, except for any particular mutations, one or both of the homodimeric starting proteins is a human antibody. Human monoclonal antibodies can be generated using transgenic mice or chromosome transfer mice, such as HuMAb mice, which possess portions of the human immune system rather than the mouse immune system. HuMAb mice contain human immunoglobulin gene minilocus that encode unrearranged human heavy chain (μ and γ) and 鎖 light chain immunoglobulin sequences, as well as targeted mutations that inactivate the endogenous μ and 鎖 chains. (Lonberg, N. et al., Nature 368, 856 859 (1994)). Thus, mice show reduced expression of mouse IgM or kappa, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation, resulting in high affinity human IgG. , kappa monoclonal antibody (Lonberg, N. et al. (1994), supra: Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and commentary in Harding, F. and Lonberg, N. Ann. NY Acad. Sci 764 536-546 (1995)). Preparation of HuMAb mice can be performed by Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996) In detail. Similarly, U.S. Pat. No. 5,545,806, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,633,425, U.S. 5,789,650, U.S. Pat. See WO 92/22645, WO 92/03918, and WO 01/09187. Splenocytes from these transgenic mice can be used to produce hybridomas that secrete human monoclonal antibodies by known techniques.

  In addition, human antibodies of the invention or antibodies of the invention from other species include phage display, retroviral display, ribosome display, mammalian display, and other techniques using techniques well known in the art. And the resulting molecule can be subjected to further maturation, such as affinity maturation, as the techniques of maturation are well known in the art.

  In a further aspect of the invention, the antibody or part thereof, eg one or more of the CDRs, see WO 2010 0012 51, but of a species of cartilaginous fish, such as those of the family Camelidae, or sharks. Or heavy chain or domain antibody.

  In one embodiment of the method of the invention, the first and second homodimeric proteins provided in steps a) and b) are purified.

  In one embodiment, the first and / or second homodimeric protein is attached to or contains an acceptor group for the drug, prodrug or toxin. Such acceptor group may, for example, be a non-naturally occurring amino acid.

  As mentioned above, the sequences of the first and second CH3 regions of the homodimeric starting protein are different and the heterodimer interaction between the first CH3 region and the second CH3 region is said It is likely to be stronger than each of the homodimeric interactions of one CH3 region and a second CH3 region.

  In one embodiment, the increase in the strength of the heterodimeric interaction in comparison to each of the homodimeric interactions is by CH3 modification other than the introduction of a covalent bond, a cysteine residue or a charged residue .

  In some embodiments, the products of the invention are very stable and do not undergo Fab arm exchange in vivo under mild reducing conditions in vitro or, importantly, upon administration to humans. Thus, in one embodiment, the heterodimeric interaction between the first protein and the second protein in the resulting heterodimeric protein is 0.5 mM under the conditions described in Example 13 It is such that Fab arm exchange can not occur in GSH.

  In another embodiment, the heterodimeric interaction between the first protein and the second protein in the resulting heterodimeric protein is carried out in vivo in mice under the conditions described in Example 14 It is such that Fab arm exchange does not occur.

  In another embodiment, the heterodimeric interaction between the first protein and the second protein in the resulting heterodimeric protein is, for example, 2 as determined as described in Example 30. Of the two homodimeric interactions, it is more than 2 times stronger, for example more than 3 times stronger, for example more than 5 times stronger than the strongest one.

  In a further embodiment, the sequences of the first CH3 region and the second CH3 region, when assayed as described in Example 30, result in first and second proteins in the resulting heterodimeric protein And the dissociation constant of the heterodimer interaction between is less than 0.05 micromolar.

  In a further embodiment, the sequences of the first CH3 region and the second CH3 region have a dissociation constant of both homodimer interactions of greater than 0.01 micromolar when assayed as described in Example 21. For example, more than 0.05 micromole, preferably 0.01 to 10 micromole, for example 0.05 to 10 micromole, more preferably 0.01 to 5 micromole, for example 0.05 to 5 micromole, still more preferably 0.01 to 1 micromole, for example 0.05 Such as to be 1 micromolar, 0.01 to 0.5 micromolar or 0.01 to 0.1 micromolar. In aspects where the homodimeric starting protein is relatively stable, it can have the advantage of being easier to produce large amounts of starting protein and to avoid, for example, aggregation or misfolding.

  In some embodiments, stable heterodimeric proteins can be prepared using the methods of the invention based on two homodimeric starting proteins that contain asymmetric mutations that are only slightly conserved in the CH3 region. It can be obtained in high yield.

  Thus, in one embodiment, the sequences of the first CH3 region and the second CH3 region comprise amino acid substitutions at non-identical positions.

  Amino acid substituents may be naturally occurring amino acids or non-naturally occurring amino acids. Examples of unnatural amino acids are, for example, Xie J and Schultz PG, Current Opinion in Chemical Biology (2005), 9: 548-554, and Wang Q. et al., Chemistry & Biology (2009), 16: 323-336. Is disclosed in

  In one embodiment, the amino acid is a naturally occurring amino acid.

  In one embodiment, in comparison to the wild type CH3 region, the first homodimeric protein has only one amino acid substitution in the CH3 region and the second homodimeric protein is in the CH3 region. Have only one amino acid substitution.

  In one embodiment, the first homodimeric protein has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407 and 409 and a second homodimer The protein has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407 and 409, wherein said first homodimeric protein and said second homodimeric protein The monomeric proteins are not substituted at the same position.

  In one embodiment, the first homodimeric protein has an amino acid substitution at position 366 and the second homodimeric protein is selected from the group consisting of 368, 370, 399, 405, 407 and 409 Have an amino acid substitution at the In one embodiment, the amino acid at position 366 is selected from Arg, Lys, Asn, Gln, Tyr, Glu and Gly.

  In one embodiment, the first homodimeric protein has an amino acid substitution at position 368 and the second homodimeric protein is selected from the group consisting of 366, 370, 399, 405, 407 and 409 Have an amino acid substitution at the

  In one embodiment, the first homodimeric protein has an amino acid substitution at position 370 and the second homodimeric protein is selected from the group consisting of 366, 368, 399, 405, 407 and 409 Have an amino acid substitution at the

  In one embodiment, the first homodimeric protein has an amino acid substitution at position 399 and the second homodimeric protein is selected from the group consisting of 366, 368, 370, 405, 407 and 409 Have an amino acid substitution at the

  In one embodiment, the first homodimeric protein has an amino acid substitution at position 405 and the second homodimeric protein is selected from the group consisting of 366, 368, 370, 399, 407 and 409 Have an amino acid substitution at the

  In one embodiment, the first homodimeric protein has an amino acid substitution at position 407 and the second homodimeric protein is selected from the group consisting of 366, 368, 370, 399, 405 and 409 Have an amino acid substitution at the

  In one embodiment, the first homodimeric protein has an amino acid substitution at position 409 and the second homodimeric protein is selected from the group consisting of 366, 368, 370, 399, 405 and 407. Have an amino acid substitution at the

  Thus, in one embodiment, the sequences of the first CH3 region and the second CH3 region are asymmetric mutations, ie mutations at different positions in the two CH3 regions, eg a mutation at position 405 in one of the CH3 regions and the other And a mutation at position 409 in the CH3 region of

  In one embodiment, the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has 366, 368, 370, 399, 405 and 407. Having an amino acid substitution at a position selected from the group consisting of

  In one such embodiment, the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein has an amino acid other than Phe at position 405 . In this further embodiment, the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein has an amino acid other than Phe, Arg or Gly at position 405 Have.

  In another embodiment, the first homodimeric protein comprises an amino acid other than Phe at position 405 and Lys, Leu or Met at position 409, and the second homodimeric protein is an amino acid other than Phe at position 405 and At position 409 contains Lys. In this further embodiment, the first homodimeric protein comprises an amino acid other than Phe at position 405 and Lys, Leu or Met at position 409, and the second homodimeric protein at position 405 is Phe, Arg or Gly. Amino acids other than and Lys at position 409.

  In another embodiment, the first homodimeric protein comprises an amino acid other than Phe at position 405 and Lys, Leu or Met at position 409 and the second homodimeric protein is at Leu and Leu at position 405 Contains Lys. In this further embodiment, the first homodimeric protein comprises Phe at position 405 and Arg at position 409 and the second homodimeric protein is an amino acid other than Phe, Arg or Gly at position 405 and at position 409 Contains Lys.

  In another embodiment, the first homodimeric protein comprises Phe at position 405 and Arg at position 409, and the second homodimeric protein comprises Leu at position 405 and Lys at position 409.

  In a further embodiment, the first homodimeric protein comprises an amino acid other than Lys, Leu or Met at position 409, the second homodimeric protein is Lys at position 409, Thr at position 370 and Leu at position 405. including.

  In a further embodiment, the first homodimeric protein comprises Arg at position 409, and the second homodimeric protein comprises Lys at position 409, Thr at position 370 and Leu at position 405.

  In a further embodiment, the first homodimeric protein comprises Lys at position 370, Phe at position 405 and Arg at position 409, and the second homodimeric protein is Lys at position 409, Thr at position 370 and a position 405 contains Leu.

  In another embodiment, the first homodimeric protein comprises an amino acid other than Lys, Leu or Met at position 409, the second homodimeric protein a Lys at position 409 and: a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

  In another embodiment, the first homodimeric protein comprises Arg at position 409 and the second homodimeric protein is Lys at position 409 and: a) Ile at position 350 and Leu at position 405, or b ) Including Thr at position 370 and Leu at position 405.

  In another embodiment, the first homodimeric protein comprises Thr at position 350, Lys at position 370, Phe at position 405 and Arg at position 409, and the second homodimeric protein comprises Lys at position 409 and a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

  In another embodiment, the first homodimeric protein comprises Thr at position 350, Lys at position 370, Phe at position 405 and Arg at position 409, and the second homodimeric protein is Ile at position 350, It contains Thr at position 370, Leu at position 405 and Lys at position 409.

  In another embodiment, the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein has Tyr, Asp, Glu, Phe, Lys at position 407 , Gln, Arg, Ser or any amino acid other than Thr.

  In another embodiment, the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has Ala, Gly, His, Ile, Leu at position 407. , Met, Asn, Val or Trp.

  In another embodiment, the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein at Gly, Leu, Met, Asn or Trp at position 407 Have.

  In another embodiment, the first homodimeric protein has an amino acid other than Lys, Leu or Met at Tyr at position 407 and at position 409, and the second homodimeric protein at Tyr, Asp, at position 407 Has an amino acid other than Glu, Phe, Lys, Gln, Arg, Ser or Thr and Lys at position 409.

  In another embodiment, the first homodimeric protein has an amino acid other than Lys, Leu or Met at Tyr at position 407 and at position 409, and the second homodimeric protein is Ala, Gly at position 407, Has His, Ile, Leu, Met, Asn, Val or Trp and Lys at position 409.

  In another embodiment, the first homodimeric protein has Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has Gly, Leu, at position 407, It has Met, Asn or Trp and Lys at position 409.

  In another embodiment, the first homodimeric protein has Tyr at position 407 and Arg at position 409 and the second homodimeric protein has Tyr, Asp, Glu, Phe, Lys, Gln at position 407. , Arg, Ser or Amino acid other than Thr and Lys at position 409.

  In another embodiment, the first homodimeric protein has Tyr at position 407 and Arg at position 409 and the second homodimeric protein has Ala, Gly, His, Ile, Leu, Met at position 407. , Asn, Val or Trp and Lys at position 409.

  In another embodiment, the first homodimeric protein has Tyr at position 407 and Arg at position 409, and the second homodimeric protein has Gly, Leu, Met, Asn or Trp and position at position 407. It has Lys at 409.

In one embodiment, the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein is
(i) an amino acid other than Phe, Leu and Met at position 368, or
(ii) Trp at position 370, or
(iii) has an amino acid at position 399 other than Asp, Cys, Pro, Glu or Gln.

In one embodiment, the first homodimeric protein has Arg, Ala, His or Gly at position 409 and the second homodimeric protein is
(i) Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val or Trp at position 368, or
(ii) Trp at position 370, or
(iii) Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399.

In one embodiment, the first homodimeric protein has an Arg at position 409 and the second homodimeric protein is
(i) Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val or Trp at position 368, or
(ii) Trp at position 370, or
(iii) Phe, His, Lys, Arg or Tyr at position 399
Have.

  In addition to the amino acid substitutions specified above, the first and second homodimeric proteins can comprise further amino acid substitutions, deletions or insertions relative to the wild type Fc sequence.

In a further embodiment, the first and second CH3 regions, except for the indicated mutations, comprise the sequence set forth in SEQ ID NO: 1 (IgG1m (a)).

In a further embodiment, the first and second CH3 regions, except for the indicated mutations, comprise the sequence set forth in SEQ ID NO: 2 (IgG1m (f)).

In a further embodiment, the first and second CH3 regions, except for the indicated mutations, comprise the sequence set forth in SEQ ID NO: 3 (IgG1m (ax)).

  In a further embodiment, the provided homodimeric protein is a rat and mouse antibody that exhibits favorable pairing as described by Lindhofer et al. (1995) J Immunol 155: 21 (see above), Or may be so-called knob in hole variant antibodies as described in US Pat. No. 5,731,168 (see above). However, in some cases, the latter homodimeric starting protein may be more difficult to produce due to the very weak homodimeric CH3-CH3 interaction. Thus, variants described herein having mutations at positions 350, 370, 405 and 409 may be preferred.

  The sequence of the hinge region of the homodimeric starting protein may vary. However, the resulting heterodimeric protein may be more stable under certain circumstances if the hinge region is not IgG4-like, and preferably if it is IgG1-like.

  Thus, in one embodiment, neither the first homodimeric protein nor the second homodimeric protein comprises a Cys-Pro-Ser-Cys sequence in the (core) hinge region.

  In a further embodiment, both the first homodimeric protein and the second homodimeric protein comprise a Cys-Pro-Pro-Cys sequence in the (core) hinge region.

  In many embodiments where the first and second homodimeric proteins are antibodies, the antibody further comprises a light chain. As explained above, the light chains may be different, ie different in sequence, each forming a functional antigen binding domain with only one of the heavy chains. However, in another embodiment, the first and second homodimeric proteins are heavy chain antibodies, which do not require a light chain for antigen binding, see, eg, Hamers-Casterman (1993) Nature 363: See 446.

  As mentioned above, step c) of the method of the invention comprises subjecting the first protein to a second under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization. Incubate with the protein. Examples of suitable conditions are given herein. The minimum requirements for cysteine in the hinge region to undergo disulfide bond isomerization may differ depending on the homodimeric starting protein, in particular depending on the exact sequence in the hinge region. Each homodimeric interaction of the first and second CH3 regions is sufficiently weak to allow cysteines in the hinge region to undergo isomerization of disulfide bonds under given conditions is important.

  In one embodiment, the reducing conditions in step c) include reducing agents such as 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris (2-carboxyethyl) phosphine (TCEP), a reducing agent selected from the group consisting of L-cysteine and β-mercaptoethanol, preferably a reduction selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine Includes the addition of agents.

In one aspect, reduction conditions that allow for controlled Fab arm exchange are described in terms of the required redox potential. The tripeptide glutathione (GSH) is the major low molecular weight thiol in cells and controls the thiol disulfide redox state, which is essential for normal redox signaling in vivo. Kinetics of cellular redox balance are achieved by maintaining the thiol versus disulfide state of reduced GSH and its oxidized GSSG. The value of reduction potential can be measured as in Rost and Rapoport, Nature 201: 185 (1964) and Aslund et al., J. Biol. Chem. 272: 30780-30786 (1997). The redox potential E _h considering the stoichiometry of two GSHs oxidized per GSSG is a quantitative measure of the redox state. E _h is calculated by the Nernst equation: E _h = E _o + (RT / nF) ln ([GSSG (ox)] / [GSH (red)] ² ). E o is the standard potential of the redox couple at a defined pH, R is the gas constant, T is the absolute temperature, F is the Faraday constant, and n is the number of electrons transferred. The in vivo estimates for the Eh of the GSH / GSSG pair are in the range of -260 to -200 mV (Aw, T., News Physiol. Sci. 18: 201-204 (2003)). As a result, the terminally differentiated cells maintain Eh at around -200 mV, while actively proliferating cells maintain a further reduced Eh at around -260 mV.

  The standard redox potential of DTT is -330 mV (Cleland et al. Biochemistry 3: 480-482 (1964)). TCEP has been shown to reduce DTT in solution, and therefore has a redox potential more negative than DTT. However, the correct value has not been reported. The reducing conditions enabling controlled Fab arm exchange conditions can therefore be described in terms of the required redox potential Eh, which is achieved under normal plasma conditions in vivo The value is optimally below the redox potential above which reduces antibody disulfide bonds located in the hinge region and other than those involved in the formation of heavy chain interchain disulfide bonds.

  Thus, in a further embodiment, step c) is less than -50 mV, such as less than -150 mV, preferably -150 to -600 mV, such as -200 to -500 mV, more preferably -250 to -450 mV For example, under reducing conditions with redox potential ranging from -250 to -400 mV, even more preferably from -260 to -300 mV.

  In a further embodiment, step c) comprises incubation for at least 90 minutes at a temperature of at least 20 ° C. in the presence of at least 25 mM 2-mercaptoethylamine or in the presence of at least 0.5 mM dithiothreitol. The incubation can be performed at a pH of 5 to 8, for example at pH 7.0 or at pH 7.4.

  In a further embodiment, step d) comprises returning the conditions to be non-reducing conditions or low reducing conditions, for example by removal of the reducing agent, for example by desalting.

  In some embodiments, the methods of the invention produce antibody products in which more than 80%, such as more than 90%, such as more than 95%, such as more than 99%, of the antibody molecules are the desired bispecific antibodies.

  Post-production techniques are more flexible and easier to control than prior art methods based on co-expression.

  By virtue of the post-production technology of producing bispecific antibodies by Fab exchange under reducing conditions (such as by the addition of 2-MEA) disclosed herein, this is a bispecific antibody It is a very suitable strategy for (high-throughput) screening of multiple combinations of specificities towards the discovery of In addition, in vitro steps can be performed in the laboratory that allow for higher control, adaptability and yield of heterodimeric proteins than possible by co-expression. A further advantage of this strategy is that screening can be done in the final therapeutic format, eliminating the need for genetic manipulation at lead selection.

  As explained above, in a further aspect, the method of the invention is for “matrix” screening, ie one set has the same first CH3 region and the other set is the same. It has a second CH3 region, the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is the first Based on the two sets of antibodies, which can be used to create a number of different combinations of binding specificities that are more likely to be stronger than each of the CH3 and second CH3 regions homodimeric interactions .

Thus, in one aspect, the present invention relates to a method for the selection of heterodimeric proteins having the desired properties, comprising the following steps:
a) providing a first set of homodimeric proteins comprising an Fc region, wherein the homodimeric proteins have the same first CH3 region,
b) providing a second set of homodimeric proteins comprising an Fc region, wherein the homodimeric proteins have identical second CH3 regions,
Here, the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is the first CH3 region and the second CH3 region. Seems to be stronger than each of the domain's homodimer interactions,
c) incubating the combination of the first and second set of homodimeric proteins under reducing conditions sufficient to allow cysteines in the hinge region to cause disulfide bond isomerization And thus creating a set of bispecific antibodies,
d) optionally returning the conditions to non-reducing conditions,
e) assaying the set of heterodimeric proteins obtained for a given desired property, and
f) selecting heterodimeric proteins with the desired properties.

In one embodiment, the invention relates to a method for the selection of bispecific antibodies having the desired properties, comprising the steps of:
a) providing a first set of homodimeric antibodies comprising antibodies with different variable regions, wherein the first set of antibodies comprises the same first CH3 region,
b) providing a second set of homodimeric antibodies comprising antibodies with different variable regions or the same variable region, wherein the second set of antibodies comprises the same second CH3 region,
Here, the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is the first CH3 region and the second CH3 region. Seems to be stronger than each of the domain's homodimer interactions,
c) incubating the combination of the first and second set of antibodies under reducing conditions sufficient to allow cysteines in the hinge region to cause isomerization of the disulfide bond, thus Creating a set of bispecific antibodies,
d) optionally returning the conditions to non-reducing conditions,
e) assaying the resulting set of bispecific antibodies for a given desired property, and
f) selecting bispecific antibodies with the desired properties.

  In one embodiment, the second set of homodimeric antibodies have different variable regions.

  In one embodiment, the second set of homodimeric antibodies have identical variable regions but different amino acid or structural changes outside the antigen binding region.

  The two sets can be configured in many different ways as desired. Thus, two sets may target the same or different epitopes on the same antigen. The two sets may also target different antigens, and each set may contain antibodies that bind to the same or different epitopes on the antigen in question. Furthermore, one or both sets may each contain antibodies that target different antigens.

  In another embodiment, the desired property is cell death, cell lysis, inhibition of cell proliferation, or binding to cells expressing both antigen targets.

  The screening strategy includes two panels of antibody vectors that contain varying ranges of specificity, where one panel is reduced (as by the addition of 2-MEA) with the backbone of another panel It is cloned into a backbone that can be involved in Fab arm exchange under conditions. For example, the first panel is cloned into the IgG1-F405L backbone and the second panel is cloned into the IgG1-K409R backbone (other possible backbone combinations are described in Examples 19, 28, 29, 30, See also 35, 36, 37, 38 and 39).

  Each member of the two panels of antibody vectors is then expressed individually on a small scale. For example, all antibody vectors are transiently transfected into HEK 293 cells and expressed in 2.3 mL of culture in 24 well plates. Alternatively, other suitable (small scale) production systems known in the art can be used.

  The expressed antibodies of the two antibody panels are then mixed in pairs in equal molar proportions. For example, all individual antibodies are purified by small scale protein A chromatography and antibody concentration is measured by absorbance at a wavelength of 280 nm. Alternatively, other suitable (small scale) purification methods for determining protein concentration known in the art can also be used. In another embodiment, the purification step can be omitted if the downstream application is not affected by the expression medium. The antibody concentration is then normalized to include an equimolar amount of both antibodies in the appropriate volume. For example, a panel of eight antibodies in the F405L backbone is eight in the K409R backbone such that 80 μg / mL of antibody A (F405L) and 80 μg / mL of antibody B (K409R) are contained in 100 μl of 64 mixtures. Mix individually with the species antibody. Alternatively, if the strategy involves downstream purification steps specific for bispecific antibodies, the step of normalizing antibody amounts can be omitted.

  To the mixture of antibodies, add an appropriate amount of reducing agent and incubate at the permissive temperature for an appropriate time. For example, 25 μl of 125 mM 2-MEA is added to 100 μl containing 80 μg / mL of antibody A (F405L) and 80 μg / mL of antibody B (K409R) (final concentration 25 mM 2-MEA), overnight at 25 ° C. Incubate.

  The reducing agent is then immediately removed from the mixture (which at this point contains the bispecific antibody) to promote the oxidation of the disulfide bond and avoid interference with the reducing agent in the screening assay. For example, 2-MEA is removed by performing a buffer exchange of 64 mixtures using Zeba Spin 96 well desalting plates (Pierce Biotechnology, # 89807). Alternatively, other methods suitable for removing reducing agents known in the art can be used.

  The bispecific antibodies are then characterized biochemically or functionally to identify lead candidates. For example, 64 bispecific antibodies are assayed for growth inhibition of the appropriate cell line or binding to the appropriate cell line. The identified lead candidates are then produced on a larger scale and characterized in more detail.

Production by Co-expression The heterodimeric proteins of the invention can also be obtained by co-expression in a single cell of constructs encoding the first and second polypeptides.

Thus, in a further aspect, the present invention relates to a method for producing a heterodimeric protein comprising the following steps:
a) providing a first nucleic acid construct encoding a first polypeptide comprising an immunoglobulin first Fc region, wherein the first Fc region comprises a first CH3 region,
b) providing a second nucleic acid construct encoding a second polypeptide comprising an immunoglobulin second Fc region, wherein the second Fc region comprises a first CH3 region,
Here, the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is the first CH3 region and the second CH3 region. And each of the first homodimeric proteins has an amino acid at position 409 other than Lys, Leu or Met, and the second homodimeric protein is more likely than each of the domain homodimeric interactions. The homodimeric protein of has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 407,
And / or where the sequences of the first and second CH3 regions are 0.01 to 10 microns when the dissociation constant of each homodimer interaction of the CH3 region is assayed as described in Example 21 Moles, for example 0.05 to 10 micromoles, more preferably 0.01 to 5 micromoles, for example 0.05 to 5 micromoles, even more preferably 0.01 to 1 micromoles, for example Such as being 0.1 micromolar,
c) co-expressing said first and second nucleic acid constructs in a host cell, and
d) obtaining the heterodimeric protein from cell culture.

  Suitable expression vectors, including promoters, enhancers and the like, and host cells suitable for the production of antibodies are well known in the art. Examples of host cells include yeast cells, such as CHO or HEK cells, bacterial cells and mammalian cells.

In one embodiment of this method, the first CH3 region has an amino acid other than Lys, Leu or Met at position 409, and the second CH3 region has an amino acid other than Phe at position 405,
And / or the sequences of the first and second CH3 regions have a dissociation constant of each homodimeric interaction of each of the CH3 regions, as assayed in the manner described in Example 21, from 0.01 to 10 micromolar, for example 0.05 to 10 micromole, more preferably 0.01 to 5 micromole, for example 0.05 to 5 micromole, even more preferably 0.01 to 1 micromole, for example 0.05 to 1 micromole, 0.01 to 0.5 micromole or 0.01 to 0.1 micromole It is like being.

In another aspect of this method:
The first CH3 region has an amino acid other than Lys, Leu or Met at position 409 and the second CH3 region has an amino acid other than Phe at position 405, such as an amino acid other than Phe, Arg or Gly at position 405 And
Or The first CH3 region has an amino acid other than Lys, Leu or Met at position 409 and the second CH3 region at Tyr 407 other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr It has an amino acid.

  In some embodiments, the first and second polypeptides are full-length heavy chains of two antibodies that bind to different epitopes (ie, the first and second nucleic acid constructs bind to two different epitopes) Encoding the full-length heavy chain of the antibody), thus the heterodimeric protein is a bispecific antibody. The bispecific antibody can be a heavy chain antibody, or the host cell can further express one or more nucleic acid constructs encoding a light chain. If only one light chain construct is coexpressed with the heavy chain construct, the functional bispecific antibody is such that the light chain sequence can form a functional antigen binding domain with each of the heavy chains It is only formed in the case. If two or more different light chain constructs are coexpressed with the heavy chain, multiple products will be formed.

  In a further aspect, the method of co-expression according to the invention comprises any of the additional features described under the in vitro method described above.

  In a further aspect, the invention relates to an expression vector comprising the first and the second nucleic acid construct as specified hereinabove. In a further aspect, the invention relates to a host cell comprising the first and the second nucleic acid construct as hereinbefore specified.

Heterodimeric Proteins In a further aspect, the present invention relates to heterodimeric proteins obtained or obtainable by the method of the present invention.

  Furthermore, the method of the present invention may be characterized as asymmetric molecules, molecules having different characteristics with respect to each of the Fab arms or with respect to each of the CH3 domains, or molecules with different modifications throughout the molecule, such as molecules with unnatural amino acid substitutions for binding. Enable the formation of Such asymmetric molecules can be produced in any suitable combination. This is further illustrated below by some non-limiting examples.

  Bispecific antibodies can be used to pre-target a target cell of interest, including but not limited to tumor cells. Pre-targeting of target cells could be used for imaging studies or for purposes of immunotherapy.

  In one embodiment of the method of the invention, the first Fab arm of the bispecific molecule is a tumor cell surface protein or tumor cell surface carbohydrate, such as one of the tumor cell surface proteins described herein. Such that the second Fab arm recognizes a radioactive effector molecule, including but not limited to a radiolabel, coupled or linked (via a chelating agent) to the peptide or hapten, such as . One example of such a radiolabeled peptide is indium labeled diethylene triamine pentaacetic acid (anti-DTPA (In) van Schaijk et al. Clin. Cancer Res. 2005; 11: 7230s-7126s). Another example is the use of nanoparticles of polymeric micelles carrying radionuclides such as technetium-99, hapten labeled colloidal particles such as liposomes (Jestin et al. QJ Nucl Med Mol Imaging 2007; 51: 51-60).

  In another embodiment, another cytostatic molecule coupled to a hapten such as a toxin is used.

  In a further embodiment of the method of the invention, the first Fab arm of the bispecific molecule is glycosylated at position N297 (EU numbering) and the second Fab arm of the bispecific molecule is not glycosylated (eg , N297 is mutated to Q or A or E mutations (Bolt S et al., Eur J Immunol 1993, 23: 403-411)). Asymmetric glycosylation in the Fc region affects the interaction with Fcγ-receptors and not only affects the interaction with other effector functional molecules like C1q, but also antibody dependent cellular cytotoxicity of antibodies Also affect (Ha et al., Glycobiology 2011, April 5).

  In another embodiment of the method of the invention, the first Fab arm of the bispecific molecule interacts with the neonatal Fc receptor FcRn (Roopenian DC, et al. Nat. Rev. Immunol. 2007, 7: 715- 725), the second Fab arm has impaired binding to FcRn, for example by mutation of the FcRn interaction site on the molecule, for example by creation of the H435A mutation (Shields, RL, et al, J Biol Chem, 2001, Firan , M., et al, Int Immunol, 2001).

  In another embodiment of the method of the present invention, the first Fab arm of the bispecific molecule is staphylococcal protein A (Protein A, Deisenhofer et al, Biochemistry 20, 2361-2370, which is commonly used for the purification of antibodies. (1981) and Streptococcus protein G (Protein G, Derrick et al., Nature 359, 752-754 (1992), and the second Fab arm of the bispecific molecule interacts with Protein A or G. As a result, removal of the amount of protein A or G bond-impaired homodimer remaining after exchange to heterodimer is a bispecific molecule using protein A or G. Can be easily obtained by purification of

  In another embodiment, binding to either Fcγ-receptor or FcRn is improved or reduced with one of the two Fab arms of the bispecific molecule.

  In another embodiment, binding to C1 q is improved or reduced in one of the two Fab arms of the bispecific molecule.

  In another embodiment, the protein is engineered to enhance complement activation in one or both of the two Fab arms of the molecule.

  In another embodiment, each of the Fab arms present in the bispecific molecule is from a different IgG subclass.

  In another embodiment, each of the Fab arms present in the bispecific molecule carries a different allotypic mutation (Jefferis & Lefranc, 2009, MABs 1: 332-8).

  In another embodiment, another class of asymmetric immunotherapeutic molecules are created by displacement of one Fab of the Fab arm of the bispecific molecule by an immunostimulatory, stimulatory or inhibitory cytokine. Non-limiting examples of such cytokines are IL-2, IFN-α, IFN-β, IFN-γ, TNF-α, G-CSF, GM-CSF, IL-10, IL-4, IL-6 , IL-13. Alternatively, (growth) factors or hormone stimulators or inhibitors are included in the molecule.

  In another embodiment, one Fab of the Fab arm comprises a cytolytic peptide, ie, but not limited to antibacterial peptides such as, but not limited to, magainin, melittin, cecropin, KLAKKLAK and variants thereof. Lysable peptides (Schweizer et al. Eur. J. Pharmacology 2009; 625: 190-194, Javadpour, J. Med. Chem., 1996, 39: 3107-3113, Marks et al, Cancer Res 2005; 65: 2373- 2377, Rege et al, Cancer Res. 2007; 67: 6368-6375) or a cationic cytolytic peptide (CLYP technology, US 2009/0269341).

  In another embodiment, one or both of the Fabs in the Fab arm is replaced by a receptor for cytokines and / or growth factors to create a so-called decoy receptor, among which, Enbrel® (targeting TNF-α) Etanercept) and VEGF-trap targeting VEGF are well known examples. By combining these two decoy receptors into one molecule, superior activity was shown as compared to a single decoy receptor (Jung, J. Biol. Chem. 2011; 286: 14410-14418). ).

  In another embodiment, another class of asymmetric immunotherapeutic molecules are generated by fusion of immunoreactive, stimulatory or inhibitory cytokines to the N-terminus or C-terminus of one or both of the Fab arms present in the bispecific molecule. Be done. This can have a positive effect on the anti-tumor activity of the bispecific molecule. However, examples of such molecules, not limited to the following list, are IL-2 (Fournier et al., 2011, Int. J. Oncology, doi: 10.3892 / ijo. 2011. 976), IFN-α, IFN-β Or IFN-γ (Huan et al., 2007; J. Immunol. 179: 6881-6888, Rossie et al., 2009; Blood 114: 3864-3871), TNF-α. Alternatively, N-terminal or C-terminal fusion of cytokines, eg G-CSF, GM-CSF, IL-10, IL-4, IL-6 or IL-13, plus bispecific antibody molecule effector function Can affect the Alternatively, a (growth) factor or hormone stimulator or inhibitor is included in the molecule at the N-terminus or C-terminus.

  In another embodiment, cytolytic peptides (Schweizer et al. Eur. J. Pharmacology 2009; 625, such as antibacterial peptides such as, for example, magainin, melittin, cecropin, KLAKKLAK and variants thereof, in one or both of the Fab arms. 190-194, Javadpour, J. Med. Chem., 1996, 39: 3107-3113, Marks et al, Cancer Res 2005; 65: 2373-2377, Rege et al, Cancer Res. 2007; 67: 6368-6375. Or N-terminal or C-terminal fusion of cationic cytolytic peptides (CLYP technology, US 2009/0269341) may enhance the activity of the molecule.

  In another embodiment, another class of asymmetric immunotherapeutic molecules are monovalent antibodies, ie, molecules that interact with the target of choice in one Fab arm. In such molecules, one of the Fab arms present in the bispecific molecule is generated against the target molecule of choice, and the other Fab arm of the molecule does not possess Fab or MetMab (Genentech; WO 96 / With non-binding / non-functional Fab as described for 38 557). Alternatively, monomeric Fc fusion proteins (Peters et al., Blood 2010; 115: 2057-2064), such as those described for Factor VIII and Factor IX can be generated.

  Alternatively, combinations of any of the above described asymmetric molecules can be made by the methods of the present invention.

In a further aspect, the present invention provides a first polypeptide comprising a first Fc region of an immunoglobulin, wherein the first Fc region comprises a first CH3 region, and a second Fc region a second CH3 region A heterodimeric protein comprising a second polypeptide comprising a second Fc region of an immunoglobulin, wherein the sequences of said first and second CH3 regions are different, The heterodimeric interaction between the second CH3 region is such that it is stronger than each of the homodimeric interactions of the first CH3 region and the second CH3 region, and wherein The first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein is from the group consisting of 366, 368, 370, 399, 405 and 407 Have an amino acid substitution at the selected position,
And / or where the sequences of the first and second CH3 regions are 0.01 to 10 microns when the dissociation constant of each homodimer interaction of the CH3 region is assayed as described in Example 21. Moles, for example 0.05 to 10 micromoles, more preferably 0.01 to 5 micromoles, for example 0.05 to 5 micromoles, even more preferably 0.01 to 1 micromoles, for example 0.05 to 1 micromole, 0.01 to 0.5 micromole or 0.01 to It is such that it is 0.1 micromolar.

In one embodiment, the first CH3 region has an amino acid other than Lys, Leu or Met at position 409, and the second CH3 region has an amino acid other than Phe at position 405,
And / or the sequences of the first and second CH3 regions have a dissociation constant of each homodimeric interaction of each of the CH3 regions, as assayed in the manner described in Example 21, from 0.01 to 10 micromolar, for example 0.05 to 10 micromole, more preferably 0.01 to 5 micromole, for example 0.05 to 5 micromole, even more preferably 0.01 to 1 micromole, for example 0.05 to 1 micromole, 0.01 to 0.5 micromole or 0.01 to 0.1 micromole It is like being.

In a further embodiment of the heterodimeric protein,
The first CH3 region has an amino acid other than Lys, Leu or Met at position 409, and the second CH3 region has an amino acid other than Phe at position 405, such as other than Phe, Arg or Gly at position 405 ,
Or The first CH3 region has an amino acid other than Lys, Leu or Met at position 409 and the second CH3 region at Tyr 407 other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr It has an amino acid.

  In a further aspect, the heterodimeric protein according to the invention comprises any of the above mentioned additional features for a method of production.

  Thus, in a further embodiment of the heterodimeric protein of the invention, the first polypeptide is an antibody, preferably a full-length heavy chain of a human antibody.

  In another embodiment of the heterodimeric protein of the invention, the second polypeptide is an antibody, preferably a full-length heavy chain of a human antibody.

  In a further embodiment of the heterodimeric protein of the invention, the first and second polypeptides are both full length heavy chains of two antibodies, preferably both human antibodies binding to different epitopes, thus obtaining the hetero The dimeric protein is a bispecific antibody. The bispecific antibody may be a heavy chain antibody, or an antibody comprising two full length light chains which may be the same or different in addition to the heavy chain.

  In a further embodiment of the heterodimeric protein of the invention, the Fc region of the first polypeptide is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 (except for certain mutations), The Fc region of the second polypeptide is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4 (except for certain mutations).

  In a further embodiment of the heterodimeric protein of the invention, the Fc regions of both the first and second polypeptides are of the IgG1 isotype.

  In a further embodiment of the heterodimeric protein of the invention, one of the Fc regions of the polypeptide is of the IgG1 isotype and the other is of the IgG4 isotype.

  In a further embodiment of the heterodimeric protein of the invention, the increase in the strength of the heterodimeric interaction in comparison to each of the homodimeric interactions is the introduction of a covalent bond, a cysteine residue or a charged residue. Other than CH3 modification.

  In a further embodiment of the heterodimeric protein of the invention, the heterodimeric interaction between the first polypeptide and the second polypeptide in the heterodimeric protein is under the conditions described in Example 13. Below, 0.5 mM GSH is such that Fab arm exchange can not occur.

  In a further embodiment of the heterodimeric protein of the invention, the heterodimeric interaction between the first polypeptide and the second polypeptide in the resulting heterodimeric protein is as described in Example 14. Under the conditions described, it is such that Fab arm exchange does not occur in vivo in mice.

  In a further embodiment of the heterodimeric protein of the invention, the first CH3 region comprises Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409 and the second CH3 region is other than Phe at position 405 Contains amino acids and Lys at position 409.

  In a further embodiment of the heterodimeric protein of the invention, the first CH3 region comprises Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, and the second CH3 region is Leu and position at position 405. 409 contains Lys.

  In a further embodiment of the heterodimeric protein of the invention, the first CH3 region comprises Phe at position 405 and Arg at position 409, and the second CH3 region comprises Leu at position 405 and Lys at position 409.

  In a further embodiment of the heterodimeric protein of the invention, the first CH3 region comprises an amino acid other than Lys, Leu or Met at position 409 and the second CH3 region a Lys at position 409 as well as: a) at position 350 Ile and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

  In a further embodiment of the heterodimeric protein of the invention, the first CH3 region comprises Arg at position 409, the second CH3 region is Lys at position 409 and: a) Ile at position 350 and Leu at position 405, Or b) include Thr at position 370 and Leu at position 405.

  In a further embodiment of the heterodimeric protein of the invention, the first CH3 region comprises Thr at position 350, Lys at position 370, Phe at position 405 and Arg at position 409 and the second CH3 region at position 409 Lys and also include: a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

  In a further embodiment of the heterodimeric protein of the invention, the first CH3 region comprises Thr at position 350, Lys at position 370, Phe at position 405 and Arg at position 409 and the second CH3 region at position 350 Ile, Thr at position 370, Leu at position 405 and Lys at position 409.

  In a further embodiment of the heterodimeric protein of the invention, neither the first nor the second polypeptide comprises a Cys-Pro-Ser-Cys sequence in the hinge region.

  In a further embodiment of the heterodimeric protein of the invention, both the first and second polypeptides comprise a Cys-Pro-Pro-Cys sequence in the hinge region.

  In a further embodiment of the heterodimeric protein of the invention, the first and / or second polypeptide comprises a mutation that removes the acceptor site for Asn-linked glycosylation.

Target Antigens As described above, in an important aspect of the invention, the heterodimeric protein is a bispecific antibody which has different binding specificities, ie comprises two variable regions which bind to different epitopes .

  In principle, any combination of specificities is possible. As mentioned above, bispecific antibodies can potentially be used to overcome some of the limitations of monospecific antibodies. One possible limitation of monospecific antibodies is the lack of specificity for the desired target cell due to expression of the target antigen in other cell types where antibody binding is not desired. For example, target antigens that are overexpressed on tumor cells may be expressed in healthy tissues, which may result in undesirable side effects due to treatment with antibodies directed against the antigens. Bispecific antibodies with additional specificity for proteins expressed exclusively on target cell types could potentially improve specific binding to tumor cells.

  Thus, in one embodiment of the invention, the first and second epitopes are located on the same cell, eg a tumor cell. Suitable targets on tumor cells include, but are not limited to: erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, CD138, CXCR5 C-Met, HERV-coat protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM and MRP3. Possible combinations of tumor cell targets are: erbB1 + erbB2, erbB2 + erbB3, erbB1 + erbB3, CD19 + CD20, CD38 + CD34, CD4 + CXCR5, CD38 + RANKL, CD38 + CXCR4, CD20 + CXCR4, CD20 + CCR7, CD20 + CXCR5, CD20 + RANKL, erbB2 + AXL, erbB1 + cMet, erbB2 + c-Met, erbB2 + EpCAM, c-Met + AXL, c-Met + TF, CD38 + CD20, CD38 + CD138, but these include There is no limitation.

  In a further embodiment, the first and second epitopes can be located on the same target antigen, wherein the positions of the two epitopes on the target antigen are such that the binding of the antibody to one epitope is to the other epitope Not to interfere with the binding of the antibody. In this further embodiment, the first and second homodimeric proteins bind to two different epitopes located on the same target antigen, but have different mechanisms of action on the death of the target cells, eg tumor cells. Is an antibody having For example, in one embodiment, the target antigen is erbB2 (HER2) and the bispecific antibody combines the antigen binding sites of pertuzumab and trastuzumab. In another embodiment, the target antigen is erbB1 (EGFr) and the bispecific antibody combines the antigen binding sites of Saltuzumab and Nimotuzumab.

  Bispecific antibodies can also be used as mediators to retarget effector mechanisms to disease associated tissues such as tumors. Thus, in a further embodiment, the first or second epitope is located on a tumor cell, such as a tumor cell protein or a tumor cell carbohydrate, and the other epitope is located on an effector cell.

  In one embodiment, the effector cell is a T cell.

  Possible targets on effector cells include: FcgammaRI (CD64) expressed on monocytes and macrophages and activated neutrophils; FcgammaRIII (CD16) expressed on natural killers and macrophages; CD3 expressed on cells; PMN (polymorphonuclear neutrophils), CD89 expressed on eosinophils, monocytes and macrophages; CD32a expressed on macrophages, neutrophils, eosinophils; FcεRI expressed on basophils and mast cells. In one embodiment, the epitope is located on CD3 expressed on T cells.

  In another embodiment, the first antibody has binding specificity to a pathogenic microorganism and the second antibody is like CD3, CD4, CD8, CD40, CD25, CD28, CD16, CD89, CD32, CD64, FcεRI or CD1. Have binding specificity for effector cell proteins.

  In addition, bispecific antibodies can be used to more specifically target chemotherapeutic agents to the cells on which the agents are to act. Thus, in one embodiment, one of the homodimeric proteins recognizes a small molecule or peptide, or, for example, according to the principles described in Rader et al, (2003) PNAS 100: 5396, such It is an antibody that can form a covalent bond with a molecule. In a further embodiment of the method of the present invention, the first antibody is a tumor cell or tumor cell surface protein, such as erbB1, erbB2, erbB3, erbB4, EGFR3vIII, CEA, MUC-1, CD19, CD20, CD4, CD38, EPCAM, c. -With binding specificity for Met, AXL, L1-CAM, tissue factor, CD74 or CXCR5 (ie binding to their epitope), the second antibody is a toxin (including radiolabeled peptide), drug or prodrug Have a binding specificity for chemotherapeutic agents, such as

  Bispecific antibodies can also be used to target toxins, drugs or prodrugs containing vesicles, such as high electron density vesicles or minicells, to tumors. See, eg, MacDiarmid et al. (2009) Nature Biotech 27: 643. Minicells are cells that do not contain chromosomal DNA and that are chromosome-free products of abnormal cell division. Thus, in another embodiment, the first or second epitope is located on a tumor cell, such as a tumor cell protein or a tumor cell carbohydrate, and the other epitope is located on high electron density vesicles or minicells.

In addition, the serum half-life of antibodies can be altered by including binding specificity for serum proteins in bispecific antibodies. For example, serum half-life can be extended by including binding specificity for serum albumin in the bispecific antibody. Thus, in a further embodiment of the method of the invention, the first antibody is a tumor cell or a tumor cell protein such as erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD19, CD20, CD4, CD38, Specific binding to CD138, CXCR5, c-Met, HERV-jacket protein, periostin, Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM or MRP3, CEA The second antibody has binding specificity for a blood protein, such as serum albumin. The second binding specificity can also be used to target antibodies (beyond the blood-brain barrier) to specific tissues, such as the central nervous system or the brain. Thus, in a further embodiment of the method of the invention, the first antibody is a brain specific target, such as amyloid-beta (e.g. for the treatment of Alzheimer's disease), Her-2 (e.g. for the treatment of breast cancer metastasis in the brain) , EGFr (eg, for treatment of primary brain tumors), Nogo A (eg, for treatment of brain injury), TRAIL (eg, for treatment of HIV), α-synuclein (eg, for treatment of Parkinson's), Htt (eg, for treatment of Huntington treatment), prion (eg mad cow treatment), binding specificity to West Nile virus protein, the second antibody is a blood-brain barrier protein such as transferrin receptor (TfR), insulin Receptor, melanotransferrin receptor (MTfR), lactoferrin receptor (LfR), apolipoprotein E receptor 2 (ApoER2), LDL-receptor-related protein 1 and 2 (LRP1 and LRP2), high Glycosylation final product receptor (RAGE), diphtheria toxin-receptor = heparin-binding epidermal growth factor-like growth factor (DTR = HB-EGF), gp190 (Abbott et al, Neurobiology of Disease 37 (2010) 13- 25) with binding specificity.

  Binding specificity for blood-brain barrier proteins can also be used to target other non-antibody molecules to specific tissues, such as the central nervous system or the brain (beyond the blood-brain barrier). Thus, in a further embodiment, one of the homodimeric proteins is such as blood brain barrier protein (TfR, insulin receptor, MTfR, LfR, ApoER2, LRP1, LRP2, RAGE, DTR (= HB-EGF) or gp190) And the other homodimeric protein is a cytokine, a soluble receptor or another protein such as VIP (a vasoactive intestinal peptide), BDNF (brain-derived neurotrophic factor), FGF (fibroblast growth factor), multiple FGF, EGF (epidermal growth factor), PNA (peptide nucleic acid), NGF (neural growth factor), neurotrophin (NT) -3, NT-4 / 5, derived from glia Neurotrophic factor, ciliary neurotrophic factor, neurturin, neuregulin, interleukin, transforming growth factor (TGF) -α, TGF-β, erythropoietin, hepatocyte growth factor, platelet derived growth factor, artemin, persephin, ne Torin, cardiotrophin-1, stem cell factor, midkine, pleiotrophin, bone morphogenetic protein, saposin, semaphorin, leukocyte inhibitory factor, α-L-iduronidase, iduronic acid-2-sulfatase, N-acetyl- An Fc region linked at the N-terminus or C-terminus to another protein, such as galactosamine-6-sulfatase, arylsulfatase B, acid alpha-glucosidase or sphingomyelinase (Pardridge, Bioparmaceutical drug targeting to the brain, Journal of Drug Targeting 2010, 1-11; Pardridge, Re-engineering Biopharmaceuticals for delivery to brain with molecular Trojan horses. Bioconjugate Chemistry 2008, 19: 1327-1338).

  In addition, the second binding specificity can be used to target blood coagulation factors to specific desired sites of action. For example, a bispecific antibody having a first binding specificity for tumor cells and a second binding specificity for blood coagulation factors could direct blood coagulation to a tumor, thus arresting tumor growth. Thus, in a further embodiment of the method of the invention, the first antibody has a binding specificity for a tumor cell or a tumor cell protein such as erbB1, erbB2, erbB3, erbB4, MUC-1, CD19, CD20, CD4 or CXCR5. The second antibody has a binding specificity for a protein involved in blood coagulation, such as tissue factor.

  Further particularly interesting combinations of binding specificities include CD3 + HER2, CD3 + CD20, IL-12 + IL18, IL-1a + IL-1 b, VEGF + EGFR, EpCAM + CD3, GD2 + CD3, GD3 + CD3, HER2 + CD64, EGFR + CD64, CD30 + CD16, NG2 + CD28, HER2 + HER3, CD20 + CD28, HER2 + CD16, Bcl2 + CD3, CD19 + CD3, CEA + CD3, EGFR + CD3, IgE + CD3, EphA2 + CD3 CD33 + CD3, MCSP + CD3, PSMA + CD3, TF + CD3, CD19 + CD16, CD19 + CD16a, CD30 + CD16a, CEA + HSG, CD20 + HSG, MUC1 + HSG, CD20 + CD22, HLA-DR + CD79 , PDGFR + VEGF, IL17a + IL23, CD32b + CD25, CD20 + CD38, HER2 + AXL, CD89 + HLA class II, CD38 + CD138, TF + cMet, Her2 + EpCAM, HER2 + HER2, EGFR + EGFR, EGFR + c A combination of -Met, c-Met + non-binding arm and G protein coupled receptor is included.

  In a further embodiment, using a bispecific antibody according to the invention, essentially, Taylor et al. J. Immunol. 158: 842-850 (1997) and Taylor and Ferguson, J. Hematother. 4: 357-362. Targeting to red blood cells, as described in 1995, can remove pathogens, pathogenic autoantibodies or harmful compounds such as venom and toxins from circulating blood. The first epitope is located on erythrocyte proteins, including but not limited to erythrocyte complement receptor 1, and the second epitope is located on the compound or organic targeted for removal.

  In a further embodiment, the second Fab arm comprises a fusion protein that falls on a self antigen or binding site and attaches a self antigen such as dsDNA. Targeting of pathogens, autoantibodies or harmful compounds with the bispecific antibodies of the invention and subsequent removal via erythrocytes may thus have therapeutic utility in the treatment of various diseases and syndromes.

Binding In a further aspect of the invention, the first and / or second homodimeric protein is linked to a compound selected from the group consisting of toxins (including radioactive isotopes), prodrugs or drugs. Such compounds can make target cell death more effective, for example, in cancer treatment. The resulting heterodimeric protein is thus an immunoconjugate. The compounds may alternatively be coupled to the resulting heterodimeric protein, ie after Fab arm exchange has taken place.

  Compounds suitable for forming the immunoconjugate of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, emeticin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin di On, mitoxantrone, mithramycin, actinomycin D, 1-dehydro-testosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (eg methotrexate, 6-mercaptopurine, 6-thioguanine , Cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine, alkylating agents (eg mecro Tamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin ), Antibiotics (eg dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC), diphtheria toxin And related molecules (eg, diphtheria A chain and active fragments and hybrid molecules thereof), ricin toxin (eg, ricin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas aeruginosa exotoxin, alloline, saporin, modexin, Geranin, abrin A chain, modexin A chain, α-sarcin, Cinnamon (Aleurites fordii) protein, diantin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), bitter melon (morodica charantia) Inhibitors, curcin, crotin, sapaonaria officinalis inhibitors, xeronine, miterin, restrictocin, phenomycin, as well as enomycin toxin. Other suitable binding molecules are ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtherin toxin, Pseudomonas aeruginosa endotoxin, maytansinoid, auristatin (MMAE, MMAF), calicheamicin And duocarmycin analogues (Ducry and Stump, Bioconjugate Chem. 2010, 21: 5-13), dolastatin-10, dolastatin-15, irinotecan or its active metabolite SN38, pyrrolobenzodiazepines (PBD's).

  In a further aspect of the invention, the first and / or second homodimeric proteins are linked to an alpha emitter, including but not limited to thorium-227, radium-223, bismuth-212 and actinium-225 Be done.

  In a further embodiment of the present invention, the first and / or second homodimeric proteins can be selected from the group consisting of iodide-313, yttrium-90, fluorine-18, rhenium-186, gallium-68, technetium-99, indium-111, And is linked to a beta radionuclide, including but not limited to lutetium-177.

  In another embodiment, the compound to be bound comprises a nucleic acid or nucleic acid related molecule. In one such aspect of the invention, the nucleic acid to be linked is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (e.g. a siRNA molecule) or an immunostimulatory nucleic acid (e.g. an immunostimulatory CpG motif containing DNA molecule) .

Hunter et al., Nature 144 , 945 (1962), David et al., Biochemistry 13 , 1014 (1974), Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. Any method known in the art for conjugation can be utilized, including the method described by J. and Cytochem. 30 , 407 (1982). Conjugates can be made by chemically linking other components to the N- or C-terminal side of the protein (eg, by Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994) See for reference). Such conjugated antibody derivatives may be generated by conjugation with internal residues or sugars, as appropriate. Agents may be coupled directly or indirectly to the proteins of the invention. One example of indirect coupling of a second agent is coupling by a spacer moiety. Drug-conjugate conjugation techniques have recently been summarized by Ducrry and Stump (2010) Bioconjugate Chem. 21 : 5.

Compositions and Uses In a further main aspect, the present invention relates to a pharmaceutical composition comprising a heterodimeric protein according to the invention as described herein and a pharmaceutically acceptable carrier.

  The pharmaceutical composition may be formulated according to the prior art, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Can. The pharmaceutical composition of the present invention may, for example, be a diluent, an extender, a salt, a buffer, a surfactant (for example, a nonionic surfactant such as Tween-20 or Tween-80), a stabilizer (for example, Sugar or protein free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in pharmaceutical compositions can be included.

  Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorptions that are physiologically compatible with the compounds of the present invention Retarders etc. are included. Examples of suitable aqueous and non-aqueous carriers that can be utilized in the pharmaceutical composition of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (eg glycerol, propylene glycol, polyethylene glycol) Can be mentioned. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

  The pharmaceutical compositions of the present invention may also be water soluble anti-oxidants such as (1) ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium bisulfite etc. (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, α-tocopherol and the like; and (3) citric acid, ethylenediaminetetraacetic acid ( Metal chelating agents may also be included, such as EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

  The pharmaceutical compositions of the invention may also include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, glycerol or sodium chloride in the composition.

  The pharmaceutical compositions of the invention are also suitable for selected routes of administration, such as preservatives, wetting agents, emulsifiers, dispersants, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. One or more adjuvants can also be included. The compounds of the present invention may be formulated with carriers that prevent rapid release of the compound, such as controlled release formulations, including implants, transdermal patches and microencapsulated delivery systems. Such carriers may be biodegradable such as gelatin, glyceryl monostearate, glyceryl distearate, ethylene vinyl acetate alone or with a wax, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, etc. The polymer can be a biocompatible, biocompatible polymer, or other material known in the art. Methods for preparation of such formulations are generally known to those skilled in the art.

  Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent, optionally with one or a combination of ingredients enumerated above, as required, followed by microfiltration It can be prepared.

  The actual dosage level of the active ingredient in the pharmaceutical composition is such that it is not toxic to the patient and of the active ingredient effective to achieve the therapeutic response desired for the particular patient, composition, and mode of administration. It can be varied to get the quantity. The dosage level chosen will depend on the activity of the particular composition of the invention to be employed, the route of administration, the time of administration, the excretion rate of the particular compound to be employed, the duration of treatment, the particular composition to be employed and the like. Various other drugs, compounds and / or materials used in combination, including the age, sex, weight, condition, general health and past medical history of the patient being treated, and similar factors well known in the medical arts It will depend on pharmacokinetic factors.

  The pharmaceutical composition can be administered by any suitable route and mode. In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. As used herein, "parenterally administered" means a mode of administration, usually by injection other than enteral and topical administration, which may be epidermal, intravenous, intramuscular, intraarterial, intrathecal. Intracavitary, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendon, transtracheal, subcutaneous, subepithelial, intraarticular, subcapsular, subarachnoid, intraspinal, intraspinal, intracranial, intrathoracic, dura Includes external and intrasternal injections and infusions.

  In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

  In a main aspect, the invention relates to a heterodimeric protein according to the invention, such as a bispecific antibody according to the invention, for use as a medicament. The heterodimeric proteins of the invention can be used for several purposes. In particular, as explained above, the heterodimeric proteins of the invention can be used for the treatment of various forms of cancer, including metastatic and refractory cancers.

  Thus, in one aspect, the invention inhibits the growth and / or proliferation of tumor cells, comprising the administration of the heterodimeric protein according to the invention as described herein to an individual in need thereof. And / or a method for killing tumor cells.

  In another embodiment, the heterodimeric protein of the invention is used for the treatment of immune and autoimmune diseases, inflammatory diseases, infectious diseases, cardiovascular diseases, CNS as well as musculoskeletal diseases.

  Dosage regimens in the methods of treatment and use described above are adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the treatment situation. It is also good.

  Effective dosages and dosage regimens for heterodimeric proteins will depend on the disease or condition being treated and can be determined by one skilled in the art. An exemplary, non-limiting range of a therapeutically effective amount of a bispecific antibody of the invention is about 0.1 to 100 mg / kg, such as about 0.1 to 50 mg / kg, such as about 0.1 to 20 mg / kg. kg, such as about 0.1 to 10 mg / kg, such as about 0.5 mg / kg, such as about 0.3 mg / kg, about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg is there.

  A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can lower the dose of heterodimeric protein utilized in the pharmaceutical composition to a level lower than that required to achieve the desired therapeutic effect to achieve the desired effect. The dose could be gradually increased until achieved. In general, a suitable daily dose of a composition of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. The administration may be parenteral, such as, for example, intravenous, intramuscular or subcutaneous.

  The heterodimeric proteins of the invention may also be used to reduce the risk of developing a disease, such as cancer, to delay the onset of the onset of an event in disease progression, and / or such as to cancer. If the disease is in remission, it can also be administered prophylactically to reduce the risk of recurrence.

  Heterodimeric proteins of the invention, such as bispecific antibodies, can also be administered in combination therapy, ie, combined with other therapeutic agents suitable for the disease or condition being treated. it can. Thus, in one embodiment, the medicament comprising the heterodimeric protein is directed to a combination with one or more further therapeutic agents, such as cytotoxic agents, chemotherapeutic agents or anti-angiogenic agents. Such co-administration may be simultaneous, separate or sequential. In a further aspect, the invention is a method for treating or preventing a disease, such as cancer, such as a bispecific antibody of the invention in combination with radiation therapy and / or surgery. And providing a therapeutically effective amount of the heterodimeric protein to a subject in need thereof.

  Heterodimeric proteins, such as bispecific antibodies, of the invention can also be used for diagnostic purposes.

Example 1: Expression vector for expression of human IgG1-2F8 and IgG1-7D8
The VH and VL coding regions of HuMab 2F8 (WO 02/100348) and HuMab 7D8 (WO 04/035607) contain the genomic sequence of the expression vector pConG1f (human IgG1f allotype constant region for the production of human IgG1 heavy chain (Lonza Biologics) and pCon Kappa (containing human κ light chain constant region, Lonza Biologics) for the production of κ light chains. For IgG4 antibodies, the VH region was inserted into the pTomG4 vector (containing the genomic sequence of human IgG4 constant region in pEE12.4 vector (Lonza Biologics)). Alternatively, in follow-up of the construct, the human kappa light of HuMab 2F8 or HuMab 7D8 in heavy chain (IgG1 or IgG4) or pEE6.4 vector (Lonza Biologics) in pEE12.4 vector A vector was used containing the coding region completely codon optimized of the strand.

Example 2 Expression vector for expression of human IgG1 and IgG4 CH2-CH3 fragments containing hinge-deleted IgG1-2F8, and specific mutations. Quickchange to introduce mutations in the hinge and CH3 regions of the antibody heavy chain. Site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used according to the manufacturer's recommendations. Alternatively, the construct was completely synthesized or the VH region was cloned into a vector that already contained a substitution encoding a specific amino acid.

  Constructs encoding CH2 and CH3 fragments were constructed by PCR or total synthesis and codon optimized. These constructs had an N-terminal signal peptide and a His tag of 6 amino acids and contained amino acid numbers 341-447 of the human IgG1 / 4 constant region. The construct was cloned into pEE12.4.

To construct a hinge deleted IgG1 (Uni-G1) molecule, a synthetic DNA construct encoding the Uni-G1 format of human IgG1 isotype with EGFR specificity was generated. In this construct, the natural hinge region (defined by the hinge exons) was deleted. An additional Ser to Cys mutation at position 158 was made in the IgG1 construct to take advantage of the Cys bond between the HC and LC chains in this subtype. The protein sequence is shown below. The construct was inserted into the pEE6.4 vector and named pHG1-2F8.

Example 3: Expression vector for expression of rhesus monkey IgG4-2F8 and IgG4-7D8 A vector containing the coding regions of the IgG4 heavy and kappa light chains of Chinese rhesus monkey and the VH and VL regions of Humab 2F8 and 7D8 was synthesized, Completely codon optimized and inserted into pEE12.4 (heavy chain) and pEE6.4 (light chain). The heavy chain constant region sequences used (based on the sequence described by Scinicariello et al., Immunology 111: 66-74, 2004) were as follows (aligned to human sequences).

The rhesus monkey light chain constant region (CL) sequences used were:

Example 4: Antibody production by transient expression in HEK-293F cells
Antibodies were produced under serum-free conditions by cotransfecting relevant heavy and light chain expression vectors in HEK-293F cells (Invitrogen) using 293fectin (Invitrogen) according to the manufacturer's instructions. .

Example 5: Purification of IgG1 and IgG4 Antibodies
IgG1 and IgG4 antibodies were purified by protein A affinity chromatography. The cell culture supernatant is filtered over a 0.20 μM dead end filter, followed by loading on a 5 mL protein A column (rProtein A FF, GE Healthcare, Uppsala, Sweden) and 0.1 M citric acid-NaOH, pH 3 Elution of IgG was performed. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9, and dialyzed overnight against 12.6 mM sodium phosphate, 140 mM NaCl, pH 7.4 (B. Braun, Oss, The Netherlands). After dialysis, the sample was sterile filtered over a 0.20 μM dead end filter. The concentration of purified IgG was determined by nephelometry and absorbance at 280 nm. The purified proteins were analyzed by SDS-PAGE, IEF, mass spectrometry and sugar analysis.

Example 6: Purification of CH2-CH3 Fragment
His-tagged CH2-CH3 protein is purified by immobilized metal ion (Ni ²⁺ ) affinity chromatography (Macherey-Nagel GmbH, Duren, Germany) and used with PBS equilibrated PD-10 column (GE Healthcare) Desalted and filter sterilized on a 0.2 μM dead end filter. The concentration of purified protein was determined by absorbance at 280 nm. The quality of purified protein was analyzed by SDS-PAGE.

Example 7: Generation of bispecific antibody by GSH-induced Fab arm exchange between human IgG4 antibody and rhesus monkey IgG4 antibody As described above, in WO 2008119353 (Genmab), the bispecific antibody is under reducing conditions Produces a bispecific antibody, formed by "Fab arm" or "half molecule" exchange (swapping of heavy and attached light chains) between two monospecific IgG4 or IgG4-like antibodies by incubation with In vitro methods have been described. In this Fab arm exchange reaction, the heavy chain interchain disulfide bond in the hinge region of the monospecific antibody is reduced, and the resulting free cysteine is between the cysteine residue of another antibody molecule with different specificity and the new heavy chain It is a result of the isomerization reaction of a disulfide bond which forms a disulfide bond. The resulting product is a bispecific antibody with two Fab arms with different sequences.

  Using human IgG4-2F8 (anti-EGFR), human IgG4-7D8 (anti-CD20), rhesus IgG4-2F8 and rhesus IgG4-7D8 to test for Fab arm exchange between human IgG4 and rhesus IgG4 antibodies And all possible combinations of two antibodies. For in vitro Fab-arm exchange, antibody mixtures containing each antibody at a final concentration of 4 μg / mL in 0.5 mL PBS containing 0.5 mM reduced glutathione (GSH) were incubated at 37 ° C. for 24 hours. In order to stop the reduction reaction, 0.5 mL of PBS / 0.05% Tween 20 (PBST) was added to the reaction mixture.

  The determination of bispecific binding using a sandwich enzyme-linked immunosorbent assay (ELISA) method tested for the presence of bispecific antibodies. ELISA plates (Greiner bio-one, Frickenhausen, Germany) were coated overnight at 4 ° C. with 2 μg / mL (100 μL / well) of the recombinant extracellular domain of EGFR in PBS. Plates were washed once with PBST. Transfer a dilution series (0 to 1 μg / mL at 3-fold dilution) of antibody samples in PBST / 0.2% BSA (PBSTB) to a coated ELISA plate (100 μL / well) and plate shaker (300 rpm) And incubated for 60 minutes at room temperature (RT). The sample was discarded and the plate was washed once with PBS / 0.05% Tween 20 (PBST). Next, the plate was placed on a plate shaker (300 rpm) with 2 μg / mL of mouse anti-idiotypic monoclonal antibody 2F2 SAB1.1 (prepared against 7D8) in PBTB with 100 μL / well of Genmab). Incubated for 60 minutes. Plates were washed once with PBS / 0.05% Tween 20 (PBST). Next, plates are combined with HRP-conjugated goat anti-mouse IgG (15 G; Jackson ImmunoResearch Laboratories, Westgrove, PA; USA; 1: 5.000) (100 μL / well) in PBSTB on a plate shaker (300 rpm) at RT. Incubated for 60 minutes. Plates were washed once with PBS / 0.05% Tween 20 (PBST). ABTS (50 mg / mL; Roche Diagnostics GmbH, Mannheim, Germany) was added (100 μL / well) and incubated in the dark for 30 minutes at RT. The reaction was quenched with 2% oxalic acid (100 μL / well; Riedel de Haen Seelze, Germany). After 10 minutes at RT, the absorbance at 405 nm was measured in an ELISA plate reader.

  FIG. 1 shows that the combination of human and rhesus monkey IgG4 resulted in even higher bispecific binding (higher OD 405 nm) compared to each of the combinations of IgG4 molecules of that same species. These data indicate that Fab arm exchange is performed between human IgG4 and rhesus monkey IgG4. Furthermore, higher bispecific binding indicates that human IgG4 half molecules exhibit selective dimerization (heterodimerization) with rhesus monkey IgG4 half molecules, and 50% heterodimers and 50% homodimers. It has been suggested that instead of stochastic exchange with the dimer, this results in the equilibrium of the Fab arm exchange reaction migrating towards the bispecific heterodimer.

Example 8: Sequence analysis of human and rhesus monkey IgG4 The ability of antibodies to participate in Fab arm exchange requires only a reducing environment to be activated, in the so-called permissive (eg including CPSC) hinge region In addition, it is stated that the third constant domain (CH3) is required (Van der Neut Kolfschoten, 2007, Science). In the case of human antibodies, Fab arm exchange was found to be an intrinsic feature of IgG4, characterized by an arginine (R) at position 409 in the CH3 domain and a permissive hinge (226-CPSC-229) (WO 2008145142 (Genmab)). In contrast, human IgG1 which is not involved in Fab arm exchange has a lysine (K) at position 409 and a stable (ie non-permissive) hinge (226-CPPC-229) (EU numbering, see also FIG. ).

  The core hinge and CH3-CH3 interface amino acids of human and rhesus monkey antibodies were analyzed in an effort to elucidate the higher Fab arm exchange between human IgG4 and rhesus IgG4 compared to Fab arm exchange between IgG4 molecules of the same species (e.g. See Dall 'Acqua, et al (1998) Biochemistry 37: 9266) for an overview of the residues at the human CH3-CH3 interface). FIG. 2 shows that the core hinge sequence in Chinese rhesus monkey IgG4 is 226-CPAC-229 and that the CH3 domain contains a lysine (K) at position 409. In addition, sequence alignment shows that three more amino acid substitutions at the CH3-CH3 interface for rhesus monkey IgG4 compared to human IgG4: leucine (I) for rhesus monkey at position 350 versus threonine (T) for humans; threshing at position 370 for rhesus monkey In contrast to (T) it was shown to be characterized by lysine (K) in human; and leucine (L) in position 405 in rhesus monkey by human phenylalanine (F).

Example 9: Generation of bispecific antibodies using GSH-induced Fab arm exchange between human IgG4 and human IgG1 containing rhesus IgG4 CH3 sequences
Replacing the IgG1 core hinge sequence (CPPC) with the human IgG4 sequence (CPSC) by P228S substitution to effect Fab arm exchange in the IgG1 molecule had no effect, but CH3 IgG4 like CH3 for Fab arm exchange activity It has been described for human antibodies that it was necessary to mutate the sequence (Van der Neut Kolfschoten, 2007, Science).

  Based on the Fab-arm exchange between human IgG4 and rhesus IgG4 described in Example 7, it was analyzed whether Chinese Rhesus IgG4 CH3 sequences could involve human IgG1 in Fab-arm exchange. Therefore, in addition to the P228S mutation leading to the hinge sequence CPSC, a triple mutation T350I-K370T-F405L (hereinafter referred to as ITL) was introduced into human IgG1-2F8. Human IgG1-2F8 variants were combined with human IgG4-7D8 for GSH induced Fab arm exchange in vitro. Antibody mixtures containing each antibody at a final concentration of 4 μg / mL in 0.5 mL PBS with 0.5 mM GSH were incubated at 37 ° C. for 0 to 3 to 24 hours. In order to stop the reduction reaction, 0.5 mL of PBS / 0.05% Tween 20 (PBST) was added to the reaction mixture. Measurement of bispecific binding in ELISA was performed as described in Example 7.

  From FIG. 3, it is confirmed that introduction of CPSC hinge alone does not involve human IgG1-2F8 in GSH-induced Fab arm exchange when combined with human IgG4-7D8. Moreover, although introduction of a rhesus monkey IgG4 specific CH3 interface amino acid (ITL) into human IgG1-2F8 retained the wild type IgG1 hinge, Fab arm when combined with human IgG4-7D8 under these conditions There was no involvement in the exchange. In contrast, a variant human IgG1-2F8 backbone sequence with both CPSC sequences in the hinge and Rhesus IgG4-specific CH3 interface amino acid (ITL) is GSH-inducing with human IgG4-7D8 compared to two human IgG4 antibodies It shows an increase in bispecific binding after Fab arm exchange. These data show that the hinge containing CPSC involves human IgG1 in GSH-induced Fab arm exchange in combination with the CH3 domain containing I, T and L at positions 350, 370 and 405 respectively It is shown that the exchange reaction equilibrium shifts towards the exchanged bispecific product when combined with human IgG4.

Example 10: Production of bispecific antibodies by Fab arm exchange in vivo between human IgG4 and IgG1 or IgG4 variants
Human IgG4 and IgG1 variants were analyzed in vivo to further identify the features necessary to participate in Fab arm exchange. Four female SCID mice (Charles River, Maastricht, The Netherlands) per group were iv injected with the antibody mixture containing 600 μg of antibody (500 μg 7D8 + 100 μg 2F8) in a total volume of 300 μL. Blood samples were drawn from the saphenous vein at 3, 24, 48 and 72 hours after injection. Blood was collected in heparin-containing vials and centrifuged at 10,000 g for 5 minutes to separate plasma from cells. The generation of bispecific antibodies was followed by assessing the CD20 and EGFR bispecific reactions in ELISA using plasma samples serially diluted in PBSTB as described in Example 7. Bispecific antibodies in plasma samples were quantified by nonlinear regression curve fitting (GraphPad Software, San Diego, CA) using the in vitro exchanged antibody mixture as a reference standard.

  FIG. 4 shows that human IgG4-2F8, in which either the hinge sequence or the CH3 sequence is converted to its corresponding human IgG1 sequence (CPPC or R409K, respectively) no longer participates in Fab arm exchange in vivo. Conversely, human IgG1, in which both the hinge region and the CH3 interface sequence are converted to their corresponding human IgG4 sequences (CPSC and K409R), can be involved in Fab arm exchange in vivo. These data indicate that the hinge containing CPSC (S at position 228) is sufficient to allow Fab arm exchange by human IgG1 in vivo in combination with the CH3 domain containing arginine (R) at position 409 To indicate that

Example 11: 2-MEA induced Fab arm exchange: generation of bispecific antibodies by bypassing / breaking stabilized hinges
2-Mercaptoethylamine · HCl (2-MEA) has been described as selectively cleaving a disulfide bond in the hinge region of an antibody while maintaining the disulfide bond between heavy and light chains Reductant. Therefore, a concentration series of 2-MEA was tested for its ability to induce the generation of bispecific antibodies by Fab arm exchange between two antibodies containing CPSC or CPPC hinge regions. A series of concentrations of 2-MEA (0, 0.5, 1.0, 2.0, 5.0, 7.0, 10.0, 15.0, 25.0 and 40.0) of the antibody mixture containing each antibody at a final concentration of 0.5 mg / mL in a total volume of 100 μL of TE Incubate at 37 ° C. for 90 minutes with mM). In order to stop the reduction reaction, the reducing agent 2-MEA was removed by desalting the sample using a spin column (Microcon centrifugal filter, 30 k, Millipore) according to the manufacturer's recommendation. Bispecific binding was measured by ELISA as described in Example 7.

  The 2-MEA-induced Fab arm exchange contained a CPSC hinge region, and was found to be involved in GSH-induced Fab arm exchange, for the combined IgG4-2F8 × IgG4-7D8, and by the stabilized hinge region The combination IgG1-2F8-ITL × IgG4-7D8-CPPC which was not involved in GSH-induced Fab arm exchange (described in Example 9, FIG. 3) was tested. Surprisingly, 2-MEA was found to induce the separation of the light chain from the heavy chain as determined by non-reducing SDS-PAGE (data not shown). Nevertheless, functional bispecific antibodies were generated as shown in FIG. The maximal level of bispecific binding after Fab arm exchange between wild type human IgG4-2F8 and IgG4-7D8 was achieved at a concentration of 2.0 mM 2-MEA and described in Example 9 (FIG. 3) As compared to the levels achieved with 0.5 mM GSH. However, 2-MEA was able to induce Fab arm exchange between human antibodies IgG1-2F8-ITL and IgG4-7D8-CPPC (with stabilized hinge region) in a dose dependent manner. Although generation of little or no bispecific antibody is formed at low 2-MEA concentrations probably due to the presence of CPPC sequences in the hinge region of both antibodies, generation of bispecific antibodies is very high at higher 2-MEA concentrations It was efficient. Maximal bispecific binding was achieved with 25 mM 2-MEA, exceeding maximal binding after Fab arm exchange between the two wild type IgG4 antibodies. These maximal binding levels were comparable to those described in Example 9 (FIG. 3) for GSH treatment of the corresponding antibody with a CPSC hinge (IgG1-2F8-CPSC-ITL). As both IgG1-2F8-ITL and IgG4-7D8-CPPC contain CPPC hinges, these data suggest that 2-MEA was able to circumvent the CPSC hinge requirement for Fab arm exchange in vitro.

Example 12: Mass spectrometry to track the generation of bispecific antibodies by 2-MEA induced Fab arm exchange
Generation of bispecific antibodies by 2-MEA induced Fab arm exchange is described in Example 11, where bispecific binding was demonstrated by ELISA (FIG. 5). Samples were analyzed by electrospray ionization mass spectrometry (ESI-MS) to determine molecular weight in order to confirm that bispecific antibodies were formed. The samples were first deglycosylated by incubating 200 μg of antibody overnight at 37 ° C. with 0.005 U of N-glycanase (Catalog No. GKE-5006D; Prozyme) in 180 μL of PBS. The sample is desalted at 60 ° C. on an Aquity UPLCTM (Waters, Milford, USA) with a BEH 300 C18, 1.7 μm, 2.1 × 50 mm column and 0.05% formic acid (Fluka Riedel-de Haen, Elution was carried out with a gradient of a mixture of MQ water (eluent A) and LC-MS grade acetonitrile (eluent B) (Biosolve, Valkenswaard, The Netherlands), containing Buchs, Germany). Time-of-flight electrospray ionization mass spectra were recorded on a micrOTOFTM mass spectrometer (Bruker, Bremen, Germany) operating in positive ion mode and online. Prior to analysis, a 500-4000 m / z scale was calibrated with ES tuning mix (Agilent Technologies, Santa Clara, USA). Mass spectra were deconvoluted using a Maximal Entropy equipped with DataAnalysisTM software v. 3.4 (Bruker, Bremen, Germany). Based on the molecular weight of the antibody used for Fab arm exchange in this experiment, the bispecific antibody could be distinguished from the original antibody (Example 15, for IgG1-2F8-ITL × IgG4-7D8-CPPC) Also described in FIG. 9C). For the peaks of the bispecific antibodies, the area under the curve was determined and divided by the total area under the curve to calculate the percentage of bispecific antibody in each sample. Figure 6A shows 0 mM 2-MEA (2 peaks corresponding to parent antibody), 7 mM 2-MEA (3 peaks corresponding to parent antibody and bispecific antibody), and 40 mM 2-MEA (2 3 shows representative three mass spectrometric profiles of Fab arm exchange reaction between IgG1-2F8-ITL and IgG4-7D8-CPPC at one peak corresponding to the heavy specific antibody. The homogeneous peaks of the bispecific product suggest that light chain mispairing did not occur, which should have resulted in fragmented peaks. The quantified data are presented in FIG. 6B and indicate that Fab arm exchange between IgG1-2F8-ITL and IgG4-7D8-CPPC resulted in nearly 100% bispecific antibody. In contrast, Fab arm exchange between wild type IgG4 antibodies resulted in less than 50% bispecific product. These data confirm the results of the bispecific binding ELISA described in Example 11 (FIG. 5).

Example 13: Stability of bispecific antibodies generated by 2-MEA induced Fab arm exchange
The stability of the bispecific antibodies generated by 2-MEA induced in vitro Fab-arm exchange was tested. Therefore (as described in Example 11, FIG. 5) 2 μg of the bispecific sample generated from IgG1-2F8-ITL and IgG4-7D8-CPPC in 7.0 mM 2-MEA was bispecific Concentration series (0, 2, 20, 100 μg) of irrelevant IgG4 (IgG4-MG against acetylcholine receptor, corresponding to 0, 1, 10, 50 fold excess of IgG4-MG compared to 2 μg of the sex test sample Used for GSH-induced Fab arm exchange reactions in the presence of Fab arm exchange in this reaction will cause loss of bispecific EGFR / CD20 binding. The conditions for the GSH reduction reaction were the same as described in Example 7 (24 hours at 37 ° C. in 0.5 mL PBS / 0.5 mM GSH). To stop the reduction reaction, 0.5 mL of PBSTB was added to the reaction mixture. Bispecific binding was measured by ELISA as described in Example 7. The bispecific binding after the GSH reduction reaction is presented relative to the bispecific binding measured in the starting material (control) set at 100%.

  FIG. 7A shows that EGFR / CD20 bispecific binding changes significantly after GSH-induced Fab arm exchange in the presence of irrelevant IgG4 for bispecific samples from IgG1-2F8-ITL × IgG4-7D8-CPPC Indicates not to. This suggests that the bispecific product is stable, ie not involved in GSH induced Fab arm exchange. As a control, FIG. 7B shows that samples from IgG4-2F8 × IgG4-7D8 show reduced EGFR / CD20 bispecific binding after GSH-induced Fab arm exchange in the presence of irrelevant IgG4, It is suggested that this product is not stable. From these data, a heterodimer consisting of a human IgG1 heavy chain containing the triple mutation T350I-K370T-F405L in the CH3 domain and a human IgG4 heavy chain containing the S228P substitution that results in a stabilized hinge (CPPC) It shows that the body is stable.

Example 14: In vivo analysis of pharmacokinetics and stability of bispecific antibodies generated by 2-MEA induced Fab arm exchange
SCID mice are injected with a bispecific antibody generated by in vitro 2-MEA-induced Fab-arm exchange between IgG1-2F8-ITL × IgG4-7D8-CPPC and its stability (Fab arm in vivo Exchange) and pharmacokinetic properties (plasma clearance rate) were analyzed in comparison with the parent antibodies IgG1-2F8-ITL and IgG4-7D8-CPPC. 200 μL of purified antibody in the tail vein of 3 groups of mice (3 mice per group): (1) 100 μg of bispecific antibody; (2) 100 μg of bispecific antibody + irrelevant IgG4 (natalizumab, anti α4-integrin (3) 50 μg of IgG1-2F8-ITL + 50 μg of IgG4-7D8-CPPC were injected intravenously. Blood samples (50-100 μL) were collected by buccal puncture at predetermined time intervals (10 minutes, 3 hours, 1 day, 2 days, 7 days, 14 days, 21 days) after antibody administration. Blood was collected in heparin-containing vials and centrifuged at 14,000 g for 10 minutes. Plasma was stored at -20 ° C. prior to further analysis.

  Total IgG concentration in plasma samples was assayed by ELISA. The assay conditions for the subsequent steps were the same as for the ELISA described in Example 7. The specific compounds used for total IgG measurements were: coated with 2 μg / mL of mouse anti-human IgG (clone MH16-1; CLB; catalog number M1268); serum sample dilution (500 minutes for groups 1 and 3) And one-fifth and one-half in 2,500) and (one-and-a-half and one-hundredth in the case of group 2); 1). The presence of bispecific antibodies in plasma samples was assayed and quantified by dual specificity reactivity of CD20 and EGFR in ELISA as described in Example 10.

  FIG. 8A shows total antibody plasma concentrations. The shape of the plasma clearance curve was identical in all groups, and the plasma clearance of the bispecific antibody was the same as for the parent antibodies IgG1-2F8-ITL and IgG4-7D8-CPPC over the time interval analyzed Was suggested. FIG. 8B shows plasma concentrations of bispecific antibody over time. Addition of a 10-fold excess of irrelevant IgG4 to the bispecific antibody did not affect the bispecific antibody concentration, suggesting that no Fab arm exchange was performed in vivo. After injection of the parent antibody (IgG1-2F8-ITL + IgG4-7D8-CPPC), bispecific antibodies could not be detected in plasma, confirming that these antibodies do not participate in Fab arm exchange in vivo. These data show that bispecific antibody products produced by in vitro 2-MEA-induced Fab-arm exchange between IgG1-2F8-ITL × IgG4-7D8-CPPC are stable in vivo (Fab arm (No exchange), suggesting that they showed pharmacokinetic properties (plasma clearance rate) comparable to that of the parent monovalent antibody.

Example 15: Purity of bispecific antibodies generated by 2-MEA induced Fab arm exchange between two antibodies 2-MEA induced Fab arm exchange between human IgG1-2F8-ITL x IgG4-7D8-CPPC The batch of bispecific antibody generated by was purified on PD-10 desalting column (Catalog No. 17-0851-01; GE Healthcare). Next, the purity of the bispecific product was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance size exclusion chromatography (HP-SEC) and mass spectrometry. The functionality of the generated bispecific antibodies was confirmed by bispecific binding in ELISA (data not shown).

  The sample is run at neutral pH using the modified Laemli method (Laemli 1970 Nature 227 (5259): 680-5) and reduced on a 4-12% NuPAGE Bis-Tris gel (Invitrogen, Breda, The Netherlands) SDS-PAGE was performed under conditions and non-reducing conditions. SDS-PAGE gels were stained with Coomassie and digitally processed images were obtained using GeneGenius (Synoptics, Cambridge, UK). FIG. 9A shows that there are few half molecules (H1L1) that can be detected on non-reduced gel and that the antibody sample after Fab arm exchange consists of intact IgG (FIG. 9A-b).

HP-SEC fractions were collected on a Waters Alliance 2695 separation device (Waters, TSK HP-SEC column (G3000SW _XI ; Toso Biosciences, via Omnilabo, Breda, The Netherlands) and Waters 2487 dual λ absorbance detector (Waters). Etten-Leur, The Netherlands). The sample was run at 1 mL / min. The results were processed using Empower software 2002 version and expressed as a percentage of total peak height per peak. FIG. 9B shows that substantially no aggregates are formed and more than 98% of the sample consists of intact IgG.

  Mass spectrometry was performed as described in Example 12. FIG. 9C shows mass spectrometry profiles of bispecific products generated by Fab arm exchange between starting materials IgG1-2F8-ITL and IgG4-7D8-CPPC and IgG1-2F8-ITL × IgG4-7D8-CPPC. The product in the Fab arm exchange sample is 145,901 kDa, which is complete with the bispecific product derived from IgG1-2F8-ITL (146,259.5 / 2 = 73,130) + IgG4-7D8-CPPC (145,542.0 / 2 = 72,771) To fit. Furthermore, the bispecific antibody product showed a homogeneous peak, suggesting that light chain mispairing did not occur, which should have resulted in fragmented peaks. These data indicate that Fab arm exchange resulted in 100% bispecific antibodies. The small peak detected in addition to the major peak (K0) of the IgG4-7D8-CPPC and bispecific sample can be attributed to the presence of one (K1) or two (K2) C-terminal lysines.

  These data indicate that approximately 100% of the functional bispecific antibody sample was generated by 2-MEA induced Fab arm exchange between IgG1-2F8-ITL × IgG4-7D8-CPPC.

Example 16: Elucidation of the requirements for T350I, K370T and F405L substitution for involvement of Fab arm exchange of human IgG1
In order to further identify the determinants in the IgG1 CH3 domain that are required for IgG1 to be involved in Fab arm exchange, IgG1 containing the triple mutation T350I-K370T-F405L (ITL), double mutant T350I-K370T ( IT), compared to T350I-F405L (IL) and K370T-F405L (TL). The single mutant F405L (L) was also tested. 2-MEA was used as a reducing agent to induce Fab arm exchange in vitro (50 μg of each antibody for 90 minutes at 37 ° C. in 100 μL PBS / 25 mM 2-MEA). In the case of the single mutant F405L antibody, unpurified antibody from transient transfection supernatant was used after buffer exchange into PBS using the Amicon Ultra centrifugation device (30k, Millipore, Cat. No. UFC803096) . To stop the reduction reaction, the reducing agent 2-MEA was removed by desalting the sample using a spin column as described in Example 11. Generation of bispecific antibodies was determined by bispecific binding measured by ELISA as described in Example 7.

  A triple mutation (ITL), a double mutation (IT, IL and TL) and a single mutation (L) were introduced into IgG1-2F8. These variants are either IgG4-7D8 (wild-type) containing CPSC hinges, or IgG4-7D8 containing stabilized hinges, for Fab-arm exchange with 25 mM 2-MEA for 90 minutes at 37 ° C. It was combined with (IgG4-7D8-CPPC). FIGS. 10A-B show that IgG 1-2F8-IL variants and IgG1-2F8-TL variants, regardless of combined IgG4-7D8 (CPSC or CPPC hinge), Fab arm exchange to the same level as triple mutant ITL To indicate that In contrast, no bispecific binding was seen in combination with the IgG1-2F8-IT mutant. FIG. 10C similarly shows that the IgG1-2F8-F405L mutant showed Fab arm exchange regardless of the combined IgG4-7D8 (CPSC or CPPC hinge). These data suggest that the F405L mutation is sufficient to involve human IgG1 in Fab arm exchange under the conditions described above.

Example 17: Generation of bispecific antibodies by 2-MEA induced Fab arm exchange at different temperatures
The ability of 2-MEA to induce the production of bispecific antibodies by Fab arm exchange between two different antibodies was tested at different temperatures. 160 μg of human IgG1-2F8-ITL (0.5 mg / mL for each antibody) with 160 μg of IgG4-7D8-CPPC in 320 μl of PBS / 25 mM 2-MEA at either 0 ° C., 20 ° C. (RT) or 37 ° C. The Fab arm exchange reaction was initiated by incubating the final concentration of From these reactions, 20 μL samples were taken at different time points (0, 2.5, 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180 and 240 minutes). 20 μl of PBS was added to each sample prior to removal of reductant 2-MEA by desalting the samples using a Zeba 96 well spin desalting plate (7k, cat. No. 89808 Thermo Fisher Scientific) according to the manufacturer's recommendations. The total antibody concentration was determined by measuring absorbance at a wavelength of 280 nm using a Nanodrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands). Bispecific binding was measured using a dilution series of antibody samples (total antibody concentration 0-20 μg / mL at 25-fold dilution) in an ELISA as described in Example 7.

  Figure 11. Generation of bispecific antibodies by 2-MEA induced Fab arm exchange between human IgG1-2F8-ITL and IgG4-7D8-CPPC reaches maximal bispecific binding after 45 minutes Show that at 37 ° C. it was found to be most efficient. At room temperature, the generation of bispecific antibodies was even lower, reaching maximal bispecific binding after 240 minutes. At 0 ° C. no production of bispecific binding was observed during the time course analyzed.

Example 18: Analysis of the ability of various reducing agents to induce the generation of bispecific antibodies by Fab arm exchange in vitro
Above indicated that 0.5 mM GSH can induce Fab arm exchange in vitro between human IgG4 and IgG1-CPSC-ITL, but not between human IgG4 and IgG1-ITL containing a stable hinge. Was done (Figure 3). Furthermore, it was found that 2-MEA can induce Fab arm exchange between antibodies with stabilized hinge regions, such as IgG1-ITL × IgG4-CPPC (FIG. 5). To test whether other concentrations of GSH or 2-MEA or other reducing agents can induce Fab arm exchange in vitro between two different antibodies, 2-MEA, GSH and DTT (dithiothreitol) Concentration series were tested. Therefore, the combination of 10 μg of human IgG1-2F8-ITL and 10 μg of IgG4-7D8-CPPC (final concentration of 0.5 mg / mL for each antibody) in 20 μl of PBS with different reducing agent concentration series (0.0, 0.04 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 12.5, 25.0 and 50.0 mM) were incubated at 37 ° C. After 90 minutes, 20 μl of PBS was added to each sample and the reducing agent was removed by desalting the sample using a spin desalting plate as described in Example 17. Total antibody concentration was determined as described in Example 17. Bispecific binding was measured using a dilution series of antibody samples (total antibody concentration 0-20 μg / mL at 3-fold dilution) in an ELISA as described in Example 7.

  From FIG. 12, it is confirmed that 2-MEA induces maximal bispecific binding at a concentration of 25 mM 2-MEA. DTT reached maximal bispecific binding at 2.5 mM DTT and was found to be very effective at generating bispecific antibodies. GSH concentrations in the 0-5 mM range can induce the generation of bispecific antibodies by Fab arm exchange between IgG1-ITL and IgG4-CPPC antibodies, both containing stabilized hinge regions could not. Higher GSH concentrations (12.5-50 mM) caused the formation of antibody aggregates as determined by non-reducing SDS-PAGE (data not shown). Therefore, these samples were excluded from the analysis. These data indicate that the generation of bispecific antibodies by Fab arm exchange between two different antibodies can be induced by various reducing agents.

Example 19: Determinant of position IgG1 409 for involvement in 2-MEA induced Fab arm exchange in combination with IgG1-ITL
2-MEA can induce Fab arm exchange between human IgG1-ITL and IgG4-CPPC as described in Example 11 (FIG. 5). The CH3 interface residues of human IgG1 and IgG4 differ only at position 409: lysine (K) for IgG1 and arginine (R) for IgG4 (described in Example 8, FIG. 2). Therefore, it was tested whether substitution of lysine with arginine or any other amino acid at position 409 (K409X) could cause IgG1 to participate in 2-MEA induced Fab arm exchange with IgG1-ITL. A combination of 10 μg human IgG1-2F8-ITL and 10 μg IgG1-7D8-K409X (0.5 mg / mL final concentration for each antibody) in 20 μl PBS / 25 mM 2-MEA was incubated for 90 minutes at 37 ° C. Unpurified antibody from the supernatant of transient transfection was used after buffer exchange into PBS using Amicon Ultra centrifuge device (30 k, Millipore, Cat. No. UFC803096). After the Fab-arm exchange reaction, 20 μl of PBS was added to each sample and the reducing agent was removed by desalting the sample using a spin desalting plate as described in Example 17. Bispecific binding was measured using a dilution series of antibody samples (total antibody concentration 0-20 μg / mL at 3-fold dilution) in an ELISA as described in Example 7.

  FIG. 13A shows the results of bispecific binding by 2-MEA induced Fab arm exchange between IgG1-2F8-ITL × IgG1-7D8-K409X. In FIG. 13B, for the purified batch of bispecific antibodies derived from 2-MEA induced Fab arm exchange between IgG1-2F8-ITL and IgG4-7D8-CPPC, where the exchange was set to 100%. It is presented as bispecific binding. These data are also presented in Table 1 as follows: (-) No Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange or (++) high Fab arm Scored as an exchange. When position 409 in IgG1-7D8 was K (= wild type IgG1), L or M, it was recognized that there was no (-) Fab arm exchange. Fab arm exchange was found to be moderate (+) if position 409 in IgG1-7D8 was F, I, N or Y, position 409 in IgG1-7D8 was A, D, E, G, In the case of H, Q, R, S, T, V or W, it was found to be high (++).

TABLE 1 2-MEA induced Fab arm exchange between IgG1-2F8-ITL variants and IgG1-7D8-K409X variants
Production of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between IgG1-2F8-ITL and IgG1-7D8-K409X mutants was determined by sandwich ELISA. (-) No Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange in (+), etc. (++) high Fab arm exchange.

Example 20: Antibody deglycosylation does not affect the generation of bispecific antibodies by 2-MEA induced Fab arm exchange
The IgG4-7D8 and IgG4-7D8-CPPC samples were deglycosylated by incubating 200 μg of antibody overnight at 37 ° C. with 0.005 U of N-glycanase (Catalog No. GKE-5006D; Prozyme) in 180 μL of PBS. These samples were used directly in the Fab arm exchange reaction. Fab arm exchange was performed by incubating 50 μg of each antibody (final concentration 0.5 mg / mL for each antibody) in 100 μl PBS / 25 mM 2-MEA for 90 minutes at 37 ° C. The reducing agent 2-MEA was removed by desalting the sample using a spin column as described in Example 11. Bispecific binding was measured using a dilution series of antibody samples (total antibody concentration 0-20 μg / mL at 3-fold dilution) in a sandwich ELISA as described in Example 7.

  Mass spectrometry showed that the deglycosylation reaction yielded 100% deglycosylated antibody product (data not shown). FIG. 14 shows that Fab arm exchange with deglycosylated antibody was not different from Fab arm exchange with the corresponding glycosylated antibody (IgG4-2F8 × IgG4-7D8-deglycosylation vs. IgG4-2F8 × IgG4-7D8 and IgG1-2F8-ITL × IgG4-7D8-CPPC-deglycosylation vs. IgG1-2F8-ITL × IgG4-7D8-CPPC). These data suggest that deglycosylation did not affect the generation of bispecific antibodies by 2-MEA induced Fab arm exchange.

二重特異性抗体の作出を、実施例7に記述されているようにサンドイッチELISAでの二重特異性結合の判定により測定した。図15A/B/Cは、Fabアーム交換反応後の二重特異性結合の結果を示す。 Example 21: Quantification of non-covalent CH3-CH3 interaction
The strength of the interaction at the CH3 interface is such that both heavy chains in the parent antibody disassociate in the Fab arm exchange reaction and that they can subsequently associate in the heterodimerization reaction It should be. Therefore, the correlation (dissociation constant, K _D ) between the ability to participate in Fab arm exchange and the strength of non-covalent CH3-CH3 interactions was analyzed. GSH-induced Fab arm exchange was performed as described in Example 9 (at 0.5 mM GSH 37 ° C.) for the following human antibody combinations:

Generation of bispecific antibodies was measured by determination of bispecific binding in a sandwich ELISA as described in Example 7. FIG. 15A / B / C show the results of bispecific binding after Fab arm exchange reaction.

In order to determine the effect of the above CH3 mutation on the strength of the CH3-CH3 interaction, a fragment consisting only of the CH2-CH3 domain was generated. Absence of the hinge region in these fragments blocked the covalent inter-heavy chain disulfide bond. The fragments were analyzed by nondenaturing mass spectrometry. The samples were buffer exchanged to 100 mM ammonium acetate pH 7 using a spin filter column of 10 kDa MWCO. Aliquots (approximately 1 μL) of serially diluted samples (20 μM to 25 nM; monomer equivalents) were loaded into gold plated borosilicate capillaries for analysis on a LCT mass spectrometer (Waters). The monomer signal M _s was defined as the monomer peak area (M _S / (M _S + D _S ), where D _s = dimer signal) as a percentage of the total peak area in the spectrum. The concentration of monomer at equilibrium [M] _eq is defined as M _S. [M] ₀ , where [M] ₀ is the total protein concentration in monomer units. The dimer concentration at equilibrium [D] _eq was defined as ([M] _0- [M] _eq ) / 2. The K _D was then extracted from the slope of the plot of [D] _eq vs [M] _eq ² . The K _D of the CH3-CH3 noncovalent interactions, are presented in Table 2.

The correlation between the ability involved in Fab arm exchange and the strength of non-covalent CH3-CH3 interaction was analyzed. Figure 15D / E in the case of showing a dual ratio of specific binding after Fab arm exchange plotted against measured K _D of the corresponding CH2-CH3 fragment (IgG1 Figure 15D; in the case of IgG4 is Figure 15E). These data under the conditions tested, suggesting that there is a specific range of efficient apparent K _D values of CH3-CH3 interactions that enable Fab arm exchange.

^＊野生型IgG1またはIgG4の対応するCH2-CH3断片と比較した (Table 2) K _{D of} noncovalent CH 3 -CH 3 interaction

^* Compared to the corresponding CH2-CH3 fragment of wild type IgG1 or IgG4

Example 22: Analysis of the ability of various reducing agents to induce the production of bispecific antibodies by in vitro Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R
2-MEA and DTT were found to induce in vitro Fab arm exchange between human IgG1-ITL and IgG4-CPPC (FIG. 12). It was tested whether these reducing agents could also induce in vitro Fab arm exchange between human IgG1-2F8-F405L and IgG1-7D8-K409R. Concentration series of 2-MEA, DTT, GSH and TCEP (tris (2-carboxyethyl) phosphine) were tested. Fab arm exchange was performed as described in Example 18. The various reducing agents of the concentration series tested were: 0.0, 0.04, 0.1, 0.2, 0.5, 1.0, 5.0, 25.0, 50.0 mM 2-MEA, GSH, DTT or TCEP.

  FIG. 17 confirms that 2-MEA induces maximal Fab arm exchange at a concentration of 25 mM 2-MEA, which continued at an even higher concentration of 50.0 mM 2-MEA. DTT reached maximal Fab arm exchange at 0.5 mM DDT and was found to be very effective at generating bispecific antibodies, which also persisted over higher DTT concentrations (1.0-50.0 mM). Similarly, TCEP reached maximal Fab arm exchange at 0.5 mM and was found to be very effective at generating bispecific antibodies. At concentrations of 25.0 mM and higher, Fab arm exchange by TCEP was interrupted. GSH concentrations in the 0.0 to 5.0 mM range failed to induce the generation of bispecific antibodies by Fab arm exchange. Higher GSH concentrations (25.0-50.0 mM) caused the formation of antibody aggregates (data not shown). Therefore, these samples were excluded from the analysis. These data indicate that the generation of bispecific antibodies by Fab arm exchange between two different antibodies can be induced by various reducing agents.

Example 23: Generation of bispecific antibodies by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R Between human IgG1-2F8-F405L and IgG1-7D8-K409R The molecular weight of the sample from the Fab arm exchange reaction with the concentration series of 2-MEA was determined by ESI-MS to confirm the formation of bispecific antibodies by the 2-MEA induced Fab arm exchange of The concentration series tested were: 0.0, 0.5, 1.0, 2.0, 5.0, 7.0, 10.0, 15.0, 25.0 and 40.0 mM 2-MEA. Fab arm exchange (in PBS) and sandwich ELISA were performed as described in Example 11. ESI-MS was performed as described in Example 12.

  FIG. 18A shows that, in a dose-dependent manner, 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R efficiently results in the generation of bispecific antibodies, 15.0 mM 2-MEA Indicates that the concentration of bispecific binding is maximal at a concentration of The quantified ESI-MS data is presented in FIG. 18B and that Fab arm exchange between IgG1-2F8-F405L and IgG1-7D8-K409R resulted in nearly 100% bispecific antibody And confirmed the results of the bispecific binding ELISA.

Example 24: Purity of bispecific antibody generated by 2-MEA induced Fab arm exchange between human IgG1-2F8-F405L × IgG1-7D8-K409R Between human IgG1-2F8-F405L × IgG1-7D8-K409R A batch of bispecific antibodies, generated by 2-MEA induced Fab arm exchange of SEQ ID NO: 10, was purified using a PD-10 desalting column (Catalog No. 17-0851-01; GE Healthcare). Next, the purity of the bispecific product was analyzed by mass spectrometry as described in Example 12.

  FIG. 19 shows mass spectrometry profiles of bispecific products generated by Fab arm exchange between starting materials IgG1-2F8-F405L and IgG1-7D8-K409R and IgG1-2F8-F405L × IgG1-7D8-K409R. The product in the Fab arm exchange sample is 146,160.7 kDa, which is a bispecific product = 146,459.4 derived from IgG1-2F8-F405L (146,606.8 / 2 = 73,303.3) x IgG1-7D8-K409R (146,312.2 / 2 = 73,156.1) Compatible with kDa. Furthermore, the bispecific antibody product showed a homogeneous peak, suggesting that light chain mispairing did not occur, which should have resulted in fragmented peaks. These data show that Fab arm exchange resulted in approximately 100% bispecific antibodies.

Example 25: In vivo analysis of the stability and pharmacokinetics of a bispecific antibody generated from IgG1-2F8-F405L x IgG1-7D8-K409R by 2-MEA induced Fab arm exchange
A bispecific antibody generated by in vitro 2-MEA-induced Fab-arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R was injected into SCID mice, as described in Example 14 Stability (Fab arm exchange in vivo) and pharmacokinetic properties were analyzed. Two groups of mice (3 mice per group) were analyzed: (1) 100 μg of bispecific antibody; (2) 100 μg of bispecific antibody + irrelevant IgG4 (IgG4-637, described in WO2007068255 ) 1,000 μg. In this example, as described in Example 14, except that HRP-conjugated goat anti-human IgG (Jackson, Cat. No. 109-035-098, 1 / 10,000) was used as the detection conjugate. Total IgG concentrations in plasma samples were assayed by ELISA. The presence of bispecific antibodies in plasma samples was assayed and quantified by bispecific reactivity of CD20 and EGFR in a sandwich ELISA as described in Example 14.

  FIG. 20A shows total antibody plasma concentrations over time. The shape of the plasma clearance curve was identical in both groups. FIG. 20B shows plasma concentrations of bispecific antibody over time. Addition of a 10-fold excess of irrelevant IgG4 to the bispecific antibody did not affect the bispecific antibody concentration, suggesting that no Fab arm exchange was performed in vivo. These data show that the bispecific antibody product produced by in vitro 2-MEA-induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R was stable in vivo (Fab Suggesting that there was no arm exchange).

Example 26: CDC-mediated cell killing by bispecific antibodies generated by 2-MEA induced Fab arm exchange between human IgG1-2F8-F405L × IgG1-7D8-K409R
The CD20 antibody IgG1-7D8 can efficiently kill CD20 expressing cells by complement dependent cytotoxicity (CDC). In contrast, the EGFR antibody IgG1-2F8 does not mediate CDC to target cells expressing EGFR. Bispecific antibodies generated by 2-MEA-induced Fab-arm exchange between mutant IgG1-7D8-K409R and IgG1-2F8-F405L x IgG1-7D8-K409R still induce CDC on CD20 expressing cells It was tested whether it could be done. 10 ⁵ Daudi cells or Raji cells were preincubated for 15 minutes in a shaker at room temperature with a concentration series of antibody in 80 μL of RPMI medium supplemented with 0.1% BSA. Twenty microliters of normal human serum (NHS) was added as a source of complement (20% NHS final concentration) and incubated for 45 minutes at 37 ° C. The CDC reaction was stopped by adding 30 μL of ice cold RPMI medium supplemented with 0.1% BSA. 10 μg / mL of propidium iodide (PI) 10 μL (final concentration of 1 μg / mL) was added and dead cells and viable cells were identified by FACS analysis.

  FIG. 21 shows that CDC-mediated cell killing of CD20 expressing Daudi cells (FIG. 21A) and CD20 expressing Raji cells (FIG. 21B) by IgG1-7D8 was not affected by the introduction of the K409R mutation. Neither Daudi nor Raji cells express EGFR, causing monovalent binding of bispecific antibodies created by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R. Nevertheless, bispecific antibodies still induced CDC-mediated cell killing of CD20 expressing cells. These data suggest that the CDC ability of the parent antibody was maintained in a bispecific form.

Example 27: ADCC-Mediated Cell Killing by Bispecific Antibodies Created by 2-MEA-Induced Fab-Arm Exchange Between Human IgG1-2F8-F405L × IgG1-7D8-K409R
The EGFR antibody IgG1-2F8 can kill EGFR-expressing cells such as A431 by antibody-dependent cellular cytotoxicity (ADCC). A431 cells do not express CD20 and therefore the CD20 antibody IgG1-7D8 does not induce ADCC on these cells. Bispecific antibodies generated by 2-MEA-induced Fab-arm exchange between mutant IgG1-2F8-F405L and IgG1-2F8-F405L x IgG1-7D8-K409R can still induce ADCC against A431 cells It was tested. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of healthy donors using Leucosep® tubes (Greiner Bio-one, Cat. No. 227290) according to the manufacturer's recommendations for isolation of effector cells . Target cells were labeled by adding 100 μCi ⁵¹ Cr to 5 × 10 ⁶ A431 cells in 1 mL RPMI medium supplemented with 0.1% BSA and incubating for 60 minutes in a 37 ° C. shaking water bath. The labeled cells were washed and resuspended in RPMI supplemented with 0.1% BSA. Antibody concentration series (final concentration 0-10 μg / mL in ADCC assay at 3-fold dilution) in 100 μL of 5 × 10 ⁴ labeled target cells in RPMI supplemented with 0.1% BSA at room temperature 15 It was preincubated for a minute. The ADCC assay was initiated by adding 50 μL of effector cells (5 × 10 ⁶ cells) at an E: T ratio of 100: 1. After 4 hours at 37 ° C., ⁵¹ Cr release from triplicate experiments was measured in a scintillation counter as counts per minute (cpm). The following formula: Percentage of specific lysis = (experimental cpm-basal cpm) / (maximum cpm-basal cpm) x 100 was used to calculate the percent cytotoxicity. Maximum ⁵¹ Cr release was determined by adding 50 μL of 5% Triton X-100 to 50 μL of target cells (5 × 10 ⁴ cells), and basal release was measured in the absence of sensitized antibody and effector cells.

  FIG. 22 shows that the CD20-specific antibody IgG1-7D8 did not induce ADCC against CD20 negative A431 cells. Both IgG1-2F8 and mutant IgG1-2F8-F405L are capable of inducing ADCC against A431 cells, suggesting that the introduction of the F405L mutation in IgG1-2F8 does not affect its ADCC effector function It was In addition, bispecific antibodies derived from IgG1-2F8-F405L × IgG1-7D8-K409R induced ADCC in a dose-dependent manner to A431 cells, and ADCC effector function was maintained in a bispecific form Suggested that.

Example 28: Determinants of position IgG1 405 for participation in 2-MEA induced Fab arm exchange in combination with IgG1-K409R In Example 16, in the case of the F405L mutation when combined with IgG4-7D8, human IgG1 It was stated that it was sufficient to allow it to be involved in Fab arm exchange. To further examine the determinant at position IgG1 405 for participation in 2-MEA induced Fab arm exchange in combination with human IgG1-K409R, all possible IgG1-2F8-F405X variants (C and P ) Were combined with IgG1-7D8-K409R. The procedure was performed with purified antibody as described in Example 19.

  FIG. 23 shows the results of bispecific binding by 2-MEA induced Fab arm exchange between IgG1-2F8-F405 ×× IgG1-7D8-K409R. These data are also presented in Table 3, as follows: (-) no Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange or (++) high Fab arm Scored as an exchange. When the position 405 in IgG1-2F8 was F (= wild type IgG1), it was recognized that there was no (-) Fab arm exchange. Fab arm exchange was found to be low (+/−) when position 405 in IgG1-2F8 was G or R. Fab arm exchange is high if position 405 in IgG 1-2 F 8 is A, D, E, H, I, K, L, M, N, Q, S, T, V, W or Y (+ +) I found that. These data suggest that specific mutations at position IgG1 405, when combined with IgG1-K409R, involve IgG1 in 2-MEA induced Fab arm exchange.

Table 3. 2-MEA induced Fab arm exchange between IgG1-2F8-F405X variants and IgG1-7D8-K409R
The generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between IgG1-2F8-F405X mutants and IgG1-7D8-K409R was determined by sandwich ELISA. (-) No Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange in (+), etc. (++) high Fab arm exchange.

Example 29: Determinant of position IgG1 407 for participation in 2-MEA induced Fab arm exchange in combination with IgG1-K409R In Example 28, certain single mutations at position F405 are IgG1- It was stated that it was sufficient to allow human IgG1 to participate in Fab arm exchange when combined with K409R. Mutagenesis at position IgG1 407 was used to determine if other determinants involved in the position of the Fc: Fc interface in the CH3 domain could similarly mediate the Fab arm exchange mechanism, and this mutant was The involvement in 2-MEA induced Fab arm exchange in combination with IgG1-K409R was investigated. All possible IgG1-2F8-Y407X variants (except C and P) were combined with IgG1-7D8-K409R. The procedure was performed with purified antibody as described in Example 19.

  FIG. 24 shows the results of bispecific binding by 2-MEA induced Fab arm exchange between IgG1-2F8-Y407 ×× IgG1-7D8-K409R. These data are also presented in Table 4, without (-) Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange or (++) high Fab arm Scored as an exchange. When position 407 in IgG1-2F8 was Y (= wild type IgG1), E, K, Q or R, it was recognized that there was no (-) Fab arm exchange. Fab arm exchange is low when position 407 in IgG1-2F8 was D, F, I, S or T (+/-), position 407 in IgG1-2F8 is A, H, N or V In this case, medium (+) was found to be high (++) when position 407 in IgG1-2F8 was G, L, M or W. These data suggest that a specific single mutation at position IgG1 407, when combined with IgG1-K409R, involves IgG1 in 2-MEA induced Fab arm exchange.

TABLE 4 2-MEA induced Fab arm exchange between IgG1-2F8-Y407X variants and IgG1-7D8-K409R
The generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between IgG1-2F8-Y407X mutants and IgG1-7D8-K409R was determined by sandwich ELISA. (-) No Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange in (+), etc. (++) high Fab arm exchange.

Example 30: Quantification of noncovalent CH3-CH3 interactions in IgG1 heterodimers A specific range for the strength of CH3-CH3 homodimer interactions enabling efficient Fab arm exchange Is described in Example 21. The strength of the interaction at the CH3 interface is that both heavy chains in the parent antibody (homodimer) dissociate in the Fab arm exchange reaction and that they can subsequently associate in the heterodimerization reaction It must be like that. In order to create a stable heterodimer, the strength of the heterodimer interaction must be higher than the strength of the homodimer interaction, which is more heterodimer than homodimerization. Should be in favor of To confirm this, the strength of the CH3-CH3 interaction in the heterodimer was measured and compared to the strength in the homodimer. IgG1-K409R, the K _D of the IgG1-F405L and CH2-CH3 fragment from IgG1-ITL homodimers was determined as described in Example 21. For the determination a K _D in the heterodimer, CH2-CH3 domain fragment (G1-F405L and G1-ITL), was mixed with IgG1Δ hinge fragment 7D8-K409R comprising all antibodies domains except the hinge. Absence of the hinge region in both fragments blocked the covalent inter-heavy chain disulfide bond. The fragments were mixed and analyzed after 24 hours by nondenaturing mass spectrometry as described in Example 21. K _D values for CH3-CH3 noncovalent interactions in the mixture of CH2-CH3 fragment having a CH2-CH3 fragment or IgG1Δ hinge is displayed are presented in Table 5. These data suggest that under the conditions tested, the strength of the heterodimeric interaction is higher (lower K _D ) than the corresponding homodimeric interaction.

(Table 5)

Example 31: Biochemical analysis of bispecific antibodies generated by 2-MEA induced Fab-arm exchange generated by 2-MEA-induced Fab-arm exchange between human IgG1-2F8-F405L x IgG1-7D8-K409R The batch of bispecific antibodies was purified on a PD-10 desalting column (Catalog No. 17-0851-01; GE Healthcare). Second, the purity of the bispecific product was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance size exclusion chromatography (HP-SEC), mass spectrometry, HPLC cation exchange chromatography (HPLC-CIEX ), Analyzed by capillary isoelectric focusing (cIEF).

  SDS-PAGE was performed under non-reducing conditions (FIG. 25A) and reducing conditions (FIG. 25B) as described in Example 15. FIG. 25A shows that there are few half molecules (H1L1) that can be detected on non-reducing gel and that the antibody sample after 2-MEA induced Fab arm exchange consists of intact IgG.

  HP-SEC was performed as described in Example 15. Figures 26 (B) and 26 (A) show the HP-SEC profiles of starting materials IgG1-2F8-F405L and IgG1-7D8-K409R, respectively. A mixture of both antibodies (1: 1) and bispecific products generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R are shown in FIGS. 26C and 26D, respectively. Show. Further, FIG. 26D shows that substantially no aggregates are formed, and more than 99% of the sample consists of intact IgG.

Mass spectrometry (ESI-MS) was performed as described in Example 12. FIG. 27 (B) and FIG. 27 (A) show mass spectrometry profiles of starting materials IgG1-2F8-F405L and IgG1-7D8-K409R, respectively. A mixture of both antibodies (1: 1) and bispecific products generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R are shown in FIG. 27C and FIG. 27D, respectively. Show. The product in the 2-MEA inducible Fab arm exchange sample is 146,159.7 kDa, which is a double derived from IgG1-2F8-F405L (146,289.0 / 2 = 73,145) x IgG1-7D8-K409R (146,028.0 / 2 = 73,014) Perfectly compatible with specificity products. Furthermore, the bispecific antibody product showed a homogeneous peak, suggesting that light chain mispairing did not occur, which should have resulted in fragmented peaks. These data indicate that 2-MEA induced Fab arm exchange resulted in bispecific IgG. The small peak indicated by ( ^* ) resulted from incomplete deglycosylation prior to analysis. These data indicate that bispecific antibody samples were generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R.

Capillary isoelectric focusing (cIEF) was performed using an iCE280 analyzer (Convergent Biosciences). Figures 28A and 28B show cIEF profiles of starting materials IgG1-2F8-F405L and IgG1-7D8-K409R, respectively. The bispecific product generated by Fab arm exchange between a mixture of both antibodies (1: 1) and IgG1-2F8-F405L × IgG1-7D8-K409R is shown in FIGS. 28C and 28D, respectively. All samples were desalted prior to use. The final concentration in the assay mixture was 0.3 mg / mL IgG (0.35% methyl cellulose; 2% carrier ampholite 3-10; 6% carrier ampholite 8-10.5; 0.5% pI marker 7.65 and 0.5% pI marker 10.10). Isoelectric focusing was performed at 3000 V for 7 minutes and the entire capillary absorption image was captured by a charge coupled device camera. After calibration of peak profiles, data were analyzed by EZChrom software. The pI marker is indicated by ( ^* ). These data indicate that bispecific antibody samples were generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R.

Another technique for examining charged isoforms of monoclonal antibodies is high pressure liquid chromatography cation exchange (HPLC-CIEX). Figures 29A and 29B show the HPLC-CIEX profiles of starting materials IgG1-2F8-F405L and IgG1-7D8-K409R, respectively. A mixture of both antibodies (1: 1) and bispecific products generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R are shown in FIG. Show. The sample was diluted to 1 mg / mL in mobile phase A (10 mM NaPO4, pH 7.0) to inject the sample into HPLC. Differentially charged IgG molecules were separated by using a ProPac® WCX-10, 4 mm × 250 mm analytical column at a flow rate of 1 mL / min. Elution was performed from mobile phase A with a gradient of mobile phase B (10 mM NaPO ₄ , pH 7.0, 0.25 M NaCl), and detection was performed at 280 nm. These data indicate that bispecific antibody samples were generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R. It also shows that cation exchange is a powerful tool to separate the remaining homodimers from heterodimers. Another use of cation exchange chromatography is, therefore, for the final purification of bispecific heterodimers, i.e. to remove any homodimers which remain after exchange.

Example 32: Recombinant expression of heterodimers by co-expression of both homodimers
Encode heavy and light chains of IgG1-7D8-K409R and IgG1-2F8-F405 to illustrate that heterodimerization also occurs when two homodimers are recombinantly coexpressed Four expression vectors were cotransfected into HEK-293F cells at a ratio of 1: 1: 1: 1 (see Example 1). Antibodies were produced transiently under serum free conditions as described in Example 4. The IgG was then purified by protein A chromatography as described in Example 5. The purified IgG was deglycosylated and then analyzed by electrospray ionization mass spectrometry as described in Example 12.

  The theoretical masses of heavy and light chains of IgG1-7D8-K409R and IgG1-2F8-F405 are shown in Table 6.

TABLE 6 Theoretical masses of heavy and light chains of IgG1-7D8-K409R and IgG1-2F8-F405

  Based on these masses, the following IgG molecules could be detected theoretically (Table 7). The measured mass (FIG. 30) is shown in the last row.

TABLE 7 Theoretical detection of heavy and light chains of IgG1-7D8-K409R and IgG1-2F8-F40

  The two most abundant peaks at 146345 Da and 146159 Da corresponded to one (one from IgG1-7D8-K409R) light chain or a heterodimer incorporating both light chains, respectively. Homodimers of both heavy chains of IgG1-7D8-K409R or IgG1-2F8-F405 were detected, but only in small amounts. These data indicate that heterodimerization also occurs when two homodimers are coexpressed.

Example 33: Monitoring the kinetics of 2-MEA induced Fab arm exchange and quantifying the homodimer remaining after exchange by using HPLC-CIEX
The generation of bispecific antibodies by 2-MEA induced Fab arm exchange is described in Example 11. In this example, the exchange reaction was monitored by performing high pressure liquid chromatography cation exchange (HPLC-CIEX; as described in Example 31) at various times during the exchange reaction.

  The homodimers IgG1-2F8-F405L and IgG1-7D8-K409R were mixed at a concentration of 1 mg / mL each at a molar ratio of 1: 1. After addition of 25 mM 2-MEA, the samples were placed in an HPLC autosampler, prewarmed at 25 ° C. Figures 31A-31H show eight consecutive injections at different time intervals obtained by HPLC-CIEX extending from t = 0 minutes to t = 450 minutes, respectively, after addition of 2-MEA. This data shows that bispecific IgG was formed fairly rapidly, with the majority of homodimers being exchanged after 135 minutes. The heterogenous heterodimeric peak appearing after 45 minutes resolved to a more homogeneous peak after approximately 180 minutes, suggesting that exchange was taking place in the different phases. Furthermore, FIG. 32A shows that approximately 3% of residual homodimers were detected by the CIEX method (indicated by arrows). As indicated, this method is suitable for quantifying the content of homodimer remaining when the exchange reaction is almost complete (elution of homodimer is shown in FIG. 32B).

Example 34: Generation of bispecific antibodies by 2-MEA induced Fab arm exchange at high antibody concentrations at various 2-MEA concentrations, temperatures and incubation times
2-MEA induced Fab arm exchange was performed at high IgG concentrations. The effect of 2-MEA concentration, incubation temperature and time on the amount of exchange was investigated.

  The exchange process was performed using the combination of IgG1-7D8-K409R × IgG1-2F8-F405L. Both materials were purified by affinity chromatography with protein A. After concentration of the material to greater than 20 mg / mL, a continuous anion exchange step (in pass-through mode) was performed using HiPrep Q FF 16/10 (GE Health Care, # 28-9365-43). The final purified material was buffer exchanged into PBS.

  Bispecific exchange at a final IgG concentration of 20 mg / mL (at a final concentration of 10 mg / mL each homodimer) and 10 mg / mL (at a final concentration of 5 mg / mL each homodimer) in PBS It investigated by concentration. Separate mixtures were prepared for both IgG concentrations including 2-MEA at final concentrations of 10, 25, 50 and 100 mM. The mixture was divided into 100 μl aliquots in eppendorf tubes and stored at 15, 25 and 37 ° C. Separate tubes were used for different incubation times of 90 minutes, 5 hours and 24 hours at each temperature.

  Also, mixtures were prepared without 2-MEA for both IgG concentrations and stored at 4 ° C. as untreated controls. After the appropriate incubation time, 90 min and 5 h samples were collected for desalting to remove 2-MEA (90 min sample was initially placed on ice to stop the exchange reaction). The samples were desalted using a Zeba 96 well desalting plate (7k, Cat. No. 89808, Thermo Fisher Scientific). The 24 hour samples were desalted separately after 24 hours of incubation.

  Serial dilutions of antibody samples (total antibody concentration is 10-0.123 μg / mL in 3-fold dilutions for 90 minutes and 5-hour samples; 10-0.041 μg / mL in 3-fold dilutions for 24-hour samples) Was used in a sandwich ELISA to measure bispecific binding as described in Example 7. Control of purified batch of bispecific antibodies derived from 2-MEA induced Fab arm exchange between IgG1-2F8-ITL and IgG4-7D8-CPPC (as described in Example 15) for each plate Included. Figures 34 (A)-(F) show the results of bispecific binding measured in individual ELISA plates. Bispecific binding was calculated using the highest OD405 value (determined for concentration of 10 μg / mL in ELISA) and optionally set to 100%, compared to control. This resulted in a controlled percentage of Fab arm exchange (% cFAE) relative to controls as shown in FIGS. 34 (A)-(D) for each 2-MEA concentration.

  The data show that the maximal level of bispecific binding (89-109% relative to control) was reached at a concentration of 100 mM 2-MEA for both IgG concentrations under all temperature-time conditions Be With 50 mM 2-MEA, maximal binding (88-107%) was reached at 25 ° C. and 37 ° C. and also at 15 ° C. after 24 hours of incubation. With lower concentrations of 25 mM and 10 mM 2-MEA, exchange is more efficient at higher temperatures and increases with extended incubation time, with a 24 hour incubation of 25 mM 2-MEA at 37 ° C. Reached the largest exchange. None of the conditions tested with 10 mM 2-MEA produced 100% bispecific product. The exchange process was slightly faster at an IgG concentration of 10 mg / mL compared to 20 mg / mL total IgG.

  The samples were analyzed by cation exchange (HPLC-CIEX) analysis to confirm that the bispecific antibody was formed and to examine the bispecific product in more detail. The HPLC-CIEX analysis was performed as described in Example 31 on the samples at an IgG concentration of 20 mg / mL and all 2-MEA concentrations after 5 and 24 hours of incubation.

  The CIEX chromatograms in FIG. 35 (A)-(D) show that the highest yields of bispecific product were obtained at 50 and 100 mM 2-MEA, confirming the results of the bispecific ELISA It was However, small amounts of residual homodimers were still detected at 50 and 100 mM 2-MEA (2 to 3.5% of each homodimer for samples incubated at 25 ° C. and 37 ° C.). Exchange with higher concentrations, longer (24 hours) incubation times and increasing 2-MEA concentrations results in the appearance of additional peaks at 22-24 minutes in the CIEX profile.

  A minimal amount of additional peak was obtained if the exchange was completed within 5 hours. SDS-PAGE analysis and HP-SEC analysis were performed to identify the nature of these peaks. HP-SEC indicated that the amount of aggregates was less than 1% for all conditions, suggesting that additional peaks do not correspond to aggregates. However, non-reducing SDS-PAGE suggested that the additional peak could correspond to a heterodimer lacking one or two light chains. Small amounts of half molecules were detected as well.

  From the experiments, the exchange reaction is performed at high homodimer concentration, which makes this process attractive on a commercial scale, and that the yield of bispecific antibody depends on 2-MEA concentration, temperature and incubation time Is shown.

Example 35: Determinants of position IgG1 368 for participation in 2-MEA induced Fab arm exchange in combination with IgG1-K409R In Examples 28 and 29, certain single at position F405 and Y407 The mutation is shown to be sufficient to allow human IgG1 to participate in Fab arm exchange when combined with IgG1-K409R. As illustrated in this example, additional determinants responsible for the position of the Fc: Fc interface in the CH3 domain may also mediate the Fab arm exchange mechanism. To this effect, mutagenesis at position IgG1 368 was performed, and this mutant was examined for its involvement in 2-MEA-induced Fab arm exchange in combination with human IgG1-K409R. All possible IgG1-2F8-L368X variants (except C and P) were combined with IgG1-7D8-K409R. The procedure was performed with purified antibody as described in Example 19.

  FIG. 36 shows the results of bispecific binding by 2-MEA induced Fab arm exchange between IgG1-2F8-L368 ×× IgG1-7D8-K409R. These data are also presented in Table 8: no (-) Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange or (++) high Fab arm Scored as an exchange. When position 368 in IgG1-2F8 was L (= wild type IgG1), F or M, it was recognized that there was no (-) Fab arm exchange. Fab arm exchange was found to be low (+/-) when position 368 in IgG1-2F8 was Y. Fab arm exchange is moderate (+) when position 368 in IgG1-2F8 is K, position 368 in IgG1-2F8 is A, D, E, G, H, I, N, Q, R, In the case of S, T, V or W, it was found to be high (++). These data suggest that specific mutations at position IgG1 368, when combined with IgG1-K409R, involve IgG1 in 2-MEA induced Fab arm exchange.

TABLE 8 2-MEA induced Fab arm exchange between IgG1-2F8-L368X variants and IgG1-7D8-K409R
The generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between IgG1-2F8-L368X variants and IgG1-7D8-K409R was determined by sandwich ELISA. (-) No Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange in (+), etc. (++) high Fab arm exchange.

Example 36: Determinants of position IgG1 370 for participation in 2-MEA-induced Fab arm exchange in combination with IgG1-K409R In Examples 28, 29 and 35, certain at position F405, Y407 or L368 A single mutation of <1>, when combined with IgG1-K409R, is shown to be sufficient to allow human IgG1 to participate in Fab arm exchange. As illustrated in this example, additional determinants responsible for the position of the Fc: Fc interface in the CH3 domain may also mediate the Fab arm exchange mechanism. To this effect, mutagenesis at position IgG1 370 was performed, and this mutant was examined for its involvement in 2-MEA-induced Fab arm exchange in combination with human IgG1-K409R. All possible IgG1-2F8-K370X variants (except C and P) were combined with IgG1-7D8-K409R. The procedure was performed with purified antibody as described in Example 19.

  FIG. 37 shows the results of bispecific binding by 2-MEA induced Fab arm exchange between IgG1-2F8-K370X × IgG1-7D8-K409R. These data are also presented in Table 9: no (-) Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange or (++) high Fab arm Scored as an exchange. When position 370 in IgG1-2F8 is K (= wild type IgG1), A, D, E, F, G, H, I, L, M, N, Q, R, S, T, V, or Y In addition, it was recognized that there was no Fab arm exchange (-). Replacing K370 with W only resulted in moderate Fab arm exchange (+). These data suggest that, in combination with IgG1-K409R, only one mutation at position IgG1 370 (K370W) can involve IgG1 in 2-MEA-induced Fab arm exchange.

Table 9. 2-MEA induced Fab arm exchange between IgG1-2F8-K370X variants and IgG1-7D8-K409R
The generation of bispecific antibodies after 2-MEA induced in vitro Fab-arm exchange between IgG1-2F8-K370X variants and IgG1-7D8-K409R was determined by sandwich ELISA. (-) No Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange in (+), etc. (++) high Fab arm exchange.

Example 37: Determinants of position IgG1 399 for participation in 2-MEA-induced Fab arm exchange in combination with IgG1-K409R In Examples 28, 29, 35 and 36, position F405, Y407, L368 or K370. It is shown that certain single mutations at are sufficient to allow human IgG1 to be involved in Fab arm exchange when combined with IgG1-K409R. As illustrated in this example, additional determinants responsible for the position of the Fc: Fc interface in the CH3 domain may also mediate the Fab arm exchange mechanism. To this effect, mutagenesis at position IgG1 399 was performed, and this mutant was examined for its involvement in 2-MEA-induced Fab arm exchange in combination with human IgG1-K409R. All possible IgG1-2F8-D399X variants (except C and P) were combined with IgG1-7D8-K409R. The procedure was performed with purified antibody as described in Example 19.

  FIG. 38 shows the results of bispecific binding by 2-MEA induced Fab arm exchange between IgG1-2F8-D399X × IgG1-7D8-K409R. These data are also presented in Table 10: no (-) Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange or (++) high Fab arm Scored as an exchange. When position 399 in IgG1-2F8 was D (= wild type IgG1), E and Q, it was recognized that there was no (-) Fab arm exchange. Fab arm exchange is low when position 399 in IgG1-2F8 is V (+/-), position 399 in IgG1-2F8 is G, I, L, M, N, S, T or W It was found to be moderate (+) in the case. Fab arm exchange was found to be high (++) when position 399 in IgG1-2F8 was A, F, H, K, R or Y. These data suggest that certain mutations at position IgG1 399, when combined with IgG1-K409R, involve IgG1 in 2-MEA induced Fab arm exchange.

TABLE 10 2-MEA Inducible Fab-Arm Exchange Between IgG1-2F8-D399X Variants and IgG1-7D8-K409R 2-MEA Induction Between IgG1-2F8-D399X Variants and IgG1-7D8-K409R Generation of bispecific antibodies after in vitro Fab arm exchange of sex was determined by sandwich ELISA. (-) No Fab arm exchange, (+/-) low Fab arm exchange, (+) in Fab arm exchange in (+), etc. (++) high Fab arm exchange.

Example 38: Determination of a range of conditions in which 2-MEA induced Fab arm exchange is performed at the optimal lower limit to discriminate high efficiency IgG1 variants
The process of 2-MEA induced Fab arm exchange is efficiently performed at 37 ° C. when using 25 mM 2-MEA. Under these conditions, certain IgG1 variants (at positions 368, 370, 399, 405 and 407 and / or 409 as described in Examples 19, 28, 29 and 35-37, respectively) Most of the IgG1) with a single mutation show high levels of 2-MEA induced Fab arm exchange (80% to 100%). In order to identify experimental conditions that may allow discrimination between IgG1 variants with the highest efficiency, a combination of four different variants (IgG1-2F8-F405S × IgG1-7D8-K409A, IgG1-2F8-D399R × IgG1- 2-MEA induced Fab arm exchange for 7D8-K409G, IgG1-2F8-L368R × IgG1-7D8-K409H and IgG1-2F8-F405L × IgG1-7D8-K409R) at 15 ° C. and 20 ° C. respectively over time Examined. The procedure was performed as described in Example 19, apart from changes in temperature, duration and antibody dilution (20, 2, 0.2 and 0.02 μg / mL).

  At 20 ° C., 2-MEA-induced Fab arm exchange of the four mutant combinations was performed at different rates compared to the largest exchange (positive control). After 105 minutes of incubation, IgG1-2F8-L368R × IgG1-7D8-K409H reaches maximal exchange levels, while IgG1-2F8-F405S × IgG1-7D8-K409A, IgG1-2F8-D399R × IgG1-7D8 -K409G and IgG1-2F8-F405L x IgG1-7D8-K409R reached up to 90%, 85% and 85% after 200 minutes, respectively.

  Incubation of combinations of different IgG1 variants at 15 ° C. showed the most striking differences in exchange rates (shown in FIG. 39). After 60 and 105 minutes of incubation, the difference between the 2-MEA induced Fab arm exchange, the combination of the four variants, was the most extreme. 100% efficiency (IgG1-2F8-L368R × IgG1-7D8-K409H), 85% efficiency (IgG1-2F8-F405L × IgG1-7D8-K409R) compared to positive control by Fab arm exchange after incubation for 200 minutes And IgG1-2F8-D399R × IgG1-7D8-K409G) or 65% efficiency (IgG1-2F8-F405S × IgG1-7D8-K409A).

Example 39: Analysis of the 2-MEA-induced Fab arm exchange efficiency of variants under suboptimal conditions
The process of 2-MEA induced Fab arm exchange is efficiently performed at 37 ° C. when using 25 mM 2-MEA. Under these conditions, certain IgG1 variants (at positions 368, 370, 399, 405 and 407 and / or 409 as described in Examples 19, 28, 29 and 35-37, respectively) Most of the IgG1s with a single mutation show high levels of 2-MEA induced Fab arm exchange (80-100%). In Example 38 it was described that the difference in 2-MEA induced Fab arm exchange efficiency is most pronounced after so-called optimum limit conditions, ie after incubation at 15 ° C. for 60-105 minutes. A total of 24 IgG1-2F8 variants at L368, D399, F405 and Y407 showing greater than 90% 2-MEA induced Fab arm exchange with IgG1-7D8-K409R (see Table 11) (Examples 28, 29) And 35-37) were selected and subjected to Fab arm exchange analysis with IgG1-7D8-K409A, G, H or R (based on the results reported in Example 19). To categorize the combination of these variants by their efficiency in generating bispecific antibodies, 2-MEA induced Fab arm exchange was performed for 90 minutes at 15 ° C. (conditions of the lower limit of conditions). Two IgG1-2F8 mutants Y407Q and D399Q (Examples 29 and 37), which showed weak 2-MEA induced Fab arm exchange after incubation with IgG1-7D-K409R, were taken as additional negative controls and used It was investigated whether incubation with another amino acid (G, H or W) at position K409 gave different results. Aside from changes in temperature and changes in antibody dilution (20, 2, 0.2 and 0.02 ug / mL), the procedure was performed as described in Example 19.

  Incubation of the combination of all the different IgG1 variants for 90 minutes at 15 ° C. revealed a range of different 2-MEA-induced Fab arm exchange efficiencies (as evident from Table 11). The results of bispecific binding at an antibody concentration of 20 μg / mL are shown in Table 11. The results were classified into four categories as specified in the text of Table 11 below; zero (-), low (+/-), medium (+) and high (++) double specificity Sexual coupling efficiency. From these results, it is clear that under the conditions of optimal lower limit, some combinations of amino acid mutations in IgG1 molecules favor 2-MEA induced Fab arm exchange.

(Table 11) Bispecific binding between permissive IgG1 variants (20 μg / mL) for 90 minutes at 15 ° C. (% relative to positive control)

  From the mutated IgG1-2F8 molecules tested (Table 11), six were selected for secondary analysis to confirm the previously obtained results (Table 11). Several variants have their high 2-MEA-induced Fab arm exchange efficiency (IgG1-2F8-L368R) as well as moderate 2-MEA-induced Fab arm exchange efficiency (IgG1-2F8-L368W, IgG1-2F8-F405I, IgG1 -2F8-F405L and IgG1-2F8-Y407W). In addition, IgG1-2F8-Y407Q was analyzed a second time. This is because it showed a positive 2-MEA induced Fab arm exchange reaction which was unexpected as IgG1-7D8-K409H. Overall, these results, presented in FIG. 40, confirm the primary analysis (Table 11) and show that the 2-MEA-induced Fab arm exchange reaction of mutant IgG1-2F8 molecules with IgG1-7D8-K409H is the highest efficiency It is shown that it showed. Furthermore, the 2-MEA-induced Fab arm exchange reaction between the mutated IgG1-2F8 molecules with IgG1-7D8-K409R reported as negative in Examples 28, 29 and 35-37 is still an IgG1 2-MEA induction It is interesting because it potentially promotes sexual Fab arm exchange.

Example 40: Use of a dual specificity format to remove unwanted agonist activities of antagonist c-Met antibodies and convert them to a monovalent, dual specificity format Developed for monoclonal antibody therapy Some bivalent antibodies exhibit undesired agonistic activity due to their binding to their target. This is also the case for most IgG1-based antibodies that target the receptor tyrosine kinase c-Met. These agonist antibodies induce receptor dimerization followed by activation of several downstream signaling pathways. As a result, proliferation and differentiation of (tumor) cells are induced. By using a monovalent antibody format, induction of receptor dimerization can be blocked. The combination of the anti-c-Met antibody Fab arm with an irrelevant antibody Fab arm yields a functionally monovalent, and thus fully antagonistic, bispecific antibody. Here, we use a portion (IgG1-069) or all (IgG1-058) of a bispecific antibody as an agonist antibody, IgG1-b12 (Burton DR, et al, "Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody ", Science. 1994 Nov 11; 266 (5187): 1024-1027). The IgG1-b12 was considered as an unrelated non-binding antibody as it was raised against the viral protein (HIV-gp120). The anti-c-Met antibody used in this example is a completely human monoclonal antibody produced in a transgenic mouse. IgG1-058 and IgG1-069 bind to different epitopes on c-Met.

The two anti-c-Met antibodies used are IgG1, kappa antibodies in which the Fc region has been modified, as further disclosed. They have the following heavy and light chain variable sequences.

Receptor Phosphorylation Monovalent bispecific c-Met antibodies were prepared as described in Example 23 using 25 mM 2-MEA, IgG1-058-F405L or IgG1--069-F405L and IgG1-b12-K409R And Fab arm exchange reaction with The effect of bispecific antibodies on c-Met phosphorylation was evaluated. In the dimerization of two adjacent c-Met receptors by the natural ligand HGF or by agonistic bivalent antibodies, three tyrosine residues (positions 1230, 1234 and 1235) in the intracellular domain of c-Met are cross-linked It is oxidized. This results in the phosphorylation of some other amino acids in the intracellular domain of c-Met and the activation of some signal transduction cascades. Dimerization and activation of c-Met uses an antibody specific for the receptor phosphorylated at these positions, which serves as a readout for the potential receptor activation of anti-c-Met antibodies Can be monitored by

A549 cells, CCL-185 obtained from ATCC, were grown in DMEM medium containing serum until reaching 70% confluence. Cells were trypsinized, washed and plated in 6 well culture plates at 1 ^* 10e6 cells / well in medium containing serum. After overnight incubation, cells were treated with HGF (R & D systems; catalog number 294-HG) (50 ng / mL) or a panel of antibodies (30 μg / mL) and incubated at 37 ° C. for 15 minutes. Cells were washed twice with ice cold PBS and lysed with lysis buffer (Cell Signaling; catalog number 9803) supplemented with a protease inhibitor cocktail (Roche; catalog number 11836170001). Cell lysate samples were stored at -80 ° C. Receptor activation was determined by detection of c-Met phosphorylation in a Western blot using a phospho-c-Met specific antibody. The proteins present in the cell lysate are separated on a 4-12% SDS-PAGE gel and transferred to a nitrocellulose membrane, which is then specific for phosphorylated c-Met (Y1234 / 1235) Stained with antibody (Cell Signaling, catalog number: 3129). As controls for gel loading, total β-actin and c-Met levels were determined using anti-c-Met antibody (Cell Signaling, catalog no. 3127) and anti-β-actin antibody (Cell Signaling, catalog no. 4967). Western blot results are shown in FIG.

  Tissue media control and cells treated with antibody 5D5 (Genentech; WO 96/38557) monovalent format UniBody® (Genmab, WO 2007059782 and WO 2010063785) show no c-Met receptor phosphorylation The The format of the monovalent UniBody used herein is IgG4 with the hinge region deleted and the CH3 region mutated at positions 405 and 407. In contrast, western blot analysis of cells treated with positive control HGF or agonist antibody IgG1-058 showed a clear band at the expected height of phosphorylated c-Met. The partial agonist antibody IgG1-069 showed lower but detectable receptor phosphorylation, suggesting that some cross-linking of the receptor occurred. However, neither the bispecific IgG1 058 / b12 nor the bispecific 069 / b12 antibodies induce c-Met phosphorylation at all, indicating a complete lack of agonist activity associated with the parent antibody (Figure 41).

Effect of c-Met antibody on proliferation of NCI-H441 in vitro
The potential proliferative agonist activity of the c-Met antibody was tested in vitro in the lung adenocarcinoma cell line NCI-H441 (ATCC, HTB-174TM). This cell line expresses high levels of c-Met but does not produce its ligand HGF. NCI-H441 cells were seeded in serum free RPMI (Lonza) on 96 well tissue culture plates (Greiner bio-one, Frickenhausen, Germany) (5,000 cells / well). Anti-c-Met antibody was diluted to 66, 7 nM in serum free RPMI and added to the cells. After 7 days incubation at 37 ° C./5% CO ₂ , the amount of viable cells was quantified by Alamar Blue (BioSource International, San Francisco, US) according to the manufacturer's instructions. Fluorescence was monitored using an EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with a standard alamar blue setting.

  In contrast to IgG1-069, as shown in FIG. 42, no proliferation was induced by incubation of NCI-H441 cells with bispecific IgG1-069 / b12. Also, no proliferation was induced in the UniBody-069 control, which was comparable to no treatment or IgG1-b12 treatment.

Example 41: CDC-mediated cell killing by bispecific antibodies generated by 2-MEA induced Fab arm exchange between human IgG1-2F8-F405L or IgG1-7D8-F405L and IgG1-7D8-K409R
The CD20 antibody IgG1-7D8 can efficiently kill CD20 expressing cells by complement dependent cytotoxicity (CDC). In contrast, the EGFR antibody IgG1-2F8 does not mediate CDC to target cells expressing EGFR. Both IgG1-7D8-K409R and bispecific antibodies generated by 2-MEA induced Fab arm exchange between IgG1-2F8-F405L × IgG1-7D8-K409R (as described in Example 26) CDC can be induced on CD20-expressing cells. It was also tested whether bispecific antibodies generated by 2-MEA induced Fab arm exchange between IgG1-7D8-F405L and IgG1-7D8-K409R could also induce CDC on CD20 expressing cells. 10 ⁵ Daudi cells or Raji cells were preincubated for 15 minutes in a shaker at room temperature with a concentration series of antibody in 100 μL of RPMI medium supplemented with 0.1% BSA. Twenty-five microliters of normal human serum (NHS) was added as a source of complement (20% NHS final concentration) and incubated for 45 minutes at 37 ° C. After incubation, the plate was placed on ice to stop the CDC reaction. 10 μg / mL of propidium iodide (PI) 10 μL (final concentration of 0.6 μg / mL) was added and dead cells and viable cells were identified by FACS analysis.

  Figure 43 shows that the bispecific products produced by 2-MEA induced Fab arm exchange between IgG1-7D8 and IgG1-7D8-F405L and IgG1-7D8-K409R are CD20 expressing Daudi (Figure 43A) and FIG. 43B shows the same potency in inducing CDC-mediated cell killing of CD20 expressing Raji (FIG. 43B). Neither Daudi nor Raji cells express EGFR, leading to monovalent binding of the bispecific antibody created by 2-MEA induced Fab arm exchange between IgG2-2F8-F405L × IgG1-7D8-K409R. This bispecific product also induced CDC-mediated cell killing, albeit with slightly lower efficiency. These data suggest that the CDC ability of the parent antibody was maintained in a bispecific form. The induction of CDC-mediated cell killing by the bivalent bispecific product (IgG1-7D8-F405L × IgG1-7D8-K409R) is a monovalent bispecific product (IgG2-2F8-F405L × IgG1-7D8). The efficiency was slightly higher compared to -K409R). An 11B8 antibody that targets CD20 can not induce CDC-mediated cell death and serves as a negative control.

Example 42: HER2 × HER2 bispecific antibody tested in the in vitro κ targeted ETA 'killing assay From the example, HER2 × HER2 bispecific antibody was targeted to κ Pseudomonas aeruginosa A versatile in vitro cell-based killing assay with exotoxin A (anti-κ-ETA ') shows that cytotoxic agents can be delivered to tumor cells after internalization. This assay utilizes a high affinity anti-kappa domain antibody conjugated to a truncated form of P. aeruginosa exotoxin A. Similar fusion proteins of antibody binding protein (IgG binding motif from Streptococcus protein A or protein G) and diphtheria toxin or Pseudomonas exotoxin A have been described (Mazor Y. et al., J. Immunol. Methods 2007; 321: 41-59); Kuo SR. Et al., 2009 Bioconjugate Chem. 2009; 20: 1975-1982). These molecules, in contrast to anti-kappa-ETA ', bound the Fc portion of the complete antibody. Through internalization and endocytosis sorting, anti-κ-ETA 'domain antibodies undergo proteolytic degradation and reduction of disulfide bonds, separating the catalytic domain from the binding domain. The catalytic domain is then transported from the Golgi to the endoplasmic reticulum via the KDEL retention motif and then transferred to the cytosol where it inhibits protein synthesis and induces apoptosis (Kreitman RJ. Et. Al., BioDrugs 2009; 23: 1-13).

  The anti-HER2 antibodies used in this example and in examples 43-45 below are fully human monoclonal antibodies generated in transgenic mice. They bind to different epitopes on HER2.

They are all IgG1, kappa antibodies, whose Fc region has been modified, as further disclosed. They have the following heavy and light chain variable sequences.

  The HER2 × HER2 bispecific antibody was preincubated with anti-κ-ETA ′ prior to incubation with A431 cells. A431 cells express approximately 15,000 HER2 antibodies per cell (as determined by Qifi analysis) and are not sensitive to treatment with "naked" HER2 antibodies.

  Initially, the optimal concentration of anti-κ-ETA 'for each cell line, ie, the maximum tolerated dose that did not result in nonspecific cell death induction, was determined. A431 cells (2500 cells / well) were seeded in normal cell media in 96 well tissue culture plates (Greiner bio-one) and allowed to adhere for at least 4 hours. These cells were incubated with dilution series 100, 10, 1, 0.1, 0.01, 0.001 and 0 μg / mL of anti-κ-ETA 'in normal cell culture medium. After 3 days, the amount of viable cells was quantified by Alamar Blue (BioSource International, San Francisco, US) according to the manufacturer's instructions. Fluorescence was monitored using an EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with a standard alamar blue setting. The maximum concentration (1 μg / mL for A431 cells) of anti- 場合 -ETA 'alone which did not kill the cells was used in the following experiments.

Next, the effects of HER2 × HER2 bispecific antibody and HER2 monospecific antibody preincubated with anti-κ-ETA ′ were tested for their ability to induce cell death. A431 cells were seeded as described above. Dilution series of HER2-specific antibodies (monospecific and bispecific antibodies) were made and preincubated with the indicated concentrations of anti -'- ETA 'for 30 minutes before adding them to cells. After 3 days of incubation at 37 ° C., the amount of viable cells was quantified as described above. Alamar Blue signal of anti-κ-ETA ′ treated cells preincubated with antibody was plotted relative to treated cells without antibody treatment. EC ₅₀ values and maximal cell death were calculated using GraphPad Prism 5 software. Staurosporine (23.4 μg / mL) was used as a positive control for cell death. An isotype control antibody (IgG1 / K; IgG1-3G8-QITL) was used as a negative control.

  FIG. 44 shows that all anti --- ETA ′ preincubated HER2 bispecific antibodies were able to kill A431 cells in a dose dependent manner. These results demonstrate that most of the HER2 bispecific antibodies tested were more effective than the monospecific antibodies contained in the combination in this anti-κ-ETA 'assay. Furthermore, the potency of the bispecific antibodies 005X169, 025X169 and 153X169 distinguishes the potency of the monospecific antibody lacking activity in this in vitro を targeted ETA 'killing assay, ie HER2 specific antibody 169 It has been shown that it can be increased through the combination of bispecificity with HER2 specific antibodies.

Example 43: Inhibitory modulation of HER2 receptor by incubation with bispecific antibodies targeting different HER2 epitopes
HER2 × HER2 bispecific antibodies can bind two different epitopes on two spatially distinct HER2 receptors. This allows other HER2 × HER2 bispecific antibodies to bind to the remaining epitopes on these receptors. This could cause multivalent receptor cross-linking (as compared to dimerization induced by monovalent antibodies) and as a result was able to enhance repressive modulation of the receptor. AU565 cells were incubated with the antibody and bispecific antibody for 3 days to see if HER2 × HER2 bispecific antibody induced an enhancement of repressive modulation of HER2. Total HER2 levels and antibody bound HER2 levels were determined.

  AU565 cells are seeded in 24-well tissue culture plates in normal cell culture medium (100.000 cells / well), and cultured for 3 days at 37 ° C. in the presence of 10 μg / mL of HER2 antibody or HER2 × HER2 bispecific antibody did. After washing with PBS, cells were lysed by incubating the cells for 30 minutes at room temperature with 25 μl Surefire Lysis buffer (Perkin Elmer, Turku, Finland). Total protein levels were quantified using bicinchoninic acid (BCA) protein assay reagent (Pierce) according to the manufacturer's protocol. HER2 protein levels in the lysate were analyzed using a HER2-specific sandwich ELISA. We captured HER2 using rabbit anti-human HER2 intracellular domain antibody (Cell Signaling) and bound using biotinylated goat anti-human HER2 polyclonal antibody (R & D systems, Minneapolis, USA) followed by Streptavidin-poly-HRP Detected HER2. The reaction is visualized by dilution of 2,2'-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS tablet in 50 mL of ABTS buffer [Roche Diagnostics, Almere, The Netherlands]) and oxalic acid It was stopped with (Sigma-Aldrich, Zwijndrecht, The Netherlands). The fluorescence at 405 nm was measured with a microtiter plate reader (Biotek Instruments, Winooski, USA) and the amount of HER2 was expressed as a percentage of untreated cells.

  The results are shown in FIG. The figure demonstrates that all HER2 × HER2 bispecific antibodies tested induced 40% or more of the repressive modulation of HER2. Interestingly, all HER2 × HER2 bispecific antibodies demonstrated an increase in the repressive modulation of HER2 compared to both of its monospecific counterparts.

Example 44: Colocalization of HER2xHER2 bispecific antibody and lysosomal marker LAMP1 analyzed by confocal microscopy From HER2 inhibitory modulation assay as described in Example 43: HER2xHER2 double It was suggested that specific antibodies can increase lysosomal degradation of HER2. Confocal microscopy techniques were applied to confirm these findings. AU565 cells were grown on glass coverslips (1.5 micron thickness, Thermo Fisher Scientific, Braunschweig, Germany) in standard tissue medium for 3 days at 37 ° C. Cells were preincubated with leupeptin (Sigma) for 1 hour to block lysosomal activity, followed by addition of 10 ug / mL of HER2 monospecific antibody or HER2 × HER2 bispecific antibody. The cells were incubated at 37 ° C. for an additional 3 or 18 hours. Then they were washed with PBS and incubated with 4% formaldehyde (Klinipath) for 30 minutes at room temperature. The slides are washed with blocking buffer (PBS supplemented with 0.1% saponin [Roche] and 2% BSA [Roche]) and incubated with blocking buffer containing 20 mM NH ₄ Cl for 20 minutes to remove formaldehyde It has quenched. Slides were again washed with blocking buffer and incubated with mouse anti-human CD107a (LAMP1) (BD Pharmingen) for 45 minutes at room temperature to stain lysosomes. After washing with blocking buffer, slides were incubated with a cocktail of secondary antibodies; goat anti-mouse IgG-Cy5 (Jackson) and goat anti-human IgG-FITC (Jackson) for 30 minutes at room temperature. Slides were again washed with blocking buffer and mounted overnight on microscope slides with 20 μl of mounting medium (6 grams of glycerol [Sigma] and 2.4 grams of Mowiol 4-88 [Omnilabo] dissolved in 6 mL of distilled water To this, 12 mL of 0.2 M Tris [Sigma] pH 8.5 was added, followed by incubation for 10 minutes at 50-60 ° C. The mounting medium was aliquoted and stored at -20 ° C)). Slides were imaged with a Leica SPE-II confocal microscope (Leica Microsystems) equipped with a 63 × 1.32-0.6 oil immersion objective and LAS-AF software. Pixel saturation should be avoided to allow quantification of overlapping pixel intensities. Therefore, the FITC laser intensity was reduced to 10%, the smart gain was set to 830 V, and the smart offset was set to -9.48%. By using these settings, bispecific antibodies were clearly visualized without pixel saturation, but monospecific antibodies were sometimes difficult to detect. These settings were kept the same for all confocal slides analyzed in order to compare lysosomal colocalization between monospecific and bispecific antibodies.

  The 12-bit images were analyzed for colocalization using MetaMorph® software (Meta Series 6.1 version, Molecular Devices Inc, Sunnyvale California, USA). FITC and Cy5 images were captured as a stack and background subtracted. The same threshold settings were used for all FITC images and all Cy5 images (set manually). Colocalization was depicted as pixel intensity of FITC in the overlap region (ROI). Here, the ROI is composed of all Cy5 positive regions. Images were normalized using the pixel intensity of Cy5 to compare different slides stained with several HER2 antibodies or HER2 × HER2 bispecific antibodies. The lysosomal marker LAMP1 (CD107a) was stained using goat anti-mouse IgG-Cy5. The pixel intensity of LAMP1 should not differ between the various HER2 antibodies or HER2 × HER2 bispecific antibodies tested (one cell had approximately 200.000 Cy5 pixel intensities).

Normalized values for FITC and Cy5 colocalization =
[(TPI-FITC ×% FITC-Cy5 colocalization) / 100] × [200.000 / TPI-Cy5]

  In this equation, TPI represents the total pixel intensity.

  The above equation represents the percentage of viable cells as measured by FITC pixel intensity overlapping with Cy5 for various monospecific HER2 and HER2 × HER2 bispecific antibodies. For each antibody or bispecific molecule delineated, three different images were analyzed from one slide containing approximately 1, 3 or 5 or more cells. Significant differences were noted between the different images in each slide. However, it was clear that all HER2 × HER2 bispecific antibodies show increased colocalization with the lysosomal marker LAMP1 when compared to their monospecific counterparts. These results indicate that HER2 × HER2 bispecific antibodies, once internalized, are efficiently localized towards the lysosomal compartment, making them suitable for bispecific antibody drug conjugate approaches Suggest.

Example 45: Inhibition of proliferation of AU565 cells by incubation with HER2 monospecific antibody or HER2 × HER2 bispecific antibody
HER2 bispecific antibodies were tested for their ability to inhibit the growth of AU565 cells in vitro. Due to the high HER2 expression levels on AU565 cells (approximately 1.000.000 copies per cell as determined by the Qifi kit), HER2 is constitutively active in these cells, thus a ligand-inducible heterodimeric It does not depend on establishment. In a 96-well tissue culture plate (Greiner bio-one, Frickenhausen, Germany), 9.000 AU565 per well in the presence of 10 μg / mL of HER2 antibody or HER2 × HER2 bispecific antibody in serum-free cell culture medium Cells were seeded. As a control, cells were seeded in serum free medium without antibody or bispecific antibody. After 3 days, the amount of viable cells was quantified using Alamar Blue (BioSource International, San Francisco, US) according to the manufacturer's instructions. Fluorescence was monitored using an EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with a standard alamar blue setting. Alamar Blue signal of antibody treated cells was plotted as a percentage of untreated cells.

  FIG. 47 depicts Alamar Blue fluorescence intensity of AU565 cells after incubation with HER2 antibody and HER2 × HER2 bispecific antibody. HERCEPTIN® (trastuzumab) was included as a positive control and demonstrated inhibition of proliferation as described by Juntilla TT. Et al., Cancer Cell 2009; 15: 429-440. All HER2 × HER2 bispecific antibodies were able to inhibit the growth of AU565 cells. Bispecific antibodies: IgG1-005-ITL × IgG1-169-K409R and IgG1-025-ITL × IgG1-005-K409R are more effective in this assay compared to their monospecific antibody counterparts The

Example 46: In Vitro and In Vivo Analysis of FcRn Binding by Hinge-Depleted IgG1 Bispecific Antibody and Bispecific IgG1 Antibody Containing One or Two FcRn Sites in the Fc Region In this Example: Fig. 6 illustrates the production of bispecific molecules, ie molecules having different features in each Fab arm according to the invention.

  The neonatal Fc receptor (FcRn) participates in the long plasma half life of IgG by protecting it from degradation. After internalization of the antibody, FcRn binds to the antibody Fc region in endosomes, where the interaction is stable in a mildly acidic environment (pH 6.0). By recycling to the plasma membrane, where the environment is neutral (pH 7.4), internalization is lost and the antibody is released again into the circulating blood. The Fc region of the antibody contains two FcRn binding sites, one for each heavy chain at the CH2-CH3 interface. The H435A mutation in the Fc region of the antibody abrogates binding to FcRn (Shields, RL, et al, J Biol Chem, 2001, Firan, M., et al, Int Immunol, 2001), and the hinge region is FcRn. It is believed to affect binding (Kim, JK, et al., Mol Immunol., 1995). Furthermore, the role of binding of bivalent antibodies over binding of monovalent antibodies to FcRn has been suggested in efficient recycling (Kim, J. K., et al., Scand J Immunol., 1994).

  In this example, the effect of FcRn binding titer is assessed by an asymmetric bispecific IgG1 molecule that contains a single FcRn binding site. Asymmetric bispecific hinge deletion IgG1 (Uni-G1) molecules are evaluated for additional contribution of the hinge region.

FcRn binding of bispecific IgG1 molecules or hinge-deleted IgG1 (Uni-G1) molecules containing zero, one or two FcRn binding sites was measured by human and mouse FcRn ELISA. Antibodies IgG1-2F8-ITL, IgG1-7D8-K409R and IgG1-7D8-K409R-H435A monospecific molecules were generated as described in Examples 2, 3, 4 and 5. Hinge-depleted IgG1 molecules Uni-G1-2F8-ITL, Uni-G1-7D8-K409R and Uni-G1-7D8-K409R-H435A monospecific molecules were generated as described in Example 11. Bispecific IgG1 molecules were generated by 2-MEA induced Fab arm exchange between IgG1-2F8-ITL molecules and IgG1-7D8-K409R molecules or IgG1-7D8-K409R-H435A molecules. Bispecific hinge deletion IgG1 molecules were generated by incubation of Uni-G1-2F8-ITL with Uni-G1-7D8-K409R or Uni-G1-7D8-K409R-H435A. Add a 3-fold dilution series of monospecific and bispecific IgG1 molecules and hinge-depleted IgG1 molecules to biotinylated human or mouse FcRn captured on streptavidin coated elisa plates, then for 1 hour Incubations at pH 6.0 and 7.4 were performed. Bound antibody and hinge-deleted IgG1 molecules were visualized using horseradish peroxidase-labeled goat anti-human (Fab ') ₂ as a conjugate and ABTS as a substrate. The results were measured as optical density at a wavelength of 405 nm using an EL 808-Elisa reader.

  FIG. 48 shows the results of binding of monospecific or bispecific IgG1 molecules and hinge deleted IgG1 molecules to human FcRn (A) and mouse FcRn (B) at pH 6.0 and pH 7.4. As expected, not all antibodies tested, both (bispecific) and hinge-deleted IgG1 molecules, bind efficiently to FcRn (both human and mouse) at pH 7.4. Under mildly acidic conditions (pH 6.0), monospecific IgG1-2F8-ITL and bispecific IgG1 generated from IgG1-2F8-ITL and IgG1-7D8-K409R are human and in the case of mouse FcRn It shows bivalent binding efficiency to FcRn, albeit three times higher than it, which reproduces the positive control (IgG1-2F8) for FcRn binding. This suggests that ITL mutations and K409R do not interfere with binding to FcRn.

  When the binding of IgG1 molecules to human and mouse FcRn is compared at pH 6.0, two FcRn interaction sites vs. 1 vs 0 apparent effects can be seen (Figures XXA and B, pH 6, left panel) ). IgG1-2F8-ITL, IgG1-7D8-K409R and IgG1-2F8-ITL / IgG1-7D8-K409R (two FcRn binding sites) bind equally to the control (IgG1-2F8). The molecule IgG1-7D8-K409R-H435A with zero FcRn binding site shows no binding at all. The molecule IgG1-2F8-ITL / IgG1-7D8-K409R-H435A with one FcRn binding site shows moderate binding when compared to two molecules with an FcRn binding site.

  FIG. 48 (A), pH 6.0, right panel shows binding of hinge-deleted IgG1 molecule (Uni-G1) to human FcRn. All hinge-deleted molecules have their interaction with human FcRn impaired when compared to the control IgG1 molecule (IgG1-2F8), and hinges actually evaluated for FcRn as assessed in the FcRn binding ELISA. It is suggested to affect the interaction. No apparent effect of 2 FcRn interaction sites vs. 1 vs. 0 can be observed when binding to human FcRn at pH 6.0 is composed of these hinge deleted molecules.

  However, binding of human IgG to mouse FcRn is more potent, so when comparing the binding of these hinge-deleted IgG molecules to mouse FcRn at pH 6.0, 2 FcRn interaction sites vs 1 vs 0 There are distinct effects (Figure 48 (B), pH 6.0, right panel). The binding of Uni-G1-7D8-K409R-H435A / Uni-G1-2F8-ITL (1 FcRn binding site) is as follows: Uni-G1-2F8-ITL, Uni-G1-7D8-409R and Uni-G1-2F8- It is moderate when compared to the binding of ITL / Uni-G1-7D8-K409R (2 FcRn binding sites) and Uni-G1-2F8-ITL-H435A (0 FcRn binding sites, no binding).

Example 47: Her2xCD3 bispecific antibody tested in in vitro cytotoxicity assay
CD3 is a co-receptor in the T cell receptor complex expressed on mature T cells. The combination of the CD3 specific antibody Fab arm and the tumor antigen specific antibody Fab arm in a bispecific antibody results in specific targeting of T cells to tumor cells leading to T cell mediated tumor cell lysis I will. Similarly, CD3 positive T cells could be targeted to other derailed cells in the body, to infected cells or directly to pathogens.

huCLB-T3/4 (Parren et al., Res Immunol. 1991, 142(9):749-63により記述されている配列。わずかなアミノ酸置換を導入して、配列を、最も近いヒト生殖細胞系列に似させた。)

Her2 × CD3 bispecific antibodies were generated. The heavy chain and light chain variable region sequences of Her2-specific Fab arms were as shown for antibodies 153 and 169 in Example 42. The following heavy and light chain variable region sequences for the CD3 specific Fab arm were used:
YTH 12.5 (the sequence described by Routledge et al., Eur J Immunol. 1991, 21 (11): 2717-25.)

huCLB-T3 / 4 (Parren et al., Res Immunol. 1991, 142 (9): 749-63. Sequence introduced into the nearest human germline by introducing minor amino acid substitutions. I made it similar.)

  All antibodies were expressed as IgG1, kappa, whose Fc region has been modified as described below: IgG1-Her2-153-K409R and IgG1-Her2-153-N297Q-K409R, IgG1-Her2 -169-K409R, IgG1-hu-CLB-T3 / 4-F405L and IgG1-hu-CLB-T3 / 4-N297Q-F405L, IgG1-YTH12.5-F405L and IgG1-YTH12.5-N297Q-F405L.

  Bispecific antibodies derived from these Her2 and CD3 specific antibodies were generated as described in Example 11 and tested in in vitro cytotoxicity assays using AU565 cells.

AU565 cells were cultured to near confluence. Cells were washed twice with PBS and trypsinized at 37 ° C. for 5 minutes. 12 mL of medium was added to inactivate the trypsin and cells were spun down at 800 rpm for 5 minutes. The cells were resuspended in 10 mL medium and single cell suspensions were made by passing the cells through a cell strainer. 100 μL of a suspension of 5 × 10 ⁵ cells / mL was added to each well of a 96-well culture plate and cells were incubated at 37 ° C., 5% CO 2 for at least 3 hours to allow them to adhere to the plate.

Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy volunteers using Leucosep 30 mL test tubes according to the manufacturer's protocol (Greiner Bio-one). T cells were isolated from PBMC preparations by negative selection using the Untouched Human T-cells Dynabead kit (Dynal). Isolated cells were resuspended in medium to a final concentration of 7 × 10 ⁶ cells / mL.

Media was removed from adherent AU565 cells and replaced with 50 μl / well of 2 × concentrated antibody-diluent and 50 μl / well of T cells 7 × 10 ⁶ cells / mL (effector: target ratio = 7: 1). The plates were incubated at 37 ° C., 5% CO ₂ for 3 days. The supernatant was removed and the plate was washed twice with PBS. 150 μL of culture medium and 15 μL of alamar blue were added to each well. Plates were incubated for 4 hours at 37 ° C., 5% CO ₂ and absorbance was measured (Envision, Perkin Elmer).

  From FIG. 49, control antibodies (Her2 monospecific IgG1-Herceptin, CD3 monospecific IgG1-YTH12.5 and monospecific IgG1-huCLB-T3 / 4, irrelevant antigen monospecific IgG1-b12, (CD3 × b12 bispecific antibody) did not induce T cell-mediated cytotoxicity, but the bispecific (duplicate) Her2 × CD3 antibody huCLB / Her2-153, huCLB / Her2-169 , YTH12.5 / Her2-153 and YTH12.5 / Her2-169 are shown to induce dose-dependent T cell-mediated cytotoxicity of AU565 cells. Bispecific antibodies containing Her2-169 were more potent than those containing Her2-153.

  We have created variants of IgG1-hu-CLB-T3 / 4, IgG1-YTH12.5 and Her2-153 that contained the N297Q mutation to remove glycosylation sites; glycosylation at this site is IgG-Fcγ acceptance It is important for body interaction (Bolt S et al., Eur J Immunol 1993, 23: 403-411). FIG. 49 shows N297Q mutations of Her2 × CD3 bispecific antibodies YTH12.5 / Her2-153 and huCLB / Her2-153, hence lack of Fc glycosylation is dose dependent T cell mediated of AU565 cells Indicates that it did not affect the potential to induce cell damage.

In a further aspect, the present invention relates to heterodimeric proteins obtained or obtainable by the method of the present invention and methods for producing the heterodimeric proteins of the present invention by coexpression in suitable host cells About.
The present invention also relates to the following.
[Item 1]
In vitro method for producing heterodimeric protein comprising the following steps:
a) providing a first homodimeric protein comprising an Fc region of an immunoglobulin, wherein the Fc region comprises a first CH3 region,
b) providing a second homodimeric protein comprising an immunoglobulin Fc region, wherein the Fc region comprises a second CH3 region,
Here, the sequences of the first CH3 region and the second CH3 region are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is different from the first CH3 region and the second CH3 region. Seems to be stronger than each of the homodimeric interactions of the second CH3 region,
c) incubating the first protein with the second protein under reducing conditions sufficient to allow cysteines in the hinge region to cause disulfide bond isomerization, and
d) obtaining said heterodimeric protein.
[Item 2]
The first homodimeric protein and the second homodimeric protein are (i) an Fc region, (ii) an antibody, (iii) a fusion protein comprising an Fc region, and (iv) a prodrug, a peptide, a drug Or an in vitro method according to item 1, selected from the group consisting of an Fc region linked to a toxin.
[Item 3]
The in vitro method according to item 1, wherein the first homodimeric protein is a full-length antibody.
[Item 4]
The in vitro method according to any one of the preceding items, wherein the second homodimeric protein is a full length antibody.
[Item 5]
The in vitro method according to any one of the preceding items, wherein the first and second homodimeric proteins are both antibodies and bind to different epitopes.
[Item 6]
The Fc region of the first homodimeric protein is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, and the Fc region of the second homodimeric protein is IgG1, IgG2 , In vitro method according to any of the preceding items, which is of an isotype selected from the group consisting of: IgG3, and IgG4.
[Item 7]
The in vitro method according to any one of the preceding items, wherein the Fc region of both the first homodimeric protein and the second homodimeric protein is of the IgG1 isotype.
[Item 8]
The in vitro method according to any one of the preceding items, wherein one of the Fc regions of the homodimeric protein is of the IgG1 isotype and the other is of the IgG4 isotype.
[Item 9]
Any one of the preceding items, wherein the increase in the strength of the heterodimeric interaction compared to each of the homodimeric interactions is due to CH3 modification other than the introduction of a covalent bond, a cysteine residue or a charged residue An in vitro method according to clause.
[Item 10]
The heterodimeric interaction between the first protein and the second protein in the resulting heterodimeric protein is caused by Fab arm exchange at 0.5 mM GSH under the conditions described in Example 13. The in vitro method according to any one of the preceding items which is astonishing.
[Item 11]
The heterodimeric interaction between the first protein and the second protein in the resulting heterodimeric protein causes Fab arm exchange in vivo in mice under the conditions described in Example 14 The in vitro method according to any one of the preceding items which is as such.
[Item 12]
If the heterodimeric interaction between the first protein and the second protein in the resulting heterodimeric protein is determined, for example, as described in Example 30, then two homodimers The in vitro method according to any one of the preceding items, wherein the interaction is more than 2 times stronger, eg more than 3 times stronger, eg more than 5 times stronger than the strongest one.
[Item 13]
When the sequences of the first CH3 region and the second CH3 region are assayed as described in Example 30, between the first protein and the second protein in the resulting heterodimeric protein The in vitro method according to any one of the preceding items, wherein the dissociation constant of the heterodimer interaction is such that it is less than 0.05 micromolar.
[Item 14]
When the sequences of the first CH3 region and the second CH3 region are assayed as described in Example 21, the dissociation constant of both homodimer interactions is greater than 0.01 micromolar, for example 0.05 micromolar. More preferably, 0.01 to 10 micromoles, such as 0.05 to 10 micromoles, more preferably 0.01 to 5 micromoles, such as 0.05 to 5 micromoles, even more preferably 0.01 to 1 micromoles, such as 0.05 to 1 micromole, An in vitro method according to any of the preceding items which is such that it is 0.01 to 0.5 micromolar or 0.01 to 0.1 micromolar.
[Item 15]
The in vitro method according to any one of the preceding items, wherein the sequences of the first CH3 region and the second CH3 region comprise amino acid substitutions at non-identical positions.
[Item 16]
16. The in vitro method according to item 15, wherein the amino acid substituent is a natural amino acid or a non-natural amino acid.
[Item 17]
The first homodimeric protein has only one amino acid substitution in the CH3 region and the second homodimeric protein has only one amino acid substitution in the CH3 region as compared to the wild type CH3 region. The in vitro method according to any one of the preceding items, having.
[Item 18]
The first homodimeric protein has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407 and 409, and the second homodimeric protein is , 368, 370, 399, 405, 407 and 409, having an amino acid substitution at a position selected from the group consisting of 368, 370, 399, 405, 407 and 409, wherein the first homodimeric protein and the second homodimeric protein are the same 17. The in vitro method according to any one of the preceding items, wherein the position is not substituted.
[Item 19]
From the group consisting of 366, 368, 370, 399, 405 and 407, wherein the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein 20. The in vitro method according to any one of the preceding items, having an amino acid substitution at the selected position.
[Item 20]
19, wherein the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has an amino acid other than Phe at position 405. An in vitro method according to any one of the preceding claims.
[Item 21]
The above item, wherein the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein has an amino acid other than Phe, Arg or Gly at position 405 The in vitro method according to any one of 1 to 18.
[Item 22]
The first homodimeric protein comprises Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein is an amino acid other than Phe at position 405 and position 409 20. The in vitro method according to any one of the above items 1 to 18 comprising Lys.
[Item 23]
The first homodimeric protein contains Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein is other than Phe, Arg or Gly at position 405 20. The in vitro method according to any one of the preceding items, wherein the amino acid and Lys at position 409.
[Item 24]
The first homodimeric protein contains Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has Leu at position 405 and Lys at position 409 20. The in vitro method according to any one of the above items 1-18, comprising.
[Item 25]
The first homodimeric protein comprises Phe at position 405 and Arg at position 409, and the second homodimeric protein comprises an amino acid other than Phe, Arg or Gly at position 405 and Lys at position 409, 20. The in vitro method according to any one of items 1 to 18 above.
[Item 26]
20. Any of the preceding items, wherein the first homodimeric protein comprises Phe at position 405 and Arg at position 409 and the second homodimeric protein comprises Leu at position 405 and Lys at position 409. An in vitro method according to any one of the preceding claims.
[Item 27]
The first homodimeric protein comprises an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein comprises Lys at position 409, Thr at position 370 and Leu at position 405, 20. The in vitro method according to any one of items 1 to 18 above.
[Item 28]
20. Any of the above items, wherein the first homodimeric protein comprises Arg at position 409 and the second homodimeric protein comprises Lys at position 409, Thr at position 370 and Leu at position 405. An in vitro method according to any one of the preceding claims.
[Item 29]
The first homodimeric protein contains Lys at position 370, Phe at position 405 and Arg at position 409, and the second homodimeric protein is Lys at position 409, Thr at position 370 and Leu at position 405. 20. The in vitro method according to any one of items 1-18, comprising the steps of
[Item 30]
The first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has Tyr, Asp, Glu, Phe, Lys, Gln, Arg at position 407 20. The in vitro method according to any one of the above items 1 to 18, wherein the in vitro method has an amino acid other than Ser or Thr. [Item 31]
The first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein at position 407 Ala, Gly, His, Ile, Leu, Met, Asn 20. The in vitro method according to any one of the above items 1 to 18, having Val, Tr or Trp.
[Item 32]
The first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has Gly, Leu, Met, Asn or Trp at position 407, said 20. The in vitro method according to any one of items 1-18.
[Item 33]
The first homodimeric protein has Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has Tyr, Asp, Glu, at position 407 20. The in vitro method according to any one of the preceding items, having an amino acid other than Phe, Lys, Gln, Arg, Ser or Thr and Lys at position 409.
[Item 34]
The first homodimeric protein has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has an Ala, Gly, His, at position 407 20. The in vitro method according to any of the preceding items, having Ile, Leu, Met, Asn, Val or Trp and Lys at position 409.
[Item 35]
The first homodimeric protein has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has a Gly, Leu, Met, at position 407 20. The in vitro method according to any one of the preceding items, having Asn or Trp and Lys at position 409.
[Item 36]
The first homodimeric protein has Tyr at position 407 and Arg at position 409, and the second homodimeric protein has Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser at position 407 20. The in vitro method according to any one of the preceding items, wherein it has an amino acid other than Thr and Lys at position 409.
[Item 37]
The first homodimeric protein has Tyr at position 407 and Arg at position 409, and the second homodimeric protein at position 407 Ala, Gly, His, Ile, Leu, Met, Asn, Val Or in vitro method according to any one of the preceding items, having a Trp and a Lys at position 409.
[Item 38]
The first homodimeric protein has Tyr at position 407 and Arg at position 409, and the second homodimeric protein has Gly, Leu, Met, Asn or Trp at position 407 and Lys at position 409 20. The in vitro method according to any one of the above items 1 to 18, which comprises.
[Item 39]
The first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein is
(i) an amino acid other than Phe, Leu and Met at position 368, or
(ii) Trp at position 370, or
(iii) amino acids other than Asp, Cys, Pro, Glu or Gln at position 399
20. The in vitro method according to any one of the preceding items, wherein the in vitro method comprises the steps of
[Item 40]
The first homodimeric protein has Arg, Ala, His or Gly at position 409 and the second homodimeric protein is
(i) Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val or Trp at position 368, or
(ii) Trp at position 370, or
(iii) at position 399 Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr
20. The in vitro method according to any one of the preceding items, wherein the in vitro method comprises the steps of
[Item 41]
The first homodimeric protein has an Arg at position 409 and the second homodimeric protein is (i) at position 368: Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val or Trp Or
(ii) Trp at position 370, or
(iii) Phe, His, Lys, Arg or Tyr at position 399
20. The in vitro method according to any one of the preceding items, wherein the in vitro method comprises the steps of
[Item 42]
The in vitro method according to any one of the preceding items, wherein the first and second CH3 regions comprise the sequence set forth in SEQ ID NO: 1 except for the indicated mutations.
[Item 43]
The in vitro method according to any one of the preceding items, wherein neither the first homodimeric protein nor the second homodimeric protein comprises a Cys-Pro-Ser-Cys sequence in the hinge region.
[Item 44]
The in vitro method according to any of the preceding items, wherein both the first homodimeric protein and the second homodimeric protein comprise a Cys-Pro-Pro-Cys sequence in the hinge region.
[Item 45]
The in vitro method according to any one of the preceding items, wherein the first homodimeric protein and the second homodimeric protein are human antibodies except for any mutations specified.
[Item 46]
The in vitro method according to any one of the preceding items, wherein the first homodimeric protein and the second homodimeric protein are heavy chain antibodies.
[Item 47]
47. An in vitro method according to any of the preceding items, wherein both the first homodimeric protein and the second homodimeric protein further comprise a light chain.
[Item 48]
50. An in vitro method according to item 47, wherein the light chains are different.
[Item 49]
The in vitro method according to any one of the preceding items, wherein the first and / or the second homodimeric protein comprises a mutation which removes the acceptor site of Asn-linked glycosylation.
[Item 50]
The in vitro method according to any one of the preceding items, wherein the first and second homodimeric proteins provided in steps a) and b) are purified.
[Item 51]
The in vitro method according to any one of the preceding items, wherein the first and / or the second homodimeric protein is linked to a drug, a prodrug or a toxin or comprises an acceptor group for them.
[Item 52]
56. In vitro method according to any of the above items, wherein the first and / or second epitope is located on a tumor cell.
[Item 53]
52. In vitro method according to any of the above items, wherein the first or second epitope is located on a tumor cell and the other epitope is located on an effector cell.
[Item 54]
54. The in vitro method according to any one of the above items 4-53, wherein the epitope is located on T cells as on CD3 expressed on T cells.
[Item 55]
The first or second epitope is located on a tumor cell, and the other epitope is located on a radioactive isotope, a toxin, a drug or a prodrug, which may optionally be coupled or linked to a peptide or hapten , In vitro method according to any of the above items 4-52.
[Item 56]
56. In vitro method according to any of the above items, wherein the first or second epitope is located on a tumor cell and the other epitope is located on a high electron density vesicle or minicell.
[Item 57]
52. The in vitro method according to any one of the preceding items, wherein the homodimeric protein is both an antibody and the first and second antibodies bind to different epitopes on the same tumor cell.
[Item 58]
Both homodimeric proteins are antibodies and the first antibody binds to an epitope on tumor cells and the other antibody is unrelated or inactive without any in vivo binding activity associated with the intended use 63. An in vitro method according to any one of the preceding items, which is an antibody.
[Item 59]
The reducing conditions in step c) include the addition of a reducing agent such as a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine or a chemical derivative thereof An in vitro method according to any one of the items.
[Item 60]
The in vitro method according to any of the preceding items, wherein step c) is performed under reducing conditions with a redox potential of -150 to -600 mV, for example -250 to -400 mV.
[Item 61]
Any of the preceding items, wherein step c) comprises incubation for at least 90 minutes at a temperature of at least 20 ° C. in the presence of at least 25 mM 2-mercaptoethylamine or in the presence of at least 0.5 mM dithiothreitol The in vitro method according to one of the claims.
[Item 62]
The in vitro method according to any one of the preceding items, wherein step d) comprises the removal of a reducing agent, for example by desalting.
[Item 63]
Methods for Selection of Bispecific Antibodies with Desired Properties, Including the Following Steps:
a) providing a first set of homodimeric antibodies comprising antibodies with different variable regions, wherein the first set of antibodies comprises the same first CH3 region,
b) providing a second set of homodimeric antibodies comprising antibodies with different variable regions or the same variable region, wherein the second set of antibodies comprises the same second CH3 region,
Here, the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is different from the first CH3 region and the second CH3 region. Seems to be stronger than each of the homodimeric interactions of the CH3 domain,
c) incubating the combination of said first and said second set of antibodies under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization. , Thus creating a set of bispecific antibodies,
d) optionally returning the conditions to non-reducing conditions,
e) assaying the resulting set of bispecific antibodies for a given desired property, and
f) selecting bispecific antibodies with the desired properties.
[Item 64]
76. The method of paragraph 63, wherein the second set of homodimeric antibodies have different variable regions.
[Item 65]
76. The method of paragraph 63, wherein the second set of homodimeric antibodies have identical variable regions but different amino acid or structural changes outside the antigen binding region.
[Item 66]
Methods for producing heterodimeric proteins comprising the following steps:
a) providing a first nucleic acid construct encoding a first polypeptide comprising an immunoglobulin first Fc region, wherein the first Fc region comprises a first CH3 region,
b) providing a second nucleic acid construct encoding a second polypeptide comprising an immunoglobulin second Fc region, wherein the second Fc region comprises a first CH3 region,
Here, the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is different from the first CH3 region and the second CH3 region. Seems to be stronger than each of the homodimeric interactions of the CH3 domain, and
Here, the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein comprises 366, 368, 370, 399, 405 and 407. Having an amino acid substitution at a position selected from the group consisting of
And / or
Wherein the sequences of the first and second CH3 regions are 0.01 to 10 micromolar, for example, the dissociation constant of each homodimeric interaction of the CH3 regions as assayed in Example 21, eg 0.05 to 10 micromole, more preferably 0.01 to 5 micromole, for example 0.05 to 5 micromole, even more preferably 0.01 to 1 micromole, for example 0.05 to 1 micromole, 0.01 to 0.5 micromole or 0.01 to 0.1 micromole It is like being
c) co-expressing said first and second nucleic acid constructs in a host cell, and
d) obtaining the heterodimeric protein from cell culture.
[Item 67]
The first CH3 region has an amino acid other than Lys, Leu or Met at position 409, and the second CH3 region has an amino acid other than Phe at position 405, such as other than Phe, Arg or Gly at position 405 ,
Or
The first CH3 region has an amino acid other than Lys, Leu or Met at position 409 and the second CH3 region at position 407 is other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr With amino acids,
The method according to item 66.
[Item 68]
68. The method of claim 66 or 67, wherein the first and second polypeptides are full-length heavy chains of two antibodies that bind to different epitopes.
[Item 69]
70. A method according to any of the aforementioned items 66-68, wherein step c) further comprises co-expressing one or more nucleic acid constructs encoding light chains in a host cell.
[Item 70]
70. A method according to any of the items 66-69, further comprising the features of any one or more of items 2-58.
[Item 71]
An expression vector comprising the nucleic acid construct identified in any one of paragraphs 66-70.
[Item 72]
A host cell comprising the nucleic acid construct as specified in any one of paragraphs 66-70.
[Item 73]
73. A heterodimeric protein obtained or obtainable by the method according to any one of items 1 to 72 above.
[Item 74]
A first polypeptide comprising an immunoglobulin first Fc region, wherein the first Fc region comprises a first CH3 region, and an immunoglobulin second, wherein the second Fc region comprises a second CH3 region A heterodimeric protein comprising a second polypeptide comprising an Fc region of said first and second CH3 regions, wherein the sequences of said first and second CH3 regions are different, and said first CH3 region and second CH3 region The heterodimeric interaction with the region is such that it is stronger than each of the homodimeric interactions of the first CH3 region and the second CH3 region, and
Here, the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein comprises 366, 368, 370, 399, 405 and 407. Having an amino acid substitution at a position selected from the group consisting of
And / or
Wherein the sequences of the first and second CH3 regions are 0.01 to 10 micromolar, for example, the dissociation constant of each homodimeric interaction of the CH3 regions as assayed in Example 21, eg 0.05 to 10 micromole, more preferably 0.01 to 5 micromole, for example 0.05 to 5 micromole, even more preferably 0.01 to 1 micromole, for example 0.05 to 1 micromole, 0.01 to 0.5 micromole or 0.01 to 0.1 micromole It is like being
Said heterodimeric protein.
[Item 75]
The first CH3 region has an amino acid other than Lys, Leu or Met at position 409, and the second CH3 region has an amino acid other than Phe at position 405, such as other than Phe, Arg or Gly. Or
The first CH3 region has an amino acid other than Lys, Leu or Met at position 409 and the second CH3 region at position 407 is other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr With amino acids,
73. A heterodimeric protein according to item 73.
[Item 76]
76. A heterodimeric protein according to item 74 or 75, wherein the first and second polypeptides are full-length heavy chains of two antibodies that bind to different epitopes.
[Item 77]
79. A heterodimeric protein according to item 76, further comprising two full length light chains.
[Item 78]
79. A heterodimeric protein according to any one of the preceding items 73-77, further comprising a feature of any one or more of items 2-58.
[Item 79]
79. A heterodimeric protein according to any one of items 74 to 78 for use as a medicament. [Item 80]
80. A heterodimeric protein according to item 79 for use in the treatment of cancer.
[Item 81]
79. A pharmaceutical composition comprising the heterodimeric protein of any one of paragraphs 74-78 and a pharmaceutically acceptable carrier.
[Item 82]
79. For inhibiting the growth and / or proliferation of tumor cells, comprising the administration of the heterodimeric protein according to any one of items 74 to 78 to an individual in need thereof, and / or of a tumor cell A method for killing.

Thus, in a further aspect, the present invention relates to a method for producing a heterodimeric protein comprising the following steps:
a) providing a first nucleic acid construct encoding a first polypeptide comprising an immunoglobulin first Fc region, wherein the first Fc region comprises a first CH3 region,
b) providing a second nucleic acid construct encoding a second polypeptide comprising an immunoglobulin second Fc region, wherein the second Fc region comprises a second CH3 region,
Here, the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is the first CH3 region and the second CH3 region. And each of the first homodimeric proteins has an amino acid at position 409 other than Lys, Leu or Met, and the second homodimeric protein is more likely than each of the domain homodimeric interactions. The homodimeric protein of has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 407,
And / or where the sequences of the first and second CH3 regions are 0.01 to 10 microns when the dissociation constant of each homodimer interaction of the CH3 region is assayed as described in Example 21 Moles, for example 0.05 to 10 micromoles, more preferably 0.01 to 5 micromoles, for example 0.05 to 5 micromoles, even more preferably 0.01 to 1 micromoles, for example Such as being 0.1 micromolar,
c) co-expressing said first and second nucleic acid constructs in a host cell, and
d) obtaining the heterodimeric protein from cell culture.

Claims (82)

In vitro method for producing heterodimeric protein comprising the following steps:
a) providing a first homodimeric protein comprising an Fc region of an immunoglobulin, wherein the Fc region comprises a first CH3 region,
b) providing a second homodimeric protein comprising an immunoglobulin Fc region, wherein the Fc region comprises a second CH3 region,
Here, the sequences of the first CH3 region and the second CH3 region are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is different from the first CH3 region and the second CH3 region. Seems to be stronger than each of the homodimeric interactions of the second CH3 region,
c) incubating the first protein with the second protein under reducing conditions sufficient to allow cysteines in the hinge region to cause disulfide bond isomerization, and
d) obtaining said heterodimeric protein.   The first homodimeric protein and the second homodimeric protein are (i) an Fc region, (ii) an antibody, (iii) a fusion protein comprising an Fc region, and (iv) a prodrug, a peptide, a drug The in vitro method according to claim 1, wherein the in vitro method is selected from the group consisting of Fc regions bound to toxins.   The in vitro method of claim 1, wherein the first homodimeric protein is a full length antibody.   The in vitro method according to any one of the preceding claims, wherein the second homodimeric protein is a full length antibody.   The in vitro method according to any of the preceding claims, wherein the first and second homodimeric proteins are both antibodies and bind to different epitopes.   The Fc region of the first homodimeric protein is of an isotype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, and the Fc region of the second homodimeric protein is IgG1, IgG2 The in vitro method according to any one of the preceding claims, which is of an isotype selected from the group consisting of: IgG3, and IgG4.   The in vitro method according to any one of the preceding claims, wherein the Fc regions of both the first homodimeric protein and the second homodimeric protein are of the IgG1 isotype.   The in vitro method according to any one of the preceding claims, wherein one of the Fc regions of the homodimeric protein is of the IgG1 isotype and the other is of the IgG4 isotype.   Any of the preceding claims, wherein the increase in the strength of heterodimeric interactions compared to each of the homodimeric interactions is due to CH3 modification other than the introduction of covalent bonds, cysteine residues or charged residues. The in vitro method according to one of the claims.   The heterodimeric interaction between the first protein and the second protein in the resulting heterodimeric protein is caused by Fab arm exchange at 0.5 mM GSH under the conditions described in Example 13. The in vitro method according to any one of the preceding claims, which is astonishing.   The heterodimeric interaction between the first protein and the second protein in the resulting heterodimeric protein causes Fab arm exchange in vivo in mice under the conditions described in Example 14 The in vitro method according to any one of the preceding claims, which is as such.   If the heterodimeric interaction between the first protein and the second protein in the resulting heterodimeric protein is determined, for example, as described in Example 30, then two homodimers The in vitro method according to any one of the preceding claims, wherein the interaction is more than 2 times stronger, for example more than 3 times stronger, such as more than 5 times stronger than the strongest one.   When the sequences of the first CH3 region and the second CH3 region are assayed as described in Example 30, between the first protein and the second protein in the resulting heterodimeric protein The in vitro method according to any one of the preceding claims, wherein the dissociation constant of the heterodimer interaction is such that it is less than 0.05 micromolar.   When the sequences of the first CH3 region and the second CH3 region are assayed as described in Example 21, the dissociation constant of both homodimer interactions is greater than 0.01 micromolar, for example 0.05 micromolar. More preferably, 0.01 to 10 micromoles, such as 0.05 to 10 micromoles, more preferably 0.01 to 5 micromoles, such as 0.05 to 5 micromoles, even more preferably 0.01 to 1 micromoles, such as 0.05 to 1 micromole, The in vitro method according to any one of the preceding claims, which is such that it is 0.01 to 0.5 micromolar or 0.01 to 0.1 micromolar.   The in vitro method according to any one of the preceding claims, wherein the sequences of the first CH3 region and the second CH3 region comprise amino acid substitutions at non-identical positions.   16. The in vitro method of claim 15, wherein the amino acid substituent is a naturally occurring amino acid or a non-naturally occurring amino acid.   The first homodimeric protein has only one amino acid substitution in the CH3 region and the second homodimeric protein has only one amino acid substitution in the CH3 region as compared to the wild type CH3 region. In vitro method according to any of the preceding claims.   The first homodimeric protein has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407 and 409, and the second homodimeric protein is , 368, 370, 399, 405, 407 and 409, having an amino acid substitution at a position selected from the group consisting of 368, 370, 399, 405, 407 and 409, wherein the first homodimeric protein and the second homodimeric protein are the same 17. The in vitro method according to any one of the preceding claims, wherein the in position is not substituted.   From the group consisting of 366, 368, 370, 399, 405 and 407, wherein the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein 19. An in vitro method according to any one of the preceding claims, having amino acid substitutions at selected positions.   21. The above-mentioned, wherein the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has an amino acid other than Phe at position 405. An in vitro method according to any one of the preceding claims.   Said claim that the first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein has an amino acid other than Phe, Arg or Gly at position 405 Item 19. The in vitro method according to any one of Items 1 to 18.   The first homodimeric protein comprises Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein is an amino acid other than Phe at position 405 and position 409 19. An in vitro method according to any one of the preceding claims, comprising Lys.   The first homodimeric protein contains Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein is other than Phe, Arg or Gly at position 405 19. An in vitro method according to any one of the preceding claims, comprising an amino acid and Lys at position 409.   The first homodimeric protein contains Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has Leu at position 405 and Lys at position 409 19. An in vitro method according to any one of the preceding claims, comprising.   The first homodimeric protein comprises Phe at position 405 and Arg at position 409, and the second homodimeric protein comprises an amino acid other than Phe, Arg or Gly at position 405 and Lys at position 409, 19. An in vitro method according to any one of the preceding claims.   19. The method according to any one of the preceding claims, wherein the first homodimeric protein comprises Phe at position 405 and Arg at position 409 and the second homodimeric protein comprises Leu at position 405 and Lys at position 409. An in vitro method according to any one of the preceding claims.   The first homodimeric protein comprises an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein comprises Lys at position 409, Thr at position 370 and Leu at position 405, 19. An in vitro method according to any one of the preceding claims.   19. The method according to any one of the preceding claims, wherein the first homodimeric protein comprises Arg at position 409 and the second homodimeric protein comprises Lys at position 409, Thr at position 370 and Leu at position 405. An in vitro method according to any one of the preceding claims.   The first homodimeric protein contains Lys at position 370, Phe at position 405 and Arg at position 409, and the second homodimeric protein is Lys at position 409, Thr at position 370 and Leu at position 405. 19. An in vitro method according to any one of the preceding claims, comprising   The first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has Tyr, Asp, Glu, Phe, Lys, Gln, Arg at position 407 19. An in vitro method according to any one of the preceding claims, having an amino acid other than Ser or Thr.   The first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409 and the second homodimeric protein at position 407 Ala, Gly, His, Ile, Leu, Met, Asn 19. An in vitro method according to any one of the preceding claims, having Val or Trp.   The first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has Gly, Leu, Met, Asn or Trp at position 407, said 19. An in vitro method according to any one of the preceding claims.   The first homodimeric protein has Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has Tyr, Asp, Glu, at position 407 19. An in vitro method according to any one of the preceding claims having an amino acid other than Phe, Lys, Gln, Arg, Ser or Thr and Lys at position 409.   The first homodimeric protein has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has an Ala, Gly, His, at position 407 19. An in vitro method according to any of the preceding claims, having Ile, Leu, Met, Asn, Val or Trp and Lys at position 409.   The first homodimeric protein has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein has a Gly, Leu, Met, at position 407 19. An in vitro method according to any one of the preceding claims having Asn or Trp and Lys at position 409.   The first homodimeric protein has Tyr at position 407 and Arg at position 409, and the second homodimeric protein has Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser at position 407 19. An in vitro method according to any one of the preceding claims, having an amino acid other than Thr and Lys at position 409.   The first homodimeric protein has Tyr at position 407 and Arg at position 409, and the second homodimeric protein at position 407 Ala, Gly, His, Ile, Leu, Met, Asn, Val Or an in vitro method according to any one of the preceding claims having Trp and Lys at position 409.   The first homodimeric protein has Tyr at position 407 and Arg at position 409, and the second homodimeric protein has Gly, Leu, Met, Asn or Trp at position 407 and Lys at position 409 19. An in vitro method according to any one of the preceding claims, having. The first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein is
(i) an amino acid other than Phe, Leu and Met at position 368, or
(ii) Trp at position 370, or
(iii) The in vitro method according to any one of the preceding claims, having an amino acid other than Asp, Cys, Pro, Glu or Gln at position 399. The first homodimeric protein has Arg, Ala, His or Gly at position 409 and the second homodimeric protein is
(i) Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val or Trp at position 368, or
(ii) Trp at position 370, or
(iii) at position 399 Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr
19. An in vitro method according to any one of the preceding claims, comprising The first homodimeric protein has an Arg at position 409 and the second homodimeric protein is (i) at position 368: Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val or Trp Or
(ii) Trp at position 370, or
(iii) Phe, His, Lys, Arg or Tyr at position 399
19. An in vitro method according to any one of the preceding claims, comprising   The in vitro method according to any one of the preceding claims, wherein the first and second CH3 regions comprise the sequence set forth in SEQ ID NO: 1 except for the indicated mutations.   The in vitro method according to any one of the preceding claims, wherein neither the first homodimeric protein nor the second homodimeric protein comprises a Cys-Pro-Ser-Cys sequence in the hinge region .   The in vitro method according to any one of the preceding claims, wherein both the first homodimeric protein and the second homodimeric protein comprise Cys-Pro-Pro-Cys sequences in the hinge region.   The in vitro method according to any of the preceding claims, wherein the first homodimeric protein and the second homodimeric protein are human antibodies, except for any mutations specified.   The in vitro method according to any one of the preceding claims, wherein the first homodimeric protein and the second homodimeric protein are heavy chain antibodies.   47. The in vitro method according to any one of the preceding claims, wherein both the first homodimeric protein and the second homodimeric protein further comprise a light chain.   48. The in vitro method of claim 47, wherein the light chains are different.   The in vitro method according to any one of the preceding claims, wherein the first and / or second homodimeric protein comprises a mutation that removes the acceptor site of Asn-linked glycosylation.   The in vitro method according to any one of the preceding claims, wherein the first and second homodimeric proteins provided in steps a) and b) are purified.   The in vitro method according to any one of the preceding claims, wherein the first and / or second homodimeric protein is linked to or contains an acceptor group for a drug, prodrug or toxin.   52. The in vitro method according to any one of the claims 4-51, wherein the first and / or second epitope is located on a tumor cell.   56. The in vitro method according to any one of the claims 4-52, wherein the first or second epitope is located on a tumor cell and the other epitope is located on an effector cell.   54. The in vitro method according to any one of the claims 4-53, wherein the epitope is located on T cells as on CD3 expressed on T cells.   The first or second epitope is located on a tumor cell, and the other epitope is located on a radioactive isotope, a toxin, a drug or a prodrug, which may optionally be coupled or linked to a peptide or hapten 56. An in vitro method according to any one of the claims 4-52.   56. The in vitro method according to any one of the claims 4-52, wherein the first or second epitope is located on a tumor cell and the other epitope is located on a high electron density vesicle or minicell.   53. The in vitro method according to any one of the preceding claims, wherein the homodimeric protein is both an antibody and the first and second antibodies bind to different epitopes on the same tumor cell.   Both homodimeric proteins are antibodies and the first antibody binds to an epitope on tumor cells and the other antibody is unrelated or inactive without any in vivo binding activity associated with the intended use 53. The in vitro method according to any one of the preceding claims, which is an antibody.   The reducing conditions in step c) include the addition of a reducing agent such as a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris (2-carboxyethyl) phosphine or a chemical derivative thereof An in vitro method according to any one of the claims.   The in vitro method according to any of the preceding claims, wherein step c) is performed under reducing conditions with a redox potential of -150 to -600 mV, for example -250 to -400 mV.   Any of the preceding claims, wherein step c) comprises incubation for at least 90 minutes at a temperature of at least 20 ° C in the presence of at least 25 mM 2-mercaptoethylamine or in the presence of at least 0.5 mM dithiothreitol. An in vitro method according to any one of the preceding claims.   The in vitro method according to any one of the preceding claims, wherein step d) comprises removal of the reducing agent, for example by desalting. Methods for Selection of Bispecific Antibodies with Desired Properties, Including the Following Steps:
a) providing a first set of homodimeric antibodies comprising antibodies with different variable regions, wherein the first set of antibodies comprises the same first CH3 region,
b) providing a second set of homodimeric antibodies comprising antibodies with different variable regions or the same variable region, wherein the second set of antibodies comprises the same second CH3 region,
Here, the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is different from the first CH3 region and the second CH3 region. Seems to be stronger than each of the homodimeric interactions of the CH3 domain,
c) incubating the combination of said first and said second set of antibodies under reducing conditions sufficient to allow the cysteines in the hinge region to undergo disulfide bond isomerization. , Thus creating a set of bispecific antibodies,
d) optionally returning the conditions to non-reducing conditions,
e) assaying the resulting set of bispecific antibodies for a given desired property, and
f) selecting bispecific antibodies with the desired properties.   64. The method of claim 63, wherein the second set of homodimeric antibodies have different variable regions.   64. The method of claim 63, wherein the second set of homodimeric antibodies have identical variable regions but different amino acids or structural changes outside the antigen binding region. Methods for producing heterodimeric proteins comprising the following steps:
a) providing a first nucleic acid construct encoding a first polypeptide comprising an immunoglobulin first Fc region, wherein the first Fc region comprises a first CH3 region,
b) providing a second nucleic acid construct encoding a second polypeptide comprising an immunoglobulin second Fc region, wherein the second Fc region comprises a first CH3 region,
Here, the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first CH3 region and the second CH3 region is different from the first CH3 region and the second CH3 region. It is more likely than each of the homodimeric interactions of the CH3 domain, and wherein said first homodimeric protein has an amino acid other than Lys, Leu or Met at position 409, and The second homodimeric protein has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 407,
And / or where the sequences of the first and second CH3 regions are 0.01 to 10 microns when the dissociation constant of each homodimer interaction of the CH3 region is assayed as described in Example 21 Moles, for example 0.05 to 10 micromoles, more preferably 0.01 to 5 micromoles, for example 0.05 to 5 micromoles, even more preferably 0.01 to 1 micromoles, for example Such as being 0.1 micromolar,
c) co-expressing said first and second nucleic acid constructs in a host cell, and
d) obtaining the heterodimeric protein from cell culture. The first CH3 region has an amino acid other than Lys, Leu or Met at position 409, and the second CH3 region has an amino acid other than Phe at position 405, such as other than Phe, Arg or Gly at position 405 ,
Or the first CH3 region has an amino acid other than Lys, Leu or Met at position 409 and the second CH3 region is other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 With amino acids of
68. The method of claim 66.   68. The method of claim 66 or 67, wherein the first and second polypeptides are full-length heavy chains of two antibodies that bind to different epitopes.   69. The method of any of the above claims 66-68, wherein step c) further comprises co-expressing one or more nucleic acid constructs encoding the light chain in a host cell.   70. The method of any of claims 66-69, further comprising the features of any one or more of claims 2-58.   An expression vector comprising the nucleic acid construct as defined in any one of claims 66 to 70.   A host cell comprising the nucleic acid construct as defined in any one of claims 66 to 70.   73. A heterodimeric protein obtained or obtainable by the method of any one of the preceding claims. A first polypeptide comprising an immunoglobulin first Fc region, wherein the first Fc region comprises a first CH3 region, and an immunoglobulin second, wherein the second Fc region comprises a second CH3 region A heterodimeric protein comprising a second polypeptide comprising an Fc region of said first and second CH3 regions, wherein the sequences of said first and second CH3 regions are different, and said first CH3 region and second CH3 region The heterodimeric interaction with the domain is such that it is stronger than each of the homodimeric interactions of the first CH3 domain and the second CH3 domain, and wherein the first homo The dimeric protein has an amino acid other than Lys, Leu or Met at position 409, and the second homodimeric protein is selected from the group consisting of 366, 368, 370, 399, 405 and 407 Have an amino acid substitution at the position,
And / or where the sequences of the first and second CH3 regions are 0.01 to 10 microns when the dissociation constant of each homodimer interaction of the CH3 region is assayed as described in Example 21. Moles, for example 0.05 to 10 micromoles, more preferably 0.01 to 5 micromoles, for example 0.05 to 5 micromoles, even more preferably 0.01 to 1 micromoles, for example 0.05 to 1 micromole, 0.01 to 0.5 micromole or 0.01 to Which is like 0.1 micromolar,
Said heterodimeric protein. The first CH3 region has an amino acid other than Lys, Leu or Met at position 409, and the second CH3 region has an amino acid other than Phe at position 405, such as other than Phe, Arg or Gly. Or The first CH3 region has an amino acid other than Lys, Leu or Met at position 409 and the second CH3 region at position 407 includes Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Having an amino acid other than Thr,
74. The heterodimeric protein of claim 73.   76. The heterodimeric protein of claim 74 or 75, wherein the first and second polypeptides are full-length heavy chains of two antibodies that bind to different epitopes.   77. The heterodimeric protein of claim 76, further comprising two full length light chains.   79. A heterodimeric protein according to any one of the claims 73-77, further comprising the features of any one or more of claims 2-58.   79. A heterodimeric protein according to any one of claims 74 to 78 for use as a medicament.   80. A heterodimeric protein according to claim 79 for use in the treatment of cancer.   79. A pharmaceutical composition comprising the heterodimeric protein of any one of claims 74-78 and a pharmaceutically acceptable carrier.   79. For inhibiting the growth and / or proliferation of tumor cells, comprising the administration of the heterodimeric protein according to any one of claims 74 to 78 to an individual in need thereof, and / or a tumor cell Method for the death of

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